The Structural, Biophysical, and Functional Characterization of the CSL-RITA Complex: Similarities and Differences in Notch Transcriptional Regulation

The structural, biophysical, and functional characterization of the CSL-RITA complex: similarities and differences in Notch transcriptional regulation.

A dissertation submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy

in the Department of Molecular Genetics, Biochemistry, and Microbiology

of the College of Medicine

Nassif H. Tabaja

B.S. University of Cincinnati

June 2016

Committee Chair: Dr. Rhett A. Kovall, Ph.D.

Abstract

The Notch pathway is an essential component of metazoan development and tissue homeostasis. Dysfunctional Notch signaling has been linked with cardiovascular disease, birth defects, and various cancers. Notch signaling ultimately results in changes in gene expression, which is regulated by the DNA binding protein CSL. CSL functions as both a repressor and activator of transcription from Notch target genes by interacting with transcriptional corepressors and coactivators, respectively. A new transcriptional coregulator has been identified, termed RITA, which binds CSL and facilitates its export out of the nucleus. RITA is thought to function as a corepressor by preventing the assembly of active transcription complexes at Notch target genes.

However, the molecular details of the CSL-RITA complex are unknown. In this work, using a combination of biophysical, biochemical/cellular, and structural techniques, we characterize the interaction between CSL and RITA. Chapter 1 is a brief overview of the Notch signaling pathway, from pathway discovery to present day. Chapter 2 contains the structural, biophysical, and functional characterization of the CSL-RITA complex, in which the high affinity interaction between CSL and RITA is demonstrated.

Chapter 3 consists of comparative binding analysis of known CSL binding partners using CSL point mutations shown to disrupt CSL activation. Finally, Chapter 4 is a summary of this work as it pertains to future directions in the Notch field.

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Acknowledgments

First and foremost, I am tremendously thankful for the United States and the taxpayers of this great country. In these tumultuous times, the US is a beacon of scientific progress and free thinking. As developing and war-torn countries struggle to find sustainable growth, global citizens look to countries like the US as examples of the growth potential of public capital investments. Without public capital investment into the

National Institutes of Health and the National Science Foundation, this body of work and much of modern science would not be possible.

I am also very grateful to have been surrounded by many great mentors during my many years at the University of Cincinnati. Without whom, I may never come to have known, appreciated, or struggled through the scientific process. My undergraduate cell biology professor at UC, Dr. Katherine Tepperman was the first to open my eyes to the opportunities in research. She was very passionate about research outreach and even dedicated an undergraduate seminar series to careers in science. Taking her classes was the spring board for me to pursue bench research. Thank you, Dr. Tepperman.

Because there are fewer people in graduate school, it became easier to ask and take advice. My PI, Dr. Rhett Kovall, and the senior graduate students and researchers in the lab quickly became my scientific and social mentors. Thank you to all Kovall Lab members past and present for making the lab a friendly collaborative environment. I am eternally grateful for the patience from and mentoring by, Rhett. Dr. Kovall taught me how to conduct good research and how to communicate the importance of basic science. I originally came to meet Rhett interviewing for a student lab tech position in his lab. Under his training, I gained the confidence and skills necessary to pursue a

graduate career in science. I am also very grateful for our senior researcher, Dr. Zhenyu

Yuan who has provided me with many valuable troubleshooting and optimization tips and prevented me from burning down the lab. Also, thank you to Dr. Ken Greis and his mass spectrometry core facility for their services. Countless hours and possibly mass spectrometers were saved in performing sample validation for me and many other students. Thank you to all the great instructors at UC, including my thesis committee.

While teaching the graduate students about the mechanism of genetic linkage, Dr. Anil

Menon conducted a parallel classroom experiment demonstrating its principles. Dr.

Andrew Herr Fourier transformed a rabbit into a duck to demonstrate the potential for bias in structural modeling. Dr. William Miller illustrated the modular design of taste buds and GPCR architecture by shedding light on studies of light sensing taste buds!

Thank you all for making it a passion and not a chore.

Thank you to my family for your love and support. If I was ever short or just starting at my computer while you talked (which I know I did), I apologize. My brothers and parents have been patient with me and I am very grateful. My mom and dad have finally stopped asking me what I am going to do when I grow up. Thank you, both! My brothers Justin, Mark and Paul have been my support structure for a majority of graduate school. They are the best three brothers a guy could ask for. I am very thankful to have them in my life. I am also very thankful for my wife, Faten. My second wind, she has been patient and kind. I am forever grateful to have her in my life.

Table of Contents

Abstract ...... ii Acknowledgments ...... iv List of Abbreviations ...... viii Chapter 1: The Notch Signaling Pathway ...... 1 INTRODUCTION ...... 2 Figure 1: The Notch phenotype originally identified in Drosophila melanogaster...... 4 PATHWAY COMPONENTS...... 6 Figure 2: The three central components of the Notch Signaling Pathway ...... 7 SIGNALING TO TRANSCRIPTION ...... 12 Figure 3: The Notch pathway from signaling to transcrption ...... 15 STRUCTURAL AND BIOPHYSICAL CHARACTERIZATION OF CSL TRANSCRIPTIONAL COMPLEXES ...... 16 Figure 4: Structures of CSL complexes determined by X-ray crystallography ...... 17 NOTCH TARGET GENES AND PATHWAY FUNCTIONS ...... 19 DYSFUNCTIONAL NOTCH SIGNALING AND HUMAN HEALTH ...... 21 PROJECT DIRECTIONS ...... 26 Chapter 2: Structure and biophysical characterization of the CSL-RITA interaction: a repressor of Notch target gene transcription ...... 28 ABSTRACT ...... 29 INTRODUCTION ...... 30 Figure 1: CSL-mediated repression and activation of Notch target gene transcrption ...... 32 Figure 2: RITA domain schematic, secondary structure content, and sequence alignment ...... 35 EXPERIMENTAL PROCEDURES ...... 36 RESULTS...... 40 STRUCTURE OF THE CSL-RITA COMPLEX ...... 40 Table 1: Data collection and refinement statistics ...... 42 Figure 3: CSL-RITA complex bound to DNA and structural alignemnt ...... 45 THERMODYNAMIC ANALYSIS OF CSL-RITA COMPLEX FORMATION ...... 46 Table 2: Calorimetric data for RITA binding to CSL ...... 47 Figure 4: Analysis of the CSL-RITA interaction by ITC ...... 48 Table 3: Temperature dependence of RITA binding to CSL ...... 51 Table 4:: Calorimetric data for RITA binding to CSL mutants ...... 54 Table 5:: Calorimetric data for acetylated and phosphorylated RITA binding to CSL ...... 55 CELLULAR CHARACTERIZATION OF THE CSL-RITA COREPRESSOR COMPLEX ...... 56 Figure 5: Cellular reporter assays of RITA-mediated repression in the context of CSL mutants ...... 58 Figure 6: Cellular reporter assays of RITA-mediated repression in the context of RITA mutants ...... 59 DISCUSSION ...... 60 Chapter 3: Comparative analysis of CSL binding proteins ...... 65 ABSTRACT ...... 66 INTRODUCTION ...... 67 EXPERIMENTAL PROCEDURES ...... 69 RESULTS...... 70 THERMAL STABILITY OF CSL COMPLEXES ...... 70 Figure 1: Thermal Stability Shift Assay data for CSL in the absence and presence of ligands...... 72 THERMODYNAMIC ANALYSIS OF RITA, RAM, KYOT2, EBNA2, AND MINT BINDING CSL MUTANTS...... 74

Table 1: Calorimetric data for RITA binding to CSL ...... 73 Table 2: Calorimetric data for binding to CSL mutant F261L ...... 75 Table 3: Calorimetric data for binding to CSL mutant Q333L ...... 76 THERMODYNAMIC ANALYSIS OF THE HYDROPHOBIC TETRAPEPTIDE MOTIF AND SALT BRIDGE ...... 77 Table 4: Calorimetric data for the hydrophobic tetrapeptide motif binding to CSL ...... 78 Figure 2: CSL binding partners and the hydrophobic tetrapeptide motif ...... 79 Figure 3: The CSL-KyoT2 interaction may require salt bridge interaction ...... 81 Table 5: Calorimetric data for RITA and RAM binding to CSL salt bridge mutants ...... 82 DISCUSSION ...... 83 Figure 4:Structural models and mutaional analysis ...... 84 Supplementary Figure 1: Thermodynamic data for CSL cofactors binding to CSL RAM-binding deficinet mutants ...... 86 Chapter 4: Conclusions and Future Directions ...... 90 KEEPING THE NOTCH SIGNALING PATHWAY IN CONTEXT ...... 91 CSL IN THE NUCLEUS AND THE CYTOPLASM ...... 92 RITA AND TRANSCRIPTIONAL REPRESSION ...... 93 NOTCH THERAPEUTICS: A NEW HOPE ...... 94 FUTURE DIRECTIONS IN NOTCH PATHWAY RESEARCH ...... 97 Figure 1: The expansion of knowledge ...... 99 Bibliography ...... 100 CHAPTER 1 ...... 101 CHAPTER 2 ...... 122 CHAPTER 3 ...... 127 CHAPTER 4 ...... 131 Appendix A: A phospho-dependent mechanism involving NCoR and KMT2D controls a committed chromatin state at Notch target genes ...... 133 Appendix B: Unanticipated Structural Plasticity of CSL as Revealed by the X-ray structure of the Su(H)-Hairless Repressor Complex ...... 152 Appendix C: A re-engineered humanized anti-cocaine monoclonal antibody: analysis of its temperature- and pH-stability and energetics of ligand binding ...... 205

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List of Abbreviations

ANK ankyrin repeats BTD β-trefoil Domain CD Circular Dichroism CIR CBF-1 Interacting Repressor CSL CBF-1, Su(H), Lag-1 CtBP C-terminal Binding Protein CTD C-terminal Domain CtIP CtIP Interacting Protein DNA Deoxyribonucleic Acid DSL Delta, Serrate, Lag-2 EGF Epidermal Growth Factor EMSA Electrophoretic Mobility Shift Assay ETO Eight Twenty One GST Glutathione S-Transferase HD Heterodimerization Domain HDAC Histone Deacetylase Complex HDM Histone Demethylase HEPES 4-(2-HydroxyEthyl)-1-PiperazineEthanesulfonic acid HRP Horseradish Peroxidase IG Immunoglobulin IPTG Isopropyl β-D-1-ThioGalactopyranoside ITC Isothermal Titration Calorimetry kD KiloDalton LB Luria-Bertani medium LNR Lin12/Notch Repeats MEF Mouse Embryonic Fibroblast MES 2- (n-Morpholino) Ethanesulfonic acid MINT Msx2 Interacting Nuclear Target MLL2 Myeloid/Lymphoid or Mixed-Lineage Leukemia 2 NCoR Nuclear receptor Corepressor 1 NLS Nuclear Localization Sequence NMR Nuclear Magnetic Resonance NICD Notch Intracellular domain NRR Negative Regulator Region NTD N-terminal Domain PBS Phosphate Buffered Saline PEST Proline, Glutamine, Serine, Threonine RAM RBP-Jκ Associated Molecule RMSD Root Mean Square Deviation RRM RNA Recognition Motif RU Response Units SD Standard Deviation SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SEM Standard Error of the Mean

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SHARP SMRT/HDAC Associated Repressor Protein SKIP Ski-Interacting Protein SMRT Silencing Mediator of Retinoid and Thyroid hormone receptors SPOC Spen Paralog and Ortholog C-terminal SPR Surface Plasmon Resonance SPS Sequence Paired Site T-ALL T-cell Acute Lymphoblastic Leukemia TCEP Tris(2-CarboxyEthyl)Phosphine

Δ delta g gram μ micro m milli n nano Å Angstrom °C degrees Celsius K degrees Kelvin M Molar kcal kilo-calories mol Mole, 6.02*1023 ΔG change in free energy ΔH change in enthalpy ΔS change in entropy ΔCp heat capacity Kd dissociation constant Ka association constant

Chapter 1: The Notch Signaling Pathway - Standing on the shoulders of giants and the Notched wings of flies.

“If I have seen further, it is by standing on the shoulders of giants.” – Sir Isaac Newton

Isaac Newton made this statement in 1676 (1). 340 years later, the relatively young field of molecular biology stands on the shoulders of scientific giants. To gain a better appreciation for the scientific process, its giants, and the progression of Notch from a phenotype to a field, the introduction of this thesis presents the discovery of the

Notch signaling pathway in a historical perspective. A little over 150 years ago, in 1859,

Charles Darwin described evolution and his theory of inheritance (2). However, Darwin lacked a mechanism of inheritance until Gregor Mendel completed his famous pea plant experiments in 1865, priming the future for the age of molecular genetics (3).

Introduction

In 1914, during the infancy of genetics, Drosophila geneticist John S. Dexter was making observations on an inbred stock of fruit flies gifted from fellow geneticist Thomas

H. Morgan (4). This particular inbred lineage of Drosophila melanogaster presented a

“beaded wing” phenotype that had puzzled Morgan for years. Morgan had previously observed that the phenotypes of these “beaded” flies were all wing related but presented very differently across individual flies (5,6). Also, progeny of the mutant flies were predominately female, suggesting a haploinsufficient sex-linked gene (4-6).

Dexter was able to breed these flies into a stable wing phenotype he initially termed

“Perfect Notch” (Figure 1) (4). Three years later, in 1917, Morgan would go on to identify the gene locus responsible for the mutant phenotype he termed, Notch-wing (7-

9). It wasn’t until 1985, that the Notch receptor would be cloned, both from D. melanogaster and C. elegans, and confirmed as the gene associated with the Notch- wing phenotype first observed by Dexter and Morgan (10,11). This would be seminal

work in showing that the Notch receptor was a transmembrane protein that mediated cell-to-cell communication (12).

Figure 1: The Notch phenotype originally identified in Drosophila melanogaster. The wing of a wild-type fruit fly on the left displays no phenotypic abnormalities. On the right is a sample from a mutant fly, with only one functioning Notch allele. The arrows point to obvious phenotypic abnormalities on the wing margin that look like a notches. This figure is adapted from [8].

The human Notch homolog would later be discovered in 1991 opening the door to pathway elucidation and NIH funding (13-15). The discovery of the human Notch gene described a series of mutations in the gene found in certain T-cell malignancies that resulted in truncated gene products lacking part or all of the extracellular domain

(13). In 1993, these mutations led to results in D. melanogaster and C. elegans that demonstrated nuclear localization and constitutive activity of the intracellular portion of the Notch receptor (16,17). Since the identification of the Notch-receptor and the subsequent identification of the Notch receptor gene, the Notch signaling pathway has become its own field in molecular biology. In 1985, there were approximately 70 Notch- related publications. In 2015, there were over 1,600 Notch-related publications deposited in PubMed. The field boasts almost 20,000 total PubMed entries to date.

The Notch signaling field has greatly expanded in parallel to the advances made by molecular biology techniques. As the field has expanded, the signaling pathway has been shown to be critical for the maintenance of stem cell populations and many cell- fate decisions in embryogenesis, neurogenesis, angiogenesis, cardiovascular development, skeletal development, intestinal development, and T-cell/B-cell lineage commitment (18-30). Given the central role Notch signaling plays in a wide variety of biological systems, it is not surprising that dysfunctional Notch signaling is pleiotropic resulting in diverse disease presentation. Dysfunctional Notch signaling leads to many severe developmental diseases such as Alagille syndrome, spondylocostal dysostosis, and T-cell acute lymphoblastic leukemia, to name a few (31-38). Absence of Notch signaling stalls embryonic development emphasizing the importance of the Notch pathway (39).

In this chapter, we review Notch signaling components, the pathway, and its target genes. More specifically, we introduce the literature describing Notch pathway transcriptional complexes. This review also outlines roles of Notch signaling in the development and maintenance of different biological systems. We also describe phenotypes and underlying molecular causes of various Notch related diseases along with current therapies and therapy strategies. The conclusion will focus on how the proceeding body of work will contribute to the growing body of literature in the Notch field, and ultimately contribute to the development of modulators of the Notch signaling pathway for therapeutic use.

Pathway Components

The Notch signaling pathway owes some of its discovery to the simplicity of its design. As seminal studies demonstrated, the Notch receptor essentially tethered a transcription factor to the membrane until which time a ligand receptor interaction released the intracellular portion of the Notch receptor to act on transcription in the nucleus (12,13,16,17,40). Therefore, there are at least three essential components to this path: a ligand, a receptor, and a nuclear effector (Figure 2). Ligands and receptors in the Notch pathway are all type I single-pass transmembrane proteins that are made up of mostly EGF (epidermal growth factor)-like repeats (10,12,14). Structures of ligands and receptors show both have an elongated form and that binding occurs as an antiparallel interaction (41-46). Furthermore, a human Notch structure reveals points of flexibility because of EGF-like repeat architecture (47).

Figure 2: The three central components of the Notch Signaling Pathway. (A) Domain shematic showing the basic domain composition of DSL (Delta, Serrate, Lag1) ligands. From left to right, DSL ligands consist of the MNNL (module at the N- terminus of Notch ligands) and DSL domains (green), EGF (epidermal growth factor)- like repeats (cyan), and transmembrane domain (black), as well as, an intracellular C- terminus. (B) Domain shematic showing the domain compostion of Notch. From left to right, the Notch ECD (extracellular domian) consits of tandam EGF-like repeats (cyan), and the NRR (negative regulatory region). The NRR contains three LNR (LIN12/Notch repeat) domains and a HD (heterodimerization) domain and helps protect the receptor from premature S2 cleavage. The NICD (Notch intracellular domain) consist of the RAM (RBPJκ associated molecule) domain (red), the ANK (ankyrin repeat) domain (yellow), and the PEST (proline, glutamate, serine, and threonine) domain (maroon). (C) The nuclear effector of the Notch pathway, CSL (CBF1/RBPJ , Su(H), LAG-1), is shown as the NTD (N-terminal domain) in cyan, the BTD (β-trefoil domain) in green, and the CTD (C-terminal domain) in orange.

The canonical Notch pathway ligand is DSL (Delta in mammals, Serrate in flies,

LAG-2 in worms). In mammals, there are five DSL ligands, Jagged1,2 and Dll (Delta- like)1,3,4. D. melanogaster and C. elegans genomes only encode for two DSL paralogs each, Delta and Serrate in fly and Lag2 and Apx1 in worm (48,49). The extracellular domain of all five mammalian ligands contains an amino-terminal domain, referred to as the MNNL (module at the N-terminus of Notch ligands) (48). The bottom half of the

MNNL is cysteine rich and contains a GBM (glycosphingolipid binding motif) thought to contribute to ligand activity (50-52). Downstream of the GBM is the DSL domain shown to be necessary for interaction with the Notch receptor (50,53). In some ligands, the

DSL domain is proceeded by two EGF-like repeats that contain a DOS (Delta and OSM-

11-like protein) motif that also mediates receptor interaction and is sequence and spacing sensitive (46,54-56).

The mammalian DSL ligands are categorized according to their relation to the two model Drosophila DSL ligands, Serrate and Delta (57-59). Jagged 1 and 2 belong to the Serrate family and are characterized by having a cysteine-rich domain proceeding the extracellular EGF-like repeats (59,60). The aptly named Delta-like ligands (Dll1,3,4) belong to the Delta family of ligands and lack the extracellular cysteine-rich domain

(48,59). Some of the EGF-like repeats contained in the five mammalian DSL ligands are calcium-binding and require calcium for proper ligand-receptor interaction (61,62).

Unlike the other ligands, Dll3 and Dll4 lack the requirement for calcium-binding EGF-like repeats (45). In lower animals, such as D. melanogaster and C. elegans, DSL ligands have homologous function (63-66). Expectedly, in mammals, DSL ligands are not functionally equivalent (54). For example, Dll3, lacking a conserved DSL domain and

functioning as an antagonist of Notch signaling, is the most structural and functionally different DSL ligand and does not rescue loss of Dll1 (67).

The intracellular domains of the DSL ligands are more divergent (68). With the exception of Dll3, all DSL ligands contain sites for potential ubiquitination, required for endocytosis and signaling (54). DSL ligand interactions with Notch receptor can be regulated by ubiquitination simply by limiting the number of surface presented ligands

(69,70). It has been shown that E3 ubiquitin ligases Mindbomb (MiB) and Neuralized

(Neur) promote endocytosis of ligand and activation of receptor, a seemingly contradictory mechanism that will be described in the next section (71,72). While functionally equivalent in Drosophila, MiB and Neur seem to have opposing roles in vertebrates where MiB promotes endocytosis and signaling and Neur promotes ligand recycling and degradation (70,73-76). In vertebrates and fly, loss of MiB or Neur results in dysfunctional Notch signaling and disrupted phenotypes (72,76-79). Furthermore, some DSL ligands contain a C-terminal PDZ (PSD, Dlg, ZO1) ligand motif known to bind elements of the cytoskeleton, it is unclear what role this plays in signaling (68,80-

82). As well as providing valuable insights into surfaces required for receptor-ligand interaction, published structures of DSL ligands have provided molecular explanations for mutations involved in Notch-related diseases, such as Alagille Syndrome (83-85).

To match the diversity of Notch ligands, there are; four Notch receptors in mammals, Notch1-4, two Notch receptors in worm, and one notch receptor in fly (86).

During receptor maturation in the Golgi, in an event termed S1 (site 1) cleavage, Notch is cleaved by a furin like convertase resulting in a heterodimer ready for surface presentation (87,88). It is unclear why S1 cleavage is required for Notch receptor

maturation, as Notch1 requires it for receptor activity, but Notch2 does not (89,90). Like the DSL ligands, all Notch receptors are type 1 single-pass transmembrane receptors and contain extracellular EGF-like repeats (59,86). EGF-like repeats 11-13 of Notch have been shown to be necessary for binding DSL ligands and an x-ray crystal structure confirmed important sites of calcium-binding on these repeats (14,45). Additionally, in mammals and fly, the EGF-like repeats of Notch are O-linked glycosylated by glycotransferases such as POFUT1 (protein O-fucosyltransferase 1), Fringe and Rumi

(91-97). O-linked glycosylation of both O-fucose and O-glucose has been shown to be necessary for ligand interaction and receptor activation (91,93,95,96,98-100).

Conversely, O-linked xylose results in decreased Notch receptor activity (101). These studies suggest the existence of an EGF code of differential O-linked glycosylation and effect on ligand binding. For example, Notch receptor glycosylated by Fringe increase

Notch activation by Delta-like ligands, Dll3 and 4, while reducing Notch activation by the

Serrate-like ligands, Jagged1 and 2 (102,103).

Downstream, of the EGF-like repeats is the NRR (negative regulatory region).

The NRR consists of three LNR (LIN12/Notch repeat) domains and a HD

(heterodimerization) domain. In the absence of membrane bound DSL ligand, the NRR physically obstructs premature cleavage within the HD domain of Notch by the

ADAM/TACE metalloprotease (88). Notch1 NRR structural studies demonstrate the

NRR’s function and validates mutations that lead to constitutively active Notch signaling

(89,104,105). In the intracellular portion of Notch, the NICD (Notch intercellular domain) consists of a RAM (RBPJκ associated molecule) domain (106). Downstream of which is an ANK (ankyrin repeat) domain flanked by a NLS (nuclear localization signal) on each

side. This is followed by a transactivation domain that mediates transcriptional activation. The C-terminal of Notch consists of a PEST domain, rich in proline, glutamic acid, serine, and threonine, that signals for NICD degradation and termination of Notch activity once phosphorylated (107). The four Notch paralogs do not activate Notch target gene transcription to the same extent. Cell-based biophysical studies demonstrated that Notch1, compared to Notch2, is a much more potent activator of

Notch target gene transcription (108). More recent studies demonstrate that Notch3 and

Notch4 can antagonize Notch1 activation by directly inhibiting Notch1 or competing for downstream cofactors (109,110). Similar to the DSL ligands, Notch receptor paralogs are not functionally equivalent and do not fully rescue the knockout of one another (111-

114). The molecular explanations underlying the divergent functions of the Notch homologs have yet to be fully elucidated. Tangentially, the NICD of all four Notch paralogs can associate with CREB (cyclic-AMP response-element binding) protein and antagonizes CRE-dependent transcription (115).

After ligand and receptor, the final logical component for a basic cellular signaling pathway is a nuclear effector. In the Notch signaling pathway, the nuclear effector is termed CSL CBF1/RBPJκ in mammals; SuH in fly; and Lag1 in worm) (116-118).

Across metazoa, there is one highly conserved CSL ortholog, making it a central therapeutic target for a pan-Notch inhibitor (119). The transcription factor CSL consists of three domains: the NTD (N-terminal domain), the BTD (β-trefoil domain), and the

CTD (C-terminal domain) (120). The NTD and CTD are partially homologous to the Rel family of transcription factors (121). Although Rel family transcription factors usually

bind DNA in dimers, the BTD of CSL along with an internal β-strand allows all three domains to form a single DNA-responsive element of Notch signaling (120-122).

CSL serves as a scaffold that can bind the NICD once Notch signaling has been activated and NICD translocates to the nucleus. CSL binds a select few canonical coactivators and a variety of corepressors to form regulation complexes that effect

Notch target gene transcription by binding the CSL regulatory sites of Notch target genes. The CSL promoter is a seven base pair stretch that consists of variations on a

“canonical” -GTGGGAA- motif (123). CSL promoter sequences that diverge from the

“canonical” motif have been shown to have different affinities for CSL suggesting the existence of unidentified Notch target genes (124,125). When Notch signaling is active,

CSL binds the RAM and ANK domains of NICD and recruits a transcriptional activator termed Mastermind (MAM) to form the of the CSL-NICD-MAM ternary activation complex (106,121,126). When Notch signaling is off, CSL is bound to corepressors, such as MINT/SHARP, SMRT, Hairless, Kyot2, and RITA, known antagonist of Notch target gene transcription (30,127-134). MINT, KyoT2, and Hairless have been shown to form high affinity interactions with CSL (appendix) (127,135). Additionally, the corepressor RITA has been shown to export CSL out of the nucleus (134).

Signaling to transcription

Taken together, the data in the Notch field provide for a molecular roadmap of the Notch signaling pathway. The pathway is initiated when a single-pass transmembrane DSL ligand presented on the extracellular surface of signaling cell makes contact with a Notch receptor presented on a neighboring cell (Figure 3A-B). As

described previously, Notch receptor activation requires a membrane-bound or immobilized ligand (136-138). Soluble ligand is unable to activate Notch signaling

(139,140). More importantly, it has been demonstrated that the pulling force caused by ligand endocytosis, upon ligand-receptor interaction, is necessary and sufficient to expose the HD domain in the NRR of Notch for cleavage by the ADAM/TACE metalloprotease in a process termed S2 (site 2) cleavage (141,142).

Using this same logic, DSL ligands presented on the same cell surface as Notch receptor, in cis, will not activate the Notch receptor in a process termed cis inhibition

(143-146). Because of the mechanical pulling force required to move the LNR repeats and expose the S2 cleavage site in the HD domain of the NRR, Notch receptors can only be activated in trans, or in cell-to-cell context (46). A parallel mechanism termed lateral inhibition prevents cells from being both Notch and DSL presenting cell types

(147,148). This relies on seemingly random distributions in which the neighboring cell that has the most Notch signal becomes the Notch presenting cell in the local cluster of cells through a series of feedback loops (149-153). The phenomenon of cis and lateral inhibition allows the Notch signaling pathway to form patterning of cell fate decisions based on cellular location contributing to the pathway’s diverse functions which will be discussed shortly (154,155). These studies further underscore that the Notch pathway is a cell contact based signaling pathway that functions as a biosensor for context based cellular decisions.

After Notch receptor is trans activated by DSL ligand from a neighboring cell and

S2 cleavage has occurred, a membrane bound γ-secretase complex cleaves Notch within the cell membrane to untether NICD allowing it to be translocated to the nucleus

in a process termed S3 (site 3) cleavage (156-158). In the nucleus, NICD binds the nuclear effector of the Notch pathway, CSL, and recruits the coactivator MAM (Figure

3C) (126,159). The CSL-NICD-MAM ternary activation complex binds promoter elements of Notch target genes and scaffolds the assembly of high order complexes that contain histone acetyl transferases and other chromatin remolding enzymes (160).

The local restructuring of the chromatin opens up target genes for transcription and functions as the switch to activate transcription at these sites. MAM also recruits kinases that help terminate activation by targeting MAM and the PEST domain of NICD

(107,161). PEST phosphorylation induces an E3 ligase termed FBXW7, among others, to polyubiquitinate NICD, leading to degradation by the proteasome. A variety of E3 ubiquitin ligases have been shown to mediate proteasomal degradation of NICD and

Notch signal termination (162-164). In the absence of a NICD, CSL cannot form the ternary activation complex. Instead, it interacts with corepressors that recruit chromatin modification machinery, such as histone demethylases and deacetylases, to repress transcription of Notch target genes (128,165,166).

Figure 3: The Notch Pathway from signaling to transcription. (A) The Notch pathway is a juxtacrine signaling mechanism. (B) The pathway is initiated when the extracellular domain of DSL ligand present on one cell binds a Notch receptor on an adjacent cell. This interaction induces the Notch receptor to undergo a series of cleavage events, resulting in the release of the NICD (Notch intracellular domain) from the cell membrane and its subsequent translocation into the nucleus. (C) In the nucleus, NICD binds the transcription factor CSL (CBF1/RBPJ , Su(H), LAG-1) and recruits the coactivator MAM (Mastermind). The CSL-NICD-MAM ternary complex binds promoter elements of Notch target genes and is the switch to activate transcription at these sites. In the absence of a Notch signal, CSL interacts with corepressors to repress transcription of Notch target genes.

Structural and biophysical studies of CSL transcriptional complexes

A growing number of structural and biophysical studies have been critical in understanding how CSL functions as a transcriptional regulator. ITC (isothermal titration calorimetry) has been used to quantify the binding of CSL to DNA, which revealed CSL has a nanomolar affinity for DNA containing both predicted and endogenous CSL binding sites, including variations of the “canonical” -GTGGGAA- CSL binding site preceding the HES1 gene, which will be discussed in the next section

(124,125). The structure of CSL bound to DNA illustrated the domain organization of the NTD, BTD and CTD (PDB ID: 1TTU) (Figure 4A) (120). The complex structure demonstrated that the NTD and BTD bind DNA, providing a molecular explanation for

CSL’s site-specific interactions with DNA (120). Furthermore, this structure was the first to reveal a solvent exposed hydrophobic groove on the BTD of CSL, suggesting the existence of a hydrophobic binding partner (120). The CSL-RAM complex (PDB ID:

3BRD) represents an assembly intermediate of an active CSL complex and illustrates the binding path of the RAM domain of NICD as it binds the BTD of CSL (Figure 4C)

(167). The CSL-RAM structure also demonstrated that RAM binding allosterically effects the NTD of CSL, presenting a more suitable binding surface for ANK and MAM

(167). Additional thermodynamic studies using ITC showed that the RAM domain of

NICD has high affinity for CSL whereas ANK and MAM individually have no detectable binding to CSL (167).

Figure 4: Structures of CSL complexes determined by X-ray crystallography. CSL, NICD, and MAM are all modeled as ribbon diagrams. CSL is shown as the NTD in cyan, the BTD in green, and the CTD in orange. NICD is shown in purple; MAM, in black. DNA is modelled as a stick diagram and colored according to element. (A) CSL bound to DNA binding site (PDB ID: 1TTU) (B) The CSL active ternary complex consisting of CSL-NICD-MAM bound to DNA (PDB ID: 2FO1) (C) Structure of the RAM domain of NICD bound to CSL (PDB ID: 3BRD) (D) The structure of two CSL active ternary complexes bound to a SPS site found on the HES-1 promoter.

Parallel studies into the CSL-RAM interaction revealed a series of conserved motifs in the RAM domains of Notch1-4 that are required for binding the BTD of CSL: a four-residue basic motif, a -HG- repeat motif, a hydrophobic tetrapeptide motif, and a -

GF repeat motif (168). The hydrophobic tetrapeptide motif is a highly conserved motif found on different BTD binding proteins (120,169,170). Binding the hydrophobic BTD groove, the motif consists of four hydrophobic amino acids, φWφP; where φ is any hydrophobic residue (120,167). The φWφP motif is also found on KyoT2 and been shown to be necessary for binding the BTD of CSL for KyoT2, RAM, and the viral activator EBNA2 (106,135,168,170). When CSL is bound to coregulators, RAM, MINT, or KyoT2, CSL’s affinity for DNA is unchanged (127,135,167).

The CSL-NICD-MAM ternary complex structures (PDB ID: 2FO1, 2F8X, and

3V79) show the interaction surface of MAM, which binds an extended groove formed by the CTD-ANK interface and the NTD of CSL (Figure 4B-C) (159,171,172). The mammalian CSL-NICD-MAM ternary complex structure (2F8X) revealed crystal packing that suggested the existence of dimeric CSL ternary complexes interacting via their respective N-terminal ANK domains (173). Interestingly, the CSL promoter for HES1 contains two CSL binding site in a back-to-back configuration termed a SPS (sequence paired site) (174). In agreement, the structure of two CSL ternary activation complexes bound to an oligonucleotide containing the HES1 SPS promoter (PDB ID: 3NBN), reveals that the two activation complexes make contacts through their, respectively bound, ANK domains (Figure 4D) (175).

In addition, corepressor structures of CSL-corepressor complexes bound to DNA have also been appearing in the literature with increasing frequency. The high

resolution x-ray crystal structures of CSL in complex with MINT and CSL in complex with KyoT2 (PDB ID: 4J2X) demonstrate that both corepressors share a common binding surface with the RAM domain of NICD (135). This work identified that both

MINT and KyoT2 bind the BTD of CSL, while MINT also binds the CTD (127,135).

Interestingly, studies have also demonstrated that the major antagonist of Notch signaling in Drosophila, Hairless exclusively also binds the CTD of CSL (SuH) with high affinity. These studies have shed light on how CSL functions as a transcriptional switch, going from repressor to activator and back again. The body of literature concerning structural and biophysical characterization of CSL-corepressor complexes is growing and revealing the molecular mechanisms and requirements for repression of Notch target gene transcription.

Notch target genes and pathway functions

There are a myriad of Notch target genes involved in a wide variety of cellular processes including cell proliferation, cell survival, cell migration, cell cycle progression, and cell differentiation (176-187). Collectively, the fundamental function of Notch signaling is to help in making cell fate decisions (188). Cell fates influenced by Notch are very context dependent, being influenced by factors such as cellular environment and cell pluripotency (189,190). Therefore, Notch signaling, or the absence thereof, is able to lead many different cellular outcomes (191). This positions the Notch signaling pathway to have a central role in organ development and tissue maintenance. The HES

(Hairy and Enhancer of Split) family of genes was the first gene family to be identified as

Notch target genes and encode for bHLH (basic helix-loop-helix) proteins (192). In

mammals, there are seven HES genes, HES1-7, that maintain progenitor cell populations by repressing the expression of cell-fate specific genes (193,194). In the development of the nervous system for example, HES1 prevents neuron specific gene transcription by forming heterodimers with bHLH proneural transcription factors, thus rendering them inactive (195). The HES bHLH family forms heterodimers with other bHLH transcription factors to also repress HES gene transcription (196). This forms a negative feedback loop that oscillates from low to high expression and back again upon

Notch pathway activation (193,196). This oscillatory loop has been shown to be necessary for division and segregation of somites in the developing embryo (197,198).

Pluripotent adult stem cells require the Notch signaling pathway to maintain a functional stem cell population (199). No matter the context, the Notch signaling pathway serves as a logic gate to differentiation. Hematopoiesis and intestinal tissue maintenance, two notable differentiation pathways that are unrelated, require the Notch signaling pathway for cellular differentiation and tissue maintenance (200-202).

Development of hematopoietic cells, the stem cells of the blood, occurs in the embryo and requires the Notch signaling pathway (203,204). This is demonstrated by loss-of-function mutants that lacked Notch receptor, displaying dysfunctional hematopoiesis (205). Downstream of a hematopoietic cell fate, when a cell has differentiated into a common lymphoid progenitor (CLP) cell, Notch signaling mediates the decision of whether to commit to a B-cell or T-cell fate (206). In the presence of

Notch signaling, CLP-cells commit to the T-cell lineage (206,207). In the absence of

Notch signaling, CLP cells will commit to the B-cell fate (208). Once committed to the

B-cell path, immature B-cells will mature to follicular B-cells in the absence of Notch

signaling, while committing to a marginal zone B-cell fate in the presence of Notch signaling (30,207-209).

Parallel cell-fate decisions are made in the epithelium of intestine. Like the blood, intestines have a relatively rapid turnover of cells and thus require robust stem cell populations for continual differentiation and renewal (210). Unlike hematopoietic cells, CBC (crypt base columnar) cells, an intestinal stem cell population, requires Notch activity to maintain the undifferentiated state, strikingly similar to T-cell/B-cell differentiation from CLP cells (27,200). As intestinal stem cells differentiate, Notch signaling is required for CBC cells to differentiate to absorptive progenitor cells (200).

Notch signaling in later stages of intestinal stem cell differentiation suppresses secretory cell fates and promotes an absorptive intestinal cell fate (200,211). This juxtaposition of such two divergent processes serves to illustrate how simple molecular logic gating can be used in a wide variety of contexts. The Notch signaling pathway plays similar roles in the development of almost every biological system, including, but not limited to, cell- fate decisions in: neural progenitor cells, muscle progenitor cells, osteoblasts, and hair follicle stem cells (23,190,212-214).

Dysfunctional Notch Signaling and Human Health

With all the different roles the Notch signaling pathway plays in tissue development and maintenance, it is consistent that dysfunctional signaling has been linked with congenital and adult diseases, solid and hematological cancers, and even viral infection (214-220). As mentioned in the beginning of this chapter, Notch is a haploinsufficient gene. Haploinsufficiency is an emergent property of lateral inhibition, a

stochastically guided mechanism (221). This haploinsufficiency by definition indicates gene dosage effects, which have been experimentally demonstrated in Drosophila (222-

224). Notch related diseases also exhibit gene dosage effects from excessive or insufficient Notch signaling components (13,38,225,226). Since complete loss or gain of function is not required, gene dosage affects lend themselves to therapeutic targeting. Because of the body of literature describing the Notch signaling pathway and its disease phenotypes, the ability to synthesize approaches to modulate Notch signal activity are becoming ever apparent (227-230).

Demonstrating the pleiotropic nature of the Notch pathway, Alagille syndrome is a disorder effecting the liver, heart, skeletal system, nervous system, and kidneys that presents in children (231,232). The disease is caused by mutations of the Jagged1 or

Notch2 genes that result in haploinsufficiency (34,233-235). In Alagille syndrome, the presentation of the disease is a result of the gene-dosage effect of Notch signaling

(225,236). From one perspective, Jagged1 or Notch2 is haploinsufficient possibly resulting in insufficient Notch signaling (32,225). However, from another perspective, haploinsufficiency caused by a broken allele of Jagged1 or Notch2 is caused by excess

Notch target gene repression. As the field’s understanding of Notch signaling pathway activation and repression expands, therapeutics design can be approached using both perspectives allowing for targeting of activation and repression complexes.

The Notch pathway is an essential component of cardiac development and cardiomyocyte differentiation (237). Notch signaling promotes cardiomyocyte differentiation (238). Pathway dysregulation causes a variety of congenital heart defects, such as isolated congenital heart disease and aortic valve disease (239,240).

Moreover, Notch signaling is implicated in adult onset cardiovascular disease (241).

Nonsense mutations in the Notch1 gene play a role in calcific aortic valve disease, a disease characterized by valve malformations and high rates of valve calcium deposition (241). This was hypothesized to be mediated by the role of the Notch pathway in osteoblast differentiation, further highlighting the importance of the pathway’s role in cell fate decisions.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is a disorder that affects the smooth muscle of the circulatory system. This causes the blood vessel wall to thicken and the effective diameter to decrease, leading to constricted blood flow and impaired brain development (242). The disease is characterized by strokes, cognitive impairment, migraines, and mood disorders (243-245). CADASIL is caused by mutations in the Notch3 gene that result in insufficient Notch signaling, as mouse models can be rescued by Notch3 expression

(246-249).

Given the role of the Notch pathway in the intestine, it is not surprising that excessive Notch signaling has been linked with colorectal cancer (250,251).

Furthermore, overactive Notch signaling has been shown to increase the incidence of tumor formation, metastasis, and vascularization (251,252). Elevated levels of Notch1 receptor correlates with poor clinical outcomes (253). Similarly, clinical evidence colorectal cancers associated with gene duplications of Jagged1 correlate with more negative patient outcomes, tumor progression, and increased patient mortality rates

(254). Interestingly, Jagged1 is not necessary for the maintenance of the CBC cell niche in the intestine (255). AES (Amino-terminal Enhancer of Split), has been shown

to decrease the metastasis of colon cancer by repressing Notch target gene transcription (256). Inflammatory bowel syndromes, such as ulcerative colitis, have also been associated with dysfunctional Notch signaling (257). This further highlights the importance of characterizing transcriptional regulation of Notch target genes.

The disease originally identified to be related to Notch signaling is T-ALL (T-cell acute lymphoblastic leukemia) (13). T-ALL is characterized by an overproduction of T- cells caused by excessive cell fate commitment of CLP cells to the T-cell fate (258).

Original studies, identified Notch activating mutations in tumor samples that resulted in

Notch receptor truncations lacking all or most of the extracellular domain (13,38,259).

Subsequent clinical studies have also identified activating mutations in the NRR that allow for premature S2 cleavage and stability mutations in the PEST domain that avoid degradation by the proteasome (38). Similarly, Notch2 gene truncations have been implicated in diffuse large B-cell lymphoma (260). Conversely, the Notch pathway also has been shown to have tumor suppressor roles in some lung, kidney, and thyroid cancers (226,261,262). All of these Notch mutations found in clinical emphasize the effects of gene dosage on critical functions of the Notch signaling pathway.

Viral coevolution has allowed for viruses to co-opt the Notch pathway for host infection establishment and maintenance (220). Two such viruses are KSHV (Kaposi’s sarcoma Herpes Virus) and EBV (Epstein Barr Virus) (263,264). Both viruses are comorbidities of HIV (human immunodeficiency virus) and immunocompromised patients. Most notably, both viruses contain CSL binding sites in their genomes and express novel CSL coactivators and corepressors. Endogenous host CSL is used by

KSHV and EBV for transcriptional activation of their respective viral genomes (265,266).

During the latent/lytic transition of the KSHV life cycle, viral protein RTA (Replication and Transcription Activator) is expressed interacts with CSL to start lytic activation

(264). Similarly, EBV produces EBNA2 (Epstein Barr Nuclear Antigen 2) protein that has been shown to bind CSL and activate transcription in a manner dependent on a

φWφP motif, like some other BTD binding proteins (168,265)! Structures of the CSL-

RTA and CSL-EBNA2 complexes have yet to be elucidated. Ultimately, such structures could be used to target binding surfaces the mediate the interactions between these viral proteins and CSL.

Due to the gene dosage effect, insufficient Notch signaling results in negative human health outcomes. Furthermore, excessive Notch signaling through the mammalian Notch paralogs has been demonstrated as oncogenic in some contexts and tumor suppressive in others. Because of the critical role that the Notch signaling pathway plays in human disease and cancer, development of Notch modulating therapeutics is a quickly expanding area of study (267). Therapeutic antibodies have been developed that bind either the extracellular portion of Notch or DSL in order to prevent receptor-ligand interaction (268,269). Additionally, a series of γ-secretase inhibitors have been developed and shown to inhibit Notch signaling and Notch target gene transcription (270). From the transcriptional regulation perspective, a DNMAML1

(Dominant-Negative Mastermind-like) stapled peptide has been developed that consists of the CSL interaction domain of the coactivator MAM. DNMAML1 binds CSL and obstructs MAM binding, thus preventing the formation of ternary activation complex

(119). However, γ-secretase and DNMAML both lack specificity. In one striking example, γ-secretase inhibitor treatment of breast cancer patients resulted in a

proliferation of goblet cells in the intestines of patients in a syndrome termed goblet cell hyperplasia (270). Because of the Notch pathway’s role in intestinal homeostasis, γ- secretase inhibitor treatment led to tissue rearrangement within the gut and severe diarrhea (270). In addition to targeting core Notch pathway components, an inhibitor of the FBXW7 E3 ubiquitin ligase that prevents NICD degradation has also been developed in order to increase Notch signal dosage (271). Although they cover a wide range of targets, Notch therapies are still navigating the diverse roles of the Notch pathway.

Project Directions

CSL-corepressor complexes offer more diverse therapeutic targets due to their more heterogeneous composition when compared to the sole CSL-ternary activation complex. For example, being able to target a CSL-MINT complex over a CSL-Kyot2 for disruption with a small molecule or peptide fragment allows for a more specific therapeutic target. This approach will allow for the development of therapeutics that may marginally activate Notch target gene transcription by depressing the basal activation level. As the body of literature characterizing CSL-corepressor complexes grows, the ability to impart specificity to therapeutics targeting CSL will dramatically improve.

Given that there are a wide variety of corepressors that interact with CSL and

CSL only has NICD and MAM as coactivators, the particular interest of this thesis is to reconcile the different modes of CSL-mediated repression of Notch target gene transcription. As the field has progressed, many more high-resolution X-ray structures

of Notch transcriptional regulation complexes have been solved and characterized in the literature. Characterizing the similarities and differences that exist among known

CSL binding partners will be foundational to identify targets for therapeutics that may modulates Notch target gene transcription with specificity.

In the next chapter, the structure and function the CSL-RITA complex will be described, increasing our understanding CSL-mediated transcriptional regulation of

Notch target genes. RITA-mediated repression represents a novel form of regulating

Notch target gene transcription and may have implications in hepatocellular carcinoma

(134,272). This will be followed by a small comparative biophysical study of previously identified CSL binding proteins. The final chapter will conclude with closing remarks and future directions.

Chapter 2: Structure and biophysical characterization of the CSL-RITA interaction: a repressor of Notch target gene transcription.

Nassif Tabaja1 and Rhett A. Kovall1

1Department of Molecular Genetics, Biochemistry and Microbiology, University of

Cincinnati College of Medicine, Cincinnati, Ohio, USA. Correspondence should be addressed to R. A. K. Tel.: 513-558-4631 Fax: 513-558-1885 E-mail: [email protected].

Keywords: Notch pathway, protein crystallization, isothermal titration calorimetry (ITC), protein-protein interaction, transcription corepressor

Abstract

The Notch pathway is a cell-to-cell signaling mechanism that is essential for tissue development and maintenance. Aberrant signaling has been implicated in various cardiovascular diseases, congenital defects, and cancers. Notch signaling results in transcription of target genes, which is regulated by the transcription factor

CSL. CSL functions as both a repressor and activator by interacting with transcriptional corepressor and coactivator proteins, respectively. While our group and others have characterized the structure and function of Notch activation complexes, less is known about how CSL interacts with corepressors and functions as transcriptional repressor.

More recently, a new CSL interacting protein termed RITA has been identified and shown to export CSL out of the nucleus, thereby leading to the down-regulation of

Notch target gene expression. While this represents a previously unknown mechanism to regulate transcription in the Notch pathway, the molecular details of the CSL-RITA interaction are poorly defined. Here we characterize the structure and function of the

CSL-RITA complex using a combination of structural, biophysical, and cellular approaches. We show the crystal structure of the CSL-RITA complex bound to DNA, the regions of RITA and CSL that are necessary and sufficient for complex formation by

ITC, and the functional requirements for RITA mediated repression. Taken together, these data begin to elucidate at the molecular level the mechanism of Notch regulation mediated by RITA and contribute to a growing body of literature characterizing how CSL functions as transcriptional repressor of Notch target gene transcription.

Introduction

Notch signaling is a highly conserved component of metazoan development and tissue homeostasis (1). Genetic ablation of Notch signaling is embryonic lethal (2).

Furthermore, mutations causing dysfunctional Notch signaling have been linked to cardiovascular disease, birth defects, and a variety of cancers, highlighting the pathway’s importance (3,4). Due to the negative human health outcomes associated with aberrant Notch signaling, there is focus on developing reagents that modulate

Notch signaling to be used as potential therapeutics (5,6).

The Notch signaling pathway is a juxtacrine signaling mechanism that is initiated when the extracellular domain of a DSL (Delta in mammals, Serrate in flies, LAG-2 in worms) ligand present on one cell binds a single-pass transmembrane Notch receptor on an adjacent cell (7). Pathway activation ultimately results in transcription of Notch target genes (7). In mammals, there are five DSL ligands (Jagged1,2 and Delta like ligand1,3,4) and four Notch receptors (Notch1-4) (7). Ligand-receptor interaction induces the Notch receptor to undergo a series of cleavage events, resulting in the release of the NICD (Notch intracellular domain) from the cell membrane and its subsequent translocation into the nucleus (7). In the nucleus, NICD binds the transcription factor CSL (CBF1/RBPJ in mammals, Su(H) in flies, LAG-1 in worms) and recruits the transcriptional coactivator MAM (Mastermind) (Figure 1) (8-10). CSL has three domains that mediate coactivator and corepressor contacts: the NTD (N-terminal domain), the BTD (β-trefoil domain), and the CTD (C-terminal domain) (11-13). The

NICD contains a RAM (RBP-J associated molecule) and an ANK (ankyrin repeats) domain that mediate interactions with CSL and allow for MAM binding (12-16). The

CSL-NICD-MAM ternary complex binds enhancer and promoter elements of Notch target genes and functions as the switch to activate transcription at these sites (17). In the absence of a Notch signal, CSL functions as a transcriptional repressor by binding to corepressor proteins (18-23).

When interacting with corepressors, such as KyoT2, MINT/SHARP, or Hairless, the function of CSL is to anchor the assembly of higher order repression complexes at

Notch target gene sites (17,23-25). CSL-corepressor complexes often contain histone- modifying complexes responsible for repressing transcription of Notch target genes

(20,26,27). Initial models of the Notch pathway hypothesized that CSL was bound to

DNA and activation was a function of displacing corepressors with coactivators (Figure

1) (28). By showing that levels of CSL bound to DNA at target sites increase after activation of Notch signaling, recent studies have demonstrated that the CSL-DNA interaction in vivo is very dynamic (29,30). It remains inconclusive whether NICD is competing with corepressors for binding CSL molecules already associated with DNA or if preassembled CSL transcriptional complexes are recruited to CSL binding sites on

DNA.

Figure 1: CSL-mediated repression and activation of Notch target gene transcription. (Center) In the absence of a Notch signal, CSL (cyan) is bound to corepressors such as RITA (magenta) that prevent transcriptional activation. (Left) In the case of RITA-mediated repression, RITA binds CSL in the nucleus causing CSL to localize in the cytoplasm via nuclear export. (Right) Upon activation of the Notch signaling pathway, the NICD (Notch intercellular domain) shown in red and yellow, translocates to the nucleus, recruits the coactivator MAM (Mastermind), and competes off corepressors for CSL binding.

While structure-function studies of CSL in complex with coactivators have been seminal to understanding the transcriptional activation of Notch target genes, structure- function data of CSL-corepressor complexes and how CSL functions as a transcriptional repressor are lacking. A new transcriptional coregulator, termed RITA (RBPJ interacting tubulin associated), has been identified by a yeast two-hybrid screen for

RBPJ binding partners (31). RITA is a 269 residue protein with little secondary structure (Figure 2A-B). It contains a CSL-ID (CSL interacting domain), an N-terminal

NLS (nuclear localization signal) and NES (nuclear export signal), and a C-terminal tubulin interacting domain (Figure 2A) (31). As such, RITA has been shown to bind

CSL and facilitate its export out of the nucleus (Figure 1) (31). Previous studies have shown that RITA functions as a repressor of Notch mediated transcription in cells, as well as in Xenopus embryos where it is able to reverse loss of primary neurogenesis caused by Notch activation (31). Proteomic studies have also shown that RITA is phosphorylated and acetylated in the CSL-ID (32,33). Furthermore, RITA over expression has been shown to suppress growth and promote apoptosis in hepatocellular carcinoma (34). Although it is possible that this may be due to repression of Notch target gene transcription, it remains to be determined.

The CSL-ID of RITA is highly conserved through Xenopus and contains a conserved hydrophobic tetrapeptide motif (φWφP; where φ is any hydrophobic residue) that is also present in the RAM domain of Notch1-4, the KyoT2 repressor protein, and the viral transactivator EBNA2 (Epstein-Barr virus nuclear antigen 2) (Figure 2C-D).

The φWφP motif is necessary for RAM, KyoT2, and EBNA2 binding to the BTD of CSL

(35-37). Interestingly, the φWφP motif in RITA contains a threonine (T143) in the third

position. T143 has been shown to be phosphorylated, suggesting a regulatory mechanism for RITA binding to CSL (Figure 2C) (33). Furthermore, in RAM, three additional conserved motifs have been determined to be crucial for the high affinity interaction with the BTD; these motifs consist of an amino terminal basic region, and -

HG- and -GF- dipeptide motifs (Figure 2D) (38). However, they are not present in other high affinity CSL binders including RITA.

Figure 2: RITA domain schematic, secondary structure content, and sequence alignment. (A) The major domains of human RITA, shown in pink, including the nuclear export signal (NES) in black, the RITA conserved repeats (RCR1/RCR2) shown in red, the CSL/RBPJ interacting domain (CSL-ID) shown in purple, and the tubulin interacting domain, in orange (B) Far-UV spectra (wavelengths 185-200nm) for RITA a.a.127-158 comprising the CSL-ID. The CSL-ID of RITA consists of mostly random coil as indicated by the minimum at 200 nm in the CD spectrum. Secondary-structure was determined using Dichroweb and CDSSTR with reference set 7. The normalized root mean square deviation parameter value for analysis of the RITA CD data is 0.038. (C) The primary sequences of constructs used in this study with the φWφP motif shown in green and the arginine implicated in salt-bridge formation with CSL E260 shown in red (D) Sequence alignment of known BTD binders, including the RAM domain of the human Notch orthologs, as well as, the RAM domain of fly and worm Notch. Boxed in blue, is the RAM basic motif; in yellow, the -HG- repeat motif; in green, the φWφP motif; and in magenta, the -GF- repeat motif. Boxed in red, are the basic residues of KyoT2 and RITA implicated in salt-bridge formation.

Here we characterize the structural details of the CSL-RITA complex, quantify the thermodynamic properties of the CSL-RITA interaction, and begin to understand how RITA acts on CSL to repress Notch target gene transcription. We used X-ray crystallography to determine the structure of RITA bound to CSL. In parallel, we used

ITC to measure the thermodynamic parameters of the CSL-RITA interaction.

Luciferase-based transcriptional reporter assays were used to functionally characterize

RITA-mediated repression. Taken together, our results present the first high-resolution structure-function study of the CSL-RITA interaction and contribute to a better understanding of the binding requirements for CSL-corepressor complexes.

Experimental procedures

Cloning, expression, and protein purification-The Mus musculus CSL ortholog, residues 53-474 (CSL core domain), residues 203-393 (BTD), and residues 203-474

(BTD-CTD) were each cloned into the pGEX-6P-1 vector. Expression and purification was performed as previously described in (12). Transformed bacteria were grown at

37 °C in LB medium, cooled to 20 °C, induced with 0.1 mM isopropyl β-d- thiogalactopyranoside, and grown overnight at 20 °C. The bacteria were harvested by centrifugation, resuspended in phosphate-buffered saline. The resuspended cells were lysed by sonication, cleared by centrifugation and filtration, and subsequently loaded onto a glutathione-Sepharose column. The column was washed with phosphate- buffered saline, and the GST fusion proteins were eluted using buffer containing reduced glutathione. The elutants were dialyzed, and the GST tag cleaved with

Precision Protease (GE Healthcare) per the manufacturer's protocol. All of the protein

constructs were further purified to homogeneity using ion exchange and size exclusion chromatography.

The human RITA ortholog, residues 106-173 or residues 127-158, was cloned into a modified pET 28b(+) vector. This vector encodes a fragment of SMT3

(suppressor of Mif2 temperature-sensitive mutant 3) for increased protein stability and expression, producing a His-SMT3-RITA fusion protein (39). The fusion protein was overexpressed as described above. The cleared lysate was incubated with Ni-NTA resin

(Qiagen) and loaded onto a gravity column. The column was washed, and fusion protein was eluted with imidazole containing buffer. The fusion protein was then cleaved to remove His-SMT3 from the RITA moiety using the Ulp1 protease, which leaves only an

N-terminal serine residue attached to RITA following cleavage. The RITA constructs were further purified to homogeneity using ion exchange and size exclusion chromatography. Constructs smaller than RITA 127-158 were purchased as HPLC purified synthetic peptides form Peptide 2.0 and received as lyophilized powder.

Isothermal titration calorimetry-ITC experiments were performed using a Microcal

VP-ITC micocalorimeter. For all binding reactions, syringe concentrations varied between approximately 200-250 μM RITA and cell concentrations varied between approximately 20-25 μM CSL. Titrations consisted of an initial 1µL injection followed by 39 7µL injections. ITC binding experiments were performed in 50 mM sodium phosphate pH 6.5, 150 mM NaCl at 5°C, 15°C, 25°C, or 35°C. Samples were buffer matched using size-exclusion chromatography. The collected data were analyzed using the ORIGIN software and fit to a one site binding model.

Crystallization and data collection-A 15-mer DNA duplex with single-stranded

TT/AA overhangs corresponding to a CSL binding site from the HES-1 gene was co- crystallized with mouse CSL and human RITA. CSL-RITA-DNA complexes were set up in a 1:1.1:1.1 molar ratio and screened for crystallization conditions using the Hampton

Research Index Screen and an Art Robbins Phoenix Crystallization Robot. The final optimized crystallization conditions were in a mother liquor containing 0.1M Bis-Tris pH5.5, 0.2M Ammonium Acetate, 10% 1,4-butanediol, and 16% polyethylene glycol

3350 at 4°C microbatch under paraffin oil. Crystals were cryoprotected in mother liquor solutions containing 20% 1,4-butanediol and flash frozen in liquid nitrogen. The diffraction data were collected at the Advanced Photon Source’s LS-CAT beam line.

The crystals diffracted to 2.1 Å and belong to the orthorhombic space group P21221, with unit cell dimensions of 76.78Å, 96.41Å, and 96.71Å.

Structure determination, model building, and refinement-Collected data was processed and scaled using HKL-2000 (40). Phaser was used to generate a molecular replacement solution for our CSL-RITA-DNA diffraction data (41). As a molecular replacement search model, we used the structure of mouse CSL bound to DNA (PDB

ID: 3IAG) (13). Coot was used to rebuild missing parts of the model (42).

Translation/libration/screw (TLS) parameters were calculated and used for refinement in

Phenix Refine (43). Structural validation was performed using Molprobity (44). Our final model of the CSL-RITA complex bound to DNA consists of amino acids 53-474 of

CSL, amino acids 133-148 of RITA, and the entire DNA duplex. The structure has been refined to a Rwork = 19.3% and Rfree = 23.6% and deposited in the Protein Data Bank

(PDB) (www.rcsb.org) with PDB ID: 5EG6 (45). We used PyMOL Molecular Graphics

System, Version 1.3 for structural visualization and alignments (pymol.sourceforge.net)

(46). The PDBePISA server (http://www.ebi.ac.uk/pdbe) was used to analyze protein- protein interfaces (47).

Cellular Assays-Mouse embryonic fibroblasts (MEFs) originating from CSL knockout embryos (OT11) were transduced with retroviruses that express either wild- type or mutant CSL proteins as in (48). Transduced MEFs were grown to 50% confluence in six-well plates and transiently transfected with a constitutively active

NICD1 construct, a 4xCBS luciferase reporter containing four CSL-binding sites, and

Renilla luciferase construct (phRL). The phRL vector expresses Renilla luciferase and allows for transfection efficiency normalization. Wild-type or mutant RITA was cotransfected in increasing concentrations in order to measure the repressive effects of

RITA on Notch-mediated transcriptional activation of the luciferase reporter. TransIT®-

2020 transfection reagent (Mirus) was used for all transfections along with pBlueScript

(Stratagene) in order to normalize the amount of DNA transfected in each treatment group. 48 hours post transfection, cells were harvested and prepared for measurement of firefly luciferase and Renilla luciferase activity. The Dual Luciferase Kit (Promega) was used to measure luciferase activity. For each treatment well, firefly luciferase activity from the 4xCBS reporter was first normalized to that well’s Renilla luciferase activity. Normalized data was reported as either fold activation or relative activity.

Average values, errors, and SD were determined from three independent experiments performed in duplicate.

Thermal Stability Shift Assays- Thermal Shift studies were performed in a

StepOne Real Time PCR system (Applied Biosystems) in MicroAmp™ 48-well reaction

plate (Applied Biosytems). Melt reactions consisted of a 20µL volume containing 7µM of protein sample in a buffer of 25mM MES pH 6, 500mM NaCl, 1mM EDTA, and

0.1mM TCEP. Reactions were started at 20°C and ended at 99°C over the course of an hour. Sypro Orange dye was used to track protein unfolding as indicated by increased fluorescence due to exposure of hydrophobic surfaces. Protein Thermal Shift Software v1.2 (Applied Biosystems) was used to determine Tm values.

Circular dichroism-RITA (127-158) was characterized in a buffer containing

10mM Tris-phosphoric acid pH 7.4 and 50mM NaF at a concentration of 384µM at 1.36 mg/ml. CD data were analyzed on DICHROWEB using the CDSSTR analysis program with reference set 7 (49,50). CD measurements were taken in triplicate using an Aviv

Circular Dichroism Spectrometer Model 215 at 25 °C in a 0.01 cm cuvette. Wavelength scans were performed between 185nm and 290nm using 1nm increments.

Results

Structure of the CSL-RITA-DNA Complex-The high-resolution structure of the

CSL-RITA complex (PDB ID: 5EG6) was obtained from bacterially expressed recombinant mouse CSL (RBPJ a.a.53-474) protein. RITA peptide corresponding to residues 133-151 was synthesized and HPLC purified. CSL-RITA complexes were incubated with equal molar ratios of an oligomeric 15mer DNA duplex, containing a single CSL binding site, and screened for crystals. An optimized condition based on initial screens of the CSL-RITA complex yielded CSL-RITA-DNA crystals amenable to

X-ray diffraction analysis. A CSL-RITA-DNA complex diffracting to 2.1 Å with an orthorhombic (P21221) crystal form was obtained (Table 1). Molecular replacement using published CSL-DNA structures was utilized to solve the structure (12,13). The

asymmetric unit contains a single CSL-RITA complex bound to DNA (Figure 3A).

Interestingly, the CTD of the CSL-RITA-DNA structure is shifted up by ~10Å when compared to the structure of CSL alone bound to DNA (PDB ID: 3IAG), assuming a more open conformation (Figure 3B). The functional significance of this confirmation is yet to be determined. The final model consists of residues 53-474 of CSL, residues

133-148 of RITA, and the 15mer oligomeric DNA duplex (Figure 3A). The final dataset was refined to 2.1 Å with a Rwork and Rfree of 19.3% and 23.6%, respectively (Table 1).

Table 1. Data collection and refinement statistics. Data Collection Statistics Beam Line APS LS-CAT 21-ID-F Resolution (Å) 40.83 - 2.09 (2.15 - 2.09)

Space Group P21 221 Wavelength (Å) 0.97872 Unit Cell a, b, c (Å) 76.78, 96.41, 96.71 Unit Cell , ,  () 90.00, 90.00, 90.00

Rmerge 0.07 (0.54) I/I 22.8 (4.79) Completeness (%) 89.6 (48.3) Redundancy 7.1 (5.5) Average mosaicity 0.46

Refinement Statistics

Rwork/Rfree (%) 20.5 / 24.5 Number of reflections 38,486 Number of atoms 4285 Complexes/asymmetric 1 unit Wilson B/Mean B value 25.8 / 30.9 (Å2) RMSD Bond Lengths 0.007 (Å) RMSD Bond Angles () 1.232 Ramachandran 98.1% / 0.7% (favored/outliers) Highest resolution shell shown in parentheses.

RITA binds exclusively to the BTD of CSL in a mostly elongated random coil

(Figure 3A-C). The BTD of CSL contains a β-hairpin loop where RITA’s N-terminal residues (a.a.135-138) form a small antiparallel β-strand (Figure 3A). RITA’s φWφP motif (-LWTP-; a.a.141-144) binds a nonpolar pocket on the surface of the BTD shown to bind other φWφP motifs (Figure 3C, Figure 3E). The CSL-RITA interaction buries an estimated 987Å2 of surface area, similar to other BTD binders such as RAM which buries 950Å2 and KyoT2 which buries 874Å2 when binding CSL (47). Residues 133-

148 of RITA had appreciable electron density while the remaining three C-terminal residues were not well resolved (Figure 3C). Our structural analysis suggests that previously identified CSL-ID residues outside of the 133-148 region are not required for binding CSL.

In order to determine the differences between RITA, KyoT2, and RAM binding to the BTD, we compared three previously published X-ray structures that contain the

CSL-RAM interaction – the human CSL-NICD-MAM ternary complex (PDB ID: 3V79), the worm CSL-RAM structure (PDB ID: 3BRD), and the CSL-KyoT2 structure (PDB ID:

42JX) (Figure 3E) (13,36,51). When RITA and the RAM domains are superimposed onto one another, their binding to the BTD groove of CSL is strikingly similar, especially in the region containing the φWφP motif (Figure 3E). At the N- and C-terminal residues, there is less structural alignment between RITA, KyoT2, and RAM (Figure 3E). This may be due to the fact that RITA has no sequence similarity with KyoT2 or RAM in residues that flank the φWφP motif (Figure 2D). Additionally, the C-terminus of the human Notch1 RAM structure does not follow the same binding path as the RITA and the worm RAM structures (Figure 3E). It is hypothesized that the repeat proline residues

after the φWφP for worm RAM results in its elongated conformation, whereas human

Notch1 RAM lacks this motif causing it to hinge after the φWφP residues. RITA also contains this proline repeat motif and follows a C-terminal binding path similar to the worm RAM structure. Conversely, KyoT2 does not contain a repeat proline motif in its

C-terminus yet still follows a binding path similar to the RITA and worm RAM structures

(Figures 2D and 3E). The functional significance of these two binding paths is yet to be determined. Interestingly RITA and KyoT2 both form salt bridges with the BTD of CSL.

R138 of RITA forms a salt bridge with E259 and E260 of CSL (Figure 3D). This is similar to the CSL-KyoT2 structure where KyoT2 forms a lysine salt bridge with this

E259 and E260 of CSL (36).

Figure 3: CSL-RITA complex bound to DNA and structural alignment. (A) The x-ray crystal structure of CSL-RITA-DNA (PDB ID: 5EG6) was solved using molecular replacement to a resolution of 2.1Å. The NTD is in orange; CTD, blue; and BTD green. RITA is in yellow. (B) A structural alignment of the CTD position in the CSL-RITA-DNA structure with the CTD position in the previously published CSL-DNA structure (PDB ID: 3IAG) highlighting an up to 10.5 Å shift of the CTD of CSL when bound to RITA. (C) A model of the electron cloud density of RITA at 2.1Å with emphasis on the φWφP motif. (D) The structure of the CSL-RITA complex showing the 2.9Å and 2.6Å salt-bridges between R138 of RITA (yellow) and EE259 of CSL (green) (E) A structural alignment of BTD binding proteins emphasizing the placement of the φWφP motif, including: RITA, in yellow (PDB ID: 5EG6); KyoT2, in blue (PDB ID: 42JX); worm RAM, in pink (PDB ID: 3BRD); and human RAM, in red (PDB ID: 3V79). (F) A top view of RITA (yellow) lying on the BTD (green) binding groove of CSL in relation to residues previously demonstrated to be critical for RAM binding colored in red.

Thermodynamic analysis of CSL-RITA complex formation-The binding parameters of the CSL-RITA interaction were characterized using ITC with purified CSL and RITA constructs. As shown in Figure 4B and Table 2, the previously identified CSL binding domain of RITA, residues 127-158, binds CSL with a ~1µM Kd, two orders of magnitude weaker than previously characterized CSL-RAM and CSL-Kyot2 complexes, which bind CSL with a ~10nM and ~20nM Kd respectively (13,36,38). The binding of

RITA to CSL is enthalpically driven and incurs an entropic penalty consistent with RITA being an intrinsically disordered protein (Figure 2B). The binding affinity of RITA to CSL is moderately affected by the presence of DNA containing a Hes1 CSL binding site

(Figure 4C, Table 2). This results in an approximate 2 kcal/mol shift of the enthalpic/entropic contributions to binding (Table 2). Similar to the CSL-RAM and CSL-

KyoT2 interactions, enthalpic/entropic compensation has been demonstrated, suggesting that DNA contacts with the BTD of CSL help stabilize the domain and make it more energetically favorable for additional binding interactions (13,36). Binding experiments performed with RITA and only the BTD of CSL show similar binding as for full-length CSL (Figure 4D, Table 2). Similar to the BTD CSL construct, RITA binds a

BTD-CTD CSL construct with similar affinity when compared to the CSL core domain containing the BTD, CTD, and NTD (Figure 4E, Table 2). These data suggest that RITA binds the BTD of CSL and that the presence of the CTD and NTD, as well as DNA, provide the most optimal BTD conformation for RITA binding.

TABLE 2: Calorimetric data for RITA binding to CSL

RITA -1 K ΔG° ΔH° -TΔS° Macromolecule K (M ) d Ligand (uM) (kcal/mol) (kcal/mol) (kcal/mol)

CSL 106-173 1.1 ± 0.2 x 106 0.98 -8.2 ± 0.1 -6.4 ± 0.7 -1.8 ± 0.9 CSL 127-158 9.9 ± 0.5 x 106 1.01 -8.2 ± 0.1 -6.1 ± 0.3 -2.1 ± 0.3 CSL+DNA 127-158 1.7 ± 0.2 x 106 0.59 -8.5 ± 0.1 -4.5 ± 0.1 -4.0 ± 0.2 BTD 127-158 5.2 ± 0.5 x 105 1.94 -7.8 ± 0.1 -4.0 ± 0.2 -3.8 ± 0.3 BTD-CTD 127-158 5.8 ± 0.2 x 105 1.71 -7.8 ± 0.1 -5.5 ± 0.1 -2.3 ± 0.1 CSL 133-151 1.9 ± 0.3 x 106 0.53 -8.5 ± 0.1 -7.6 ± 0.3 -0.9 ± 0.2 CSL 133-148 2.1 ± 0.3 x 106 0.49 -8.7 ± 0.1 -7.7 ± 0.3 -1.0 ± 0.4 CSL 135-148 6.6 ± 0.7 x 106 0.15 -9.3 ± 0.1 -6.4 ± 0.1 -2.9 ± 0.1 CSL 137-148 2.0 ± 0.1 x 106 0.50 -8.6 ± 0.1 -7.9 ± 0.1 -0.7 ± 0.1 CSL 137-146 2.4 ± 0.05 x 106 0.42 -8.7 ± 0.01 -8.5 ± 0.1 -0.1 ± 0.1 CSL 139-146 2.4 ± 0.3 x 104 43.2 -6.0 ± 0.1 -9.5 ± 0.8 3.5 ± 0.9 CSL LWTP NBD ------CSL RITAWTP/AAA NBD ------All experiments were performed at 25°C. Values are the mean of at least three independent experiments and errors represent the standard deviation (S.D.) of multiple experiments. NBD represents no binding detected.

Figure 4: Analysis of the CSL-RITA interaction by ITC. Representative thermograms from individual ITC experiments with various constructs of CSL and RITA. All ITC experiments were conducted with CSL in the cell at approximately 20-25µM and RITA in the syringe at approximately 200-250µM. Experimental temperature was set at 25°C and experiments were performed in triplicate (n=3). (A) A RITA construct N- and C- terminally extending out of the previously identified CSL-ID (B) The previously identified CSL-ID of RITA (C) CSL was pre-incubated with a 19-mer oligo containing one Hes1 CSL binding site at a 1:1.1 molar ration with DNA in molar excess (D) RITA is titrated into a cell containing a BTD-only construct of CSL. (E) RITA is titrated into a cell containing only the BTD-CTD domains of CSL. (F) A construct containing the most- highly conserved region in the CSL-ID of RITA (G) The minimal binding region of the CSL-ID of RITA (H) A RITA construct lacking the R138 shown in the structure of CSL- RITA to be forming a potential salt bridge with EE259 of CSL (I) ∆Cp analysis of CSL- RITA interaction ITC experiments were performed at 5°C, 15°C, 25°C, and 35°C in triplicate using 25µM CSL and 250µ M RITA. The average change in Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (-TΔS°) were plotted as a function of temperature.

We performed a series of ITC experiments at varying temperatures to calculate the change in heat capacity (∆Cp) associated with RITA binding CSL. A negative ∆Cp is suggestive of burial of nonpolar surfaces which occurs during protein complex formation

(52). The average change in free energy, enthalpy, and entropy (∆G°, ∆H°, ∆S°) were analyzed as a function of temperature (Figure 4I, Table 3). While ∆H° and ∆S° fluctuate with temperature, the change in free energy upon complex formation is temperature independent. The CSL-RITA interaction has a ∆Cp of -0.51 kcal/mol∙K, very similar to previously characterized CSL complexes such as the CSL-Kyot2 complex with a ∆Cp of

-0.57 kcal/mol∙K and CSL-RAM complex with a ∆Cp of -0.62 kcal/mol∙K (25,36).

In order to determine the region of RITA necessary and sufficient to bind CSL, we performed a series of ITC experiments with extended and serially truncated RITA constructs. The C- and N-terminally extended RITA 106-173 construct showed no significant increase in binding affinity when compared to the CSL-ID construct, RITA

127-158 (Figure 4A-B, Table 2). The truncation of RITA from its originally identified

CSL-ID a.a.127-158 to the most highly conserved residues a.a.133-151 maintained full binding affinity for CSL (Figure 4F, Table 2). In support of our structural data, C- terminal truncation up to RITA 133-148, the residues with significant electron density in the CSL-RITA structure, did not result in significant changes in affinity (Figure 3C, Table

2). Further truncations of RITA on the N-terminus to residues 137-148 and on the C- terminus to residues 137-146 resulted in no change in binding affinities (Figure 4G,

Table 2). RITA 139-146, which no longer has the necessary R138 required for salt bridge formation, had a ~100-fold loss in binding affinity, with the Kd increasing from

~0.4µM to ~47µM (Figure 4H, Table 2). Similarly, when K187 of KyoT2, which is

involved in formation of a salt bridge with CSL, is truncated, a 40-fold loss of affinity is observed (36). These data suggest that RITA 137-146 contains the necessary and sufficient residues to bind CSL with full affinity and that part of this interaction is mediated by R138 of RITA and EE259 of CSL.

Table 3: Temperature dependence of RITA binding to CSL -1 G H -TS T (ºC) K (M ) K (M) d (kcal/mol) (kcal/mol) (kcal/mol) 5 4.0  0.3 x 106 0.43 -7.8  0.2 3.3  0.3 -11.1  0.1

CSL 15 1.1  0.3 x 106 1.0 -7.9  0.2 -2.9  0.1 -5.0  0.3 + (133-148) RITA 25 2.1  0.3 x 106 0.49 -8.7  0.1 -7.7  0.3 -1.0  0.4

35 1.0  0.4 x 106 1.0 -8.5  0.1 -11.9  0.1 3.4  0.1 Values are the mean of three independent experiments and the errors represent the S.D. of multiple experiments.

To test the relative contribution of the φWφP motif of RITA, we designed a tetrapeptide (-LWTP-) containing only those residues and no binding was detected by

ITC (Table 2). Additionally, we designed a RITA construct with a mutated φWφP motif, mutating the WTP of RITA to a triple alanine repeat. The RITA WTP142AAA mutant was also unable to show binding to CSL by ITC (Table 2). RITA contains a φWφP motif also found in the RAM domain of Notch1-4 and Kyot2. However, it lacks a true hydrophobic in the third position of the φWφP motif (T143) (Figure 2D). Recent studies have identified posttranslational modifications in the CSL-ID of RITA including phosphorylation at residues T143 and T147 and acetylation at residues K131 and K136

(Figure 2C) (32,33). Phosphorylation of T143 of RITA, results in ~12-fold weaker binding, suggesting a role for T143 phosphorylation in the φWφP motif (Table 5). In contrast, RITA T147 phosphorylation results in modestly weaker binding by ITC (Table

5). Acetylation of K131 and K136 of RITA results in ~5-fold weaker binding to CSL

(Table 5). This is in agreement with our structural and biophysical characterization of the CSL-RITA interaction which demonstrates the importance of T143 which is part of the φWφP motif of RITA, LWTP (Figures 2D, 3C, and Table 2).

RITA also lacks all other identified motifs shown to be necessary for high affinity

RAM binding, including the basic motif, the -HG- repeat motif, and the -GF- repeat motif common to the RAM domains of Notch1-4 (38). Even in the absence of these additional motifs RITA is still able to bind CSL (Figure 4, Table 2). To compare RAM and RITA binding to CSL, we tested binding of RITA to CSL constructs containing mutations that target the CSL-RAM interface (Figure 3F). Previous studies have identified four key residues of the BTD that are necessary for maintaining high binding affinity to the RAM

domains of Notch1 and Notch2 (48). When these residues are mutated to arginines,

RAM affinity for CSL is severely reduced (48). Therefore, we used these BTD arginine mutants, as well as their alanine versions (F261R, V263R, A284R, Q333R and F261A,

V263A, A284V, Q333A) to perform ITC experiments with the CSL-ID of RITA (Table 4).

We were unable to detect binding of RITA to the CSL mutant F261R (Table 4) while the

CSL F261A mutant showed ~300-fold weaker binding to RITA (Table 4). CSL mutants

V263R and V263A showed a modest decrease in binding affinity for RITA (Table 4).

CSL A284R and A284V showed a significant decrease in binding affinity for RITA, increasing from a ~0.5µM Kd to a ~6µM and ~22µM Kd, respectively (Table 4). Finally, like the CSL V263 mutants, CSL mutants Q333R and Q333A showed a modest decrease in binding affinity from that of WT CSL (Table 4). Similar to what has been previously observed with RAM and KyoT2 binding, CSL residues F261 and A284, which lie in the hydrophobic pocket that binds the φWφP motif, reduced binding the most when mutated to arginine (36,48). In order to confirm that differences in binding were not due to loss of CSL stability, we demonstrated by thermal stability assays that our mutant CSL proteins show no dramatic loss in stability when compared to wild-type CSL

(data not shown).

Table 4: Calorimetric binding data for RITA and CSL mutants

-1 K ΔG° ΔH° -TΔS° ΔΔG° CSL Ligand K (M ) d (uM) (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)

F261R RITA NBD ------F261A RITA 6.9 ± 1.0 x 103 147.2 -5.2  0.1 -8.1  0.8 2.9  0.9 3.3 V263R RITA 0.9 ± 0.2 x 106 1.29 -8.0  0.2 -6.9  0.6 -1.1  0.8 0.5 V263A RITA 1.1 ± 0.9 x 106 0.92 -8.1  0.7 -5.9  0.3 -3.0 ± 1.1 0.4 A284R RITA 1.8 ± 0.4 x 105 5.88 -7.2  0.1 -12.6  1.1 5.5  1.2 1.3 A284V RITA 4.6 ± 0.8 x 104 21.9 -6.3  0.1 -4.0  0.9 -2.3  1.0 2.2 Q333R RITA 4.8 ± 0.3 x 105 2.1 -7.8 ± 0.1 -7.0  0.3 -0.8 ± 0.3 0.7 Q333A RITA 1.2 ± 0.3 x 106 0.90 -8.3  0.1 -5.7  0.2 -2.5  0.3 0.2 All experiments were performed at 25°C. Values are the mean of at least three independent experiments and errors represent the S.D. of multiple experiments. RITA ligands are in the context of the construct 133-151. NBD represents no binding detected.

Table 5: Calorimetric data for acetylated and phosphorylated RITA binding to CSL

-1 K ΔG° ΔH° -TΔS° Macromolecule RITA Ligand K (M ) d (uM) (kcal/mol) (kcal/mol) (kcal/mol)

CSL RITA 1.1 ± 0.2 x 106 0.90 -8.2 ± 0.1 -6.1 ± 0.4 -2.1 ± 0.3 5 CSL RITA(2ac) 1.9 ± 0.4 x 10 5.0 -7.2 ± 0.1 -6.8 ± 0.2 -0.4 ± 0.2 CSL RITA(pT143) 8.6 ± 0.1 x 104 12.0 -6.7 ± 0.1 -3.9 ± 0.4 -2.8 ± 0.5 CSL RITA(pT147) 5.4 ± 0.7 x 105 1.9 -7.8 ± 0.1 -5.3 ± 0.1 -2.5 ± 0.2 All experiments were performed at 25°C. Values are the mean of at least three independent experiments and errors represent the S.D. of multiple experiments. RITA ligands are in the context of the construct 130-148.

Cellular characterization of the CSL-RITA corepressor complex-We characterized the CSL-RITA interaction in cells using luciferase transcriptional reporter assays to further validate our structural and thermodynamic data. To perform these studies, mouse embryonic fibroblasts (MEFs) were cultured from CSL-null embryos and then transduced with retrovirus expressing either wild-type or mutant CSL proteins. In order to activate Notch signaling in the MEFs, we transfected a construct encoding for a constitutively active intracellular domain of the Notch1 receptor (NICD1) (53). A construct containing four consecutive CSL binding sites (4xCBS) upstream of the luciferase gene was cotransfected in order to monitor Notch activation and CSL transcriptional activity (54). As shown in Figure 5A, constitutively active NICD1 transfected into MEFs expressing wild-type CSL results in strong ~60-fold activation of the reporter compared to cells that were not transfected with NICD1. When cotransfected with a plasmid expressing wild-type RITA, activation of the reporter is significantly reduced (Figure 5A).

When analyzing CSL mutants for fold-activation relative to cells with no NICD1 cotransfected, the CSL mutants F261A and A284V showed dramatically reduced activation compared to wild type CSL while the V263A and Q333A mutants showed significant but modestly reduced activation when compared to wild type CSL (Figure

5B), in agreement with our binding data and what has been previously seen with the arginine variants of these CSL mutants (36,48). When normalized against relative activation, all four CSL mutants (F261A, V263A, A284V, Q333A) showed significantly less repression by RITA when compared to WT CSL at the highest amounts of transfected RITA (Figure 5C-F). The CSL Q333A and A284V mutants were the least

responsive to RITA mediated repression, only showing ~2.5-fold reduction of activation at the highest levels of transfected RITA (Figure 5E-F).

The RITA 106-173 construct which contains a 2xNLS and CSL-ID from full-length

RITA (RITA WT) was able to repress reporter transcription as well as full-length RITA

(Figure 6A). A mutated full-length RITA construct containing alanine substitutions in three of the four residues of the φWφP motif (WTP/AAA) showed significantly weaker repression compared to wild type (Figure 6B), in agreement with our binding data (Table

2). RITA∆NES which contains a mutated NES sequence was also able to repress transcription (Figure 6C) but not to the same level as RITA WT or RITA 106-173.

Finally, RITA∆NLS which contains a mutated NLS sequence was not able to repress transcription by more than 20% of full relative activation (Figure 6D).

Figure 5: Cellular reporter assays of RITA-mediated repression in the context of CSL mutants. To activate Notch signaling, cells were transfected with 250ng of a construct that expresses an activated form of the Notch1 receptor (NICD1) and 250ng of a construct encoding for the 4xCBS reporter. To assay for RITA-mediated repression, cells were cotransfected with increasing amounts of RITA expressing construct: 0ng (--), 50ng (+), 100ng (++), 200ng (+++), 400ng (++++). Experiments were performed in triplicate and the error bars represent the S.E.M. (A) A plot showing fold activation of transcriptional activity relative to cells not transfected with NICD1 (B) A plot showing fold activation of transcriptional activity relative to cells not transfected with NICD1 for WT and mutant CSL constructs. (C-F) Data were normalized to cells that weren’t transfected with RITA and plotted as relative activity for cells cotransfected with mutant CSL (C) F261A (D) V263A (E) A284V (F) Q333A. Statistical significance was determined by unpaired t test with *, p ≤ 0.05; **, p ≤ 0.01; and ns, not significant.

Figure 6: Cellular reporter assays of RITA-mediated repression in the context of RITA mutants. To activate Notch signaling, cells were transfected with 250ng of a construct that expresses an activated form of the Notch1 receptor (NICD1) and 250ng of a construct encoding for the 4xCBS reporter. To assay for RITA-mediated repression, cells were cotransfected with increasing amounts of RITA expressing construct: 0ng (--), 100ng (++), 200ng (+++), 400ng (++++). Plots comparing RITA WT transcriptional repression activity to (A) RITA 106-173 containing only the CSL-ID of RITA, (B) RITA WTP/AAA containing a mutated φWφP motif, (C) RITAΔNES containing a non- functional NES, and (D) RITAΔNLS containing a non-functional NLS. Experiments were performed in triplicate and the error bars represent the S.E.M. Statistical significance was determined by unpaired t test with *, p ≤ 0.05; **, p ≤ 0.01; and ns, not significant.

Discussion

The activation of the Notch signaling pathway results in transcription of target genes, mediated by the transcription factor CSL. This mechanism involves NICD binding CSL and recruiting additional coactivators. In the absence of NICD, CSL is bound to corepressors (Figure 1). In vertebrates, coactivators and corepressors share the same CSL binding surfaces (Figure 3E). Therefore, binding of these cofactors is mutually exclusive. The body of knowledge with regards to CSL activation complexes and their molecular requirements is relatively vast when compared to what is known about CSL repression complexes. While activation follows a canonical pathway that leads to ternary complex formation, repression of Notch target gene transcription by

CSL can involve different types of CSL repression complexes, such as CSL-MINT, CSL-

Hairless, CSL-KyoT2, and CSL-RITA (23,25,31,36). Understanding the molecular details of how coactivators and corepressors assemble on to CSL will be critical to building a mechanistic model of the Notch signaling pathway. Furthermore, knowledge gleaned from structure and function studies of CSL transcriptional complexes will inform and guide the discovery and design of reagents capable of modulating the transcriptional effects of the Notch signaling pathway.

Adding to past studies of CSL transcriptional complexes, here we present the

2.1Å X-ray crystal structure of the CSL-RITA complex bound to DNA (Figure 3A, Table

1). We also thermodynamically characterize the CSL-RITA interaction by ITC (Figure 4,

Tables 2, 4-5) and functionally characterize RITA mediated repression of Notch target genes in luciferase-based transcriptional reporter assays (Figures 5-6). Our structural and thermodynamic data show that the CSL-ID of RITA binds the BTD of CSL with a

sub-micromolar Kd of ~0.5µM (Figure 4, Table 2). In the presence of DNA, this interaction is modestly tighter (Figure 4C) due to the stabilizing effects of DNA binding on the BTD of CSL, suggesting that RITA may preferentially bind CSL molecules that are pre-bound to DNA. Additionally, we mutate domains of RITA to assess the effect on

RITA binding and function. We show that RITA requires its φWφP motif to form a high affinity interaction with CSL (Table 2). However, unlike the φWφP motif of KyoT2, we are unable to detect binding of only the φWφP motif of RITA to CSL by ITC (Table 2).

Unlike Ram and KyoT2, the φWφP motif of RITA contains a threonine in the third position, T143, which has been shown to be phosphorylated (33). Phosphorylation of this residue results in weaker binding to CSL suggesting that RITA binding to CSL is regulated by the activity of an unidentified kinase, possibly Aurora or Polo-like kinase

(31,33). Along with post-translational regulation of the CSL-RITA interaction, our binding data suggest that other unidentified motifs in the CSL-ID of RITA are contributing to its interaction with CSL, such as the salt-bridge motif at R138 (Figure

3D). CSL-KyoT2 binding and structural analysis reveals the same motif interacting with

EE259 of CSL (36). This -EE- repeat on the BTD surface of CSL is a highly conserved negatively charged surface feature that precedes the F261 shown to be important for

BTD binding. While past work has studied the effects of CSL mutant EEF259AAA on

RAM binding, the contributions of EE259 alone on BTD binding has yet to be determined (28). If residues EE259 of CSL are only important for some BTD binding partners, this may present a novel feature to target in order to disrupt binding of a subset of CSL binding partners. In addition, since it is yet to be determined what role the CSL-RITA interaction plays in the ability of RITA to suppress the growth of

hepatocellular carcinoma cells, development of a reagent that specifically inhibits the

CSL-RITA interaction would provide a valuable tool in determining if RITA acts through

CSL to achieve these results.

Functionally, our reporter assays suggest that RITA requires its NES to function as a repressor of Notch target gene transcription (Figure 6C). Conversely, a RITA 106-

173 construct containing a 2xNLS, but no NES, is able to repress transcriptional activity modestly more than the RITAΔNES construct (Figure 6A), suggesting that direct competition with NICD for binding CSL and export of CSL are additive causes of repression of Notch target gene transcription. Additionally, we show that a RITA construct with a mutated φWφP motif, WTP142AAA, is still able to repress transcription of the luciferase reporter, albeit not at the same levels as wild-type RITA, providing functional evidence that other regions outside of the φWφP motif are contributing to the

CSL-RITA interaction (Figure 6B). We also analyze binding and function of RITA with

CSL mutants containing substitution mutations that disrupt the CSL-RAM interaction in order to compare RITA binding of CSL to RAM binding (Figure 3F, Table 4). Similar to

RAM and KyoT2, RITA binding to CSL is also highly dependent on F261 and A284 due to their role in forming the binding surface for φWφP motif (Figure 3F, Table 4). Unlike

RAM, RITA is able to tolerate mutation of V263 and Q333 without severely reducing binding, similar to KyoT2 binding these CSL mutants (Table 4) (36,48). These results further support the hypothesis that other critical motifs outside of the φWφP motif are mediating CSL-RITA interaction and that these motifs are distinct from the known motifs of the RAM domain of NICD.

The RAM domain of Notch1-4 contains additional motifs not found in the CSL-ID of RITA, such as a basic motif, a –HG- repeat motif, and a -GF- repeat motif that flank

φWφP motif of RAM (Figure 2D) (38). It has been shown that when these motifs are mutated RAM binding affinity for CSL is greatly reduced (38). While corepressors such as KyoT2 and RITA do not contain these motifs, detailed analysis of their binding data and respective structures complexed to CSL reveal a potential novel motif that is exclusive to KyoT2 and RITA and not present in the RAM domain of NICD (Figure 2D and 3D). Further analysis of known BTD binders of CSL will help quantify the importance of this common feature. Our work contributes to a growing body of literature describing the structure and function of CSL repression complexes. We demonstrate that RITA shares properties with RAM for binding CSL but also differs in its binding requirements for interaction with the BTD of CSL. A comprehensive comparative analysis of these proteins may reveal additional motifs and requirements for coactivators and corepressors to bind CSL, helping inform the design of reagents that can modulate Notch target gene transcription and disrupt subsets of CSL transcriptional regulation complexes.

Acknowledgments

Project support was provided by the supported by National Institutes of Health Grants

5R01CA178974-03 and 5T32CA117846-09. This research used resources of the

Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User

Facility operated for the DOE Office of Science by Argonne National Laboratory under

Contract No. DE-AC02-06CH11357. Atomic coordinates and structure factors (PDB ID:

5EG6) were deposited in the Protein Data Bank (www.rcsb.org).

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Author contributions

R.A.K. conceived of the study. R.A.K. and N.T. coordinated the research and contributed to data interpretation. N.T. designed DNA constructs, expressed and purified proteins, conducted experiments, solved the crystal structure of the CSL-RITA complex bound to DNA, and analyzed data. Both authors edited and approved the final draft of the manuscript.

Chapter 3: Comparative analysis of CSL binding proteins: RITA, KyoT2, MINT,

RAM, and EBNA2.

Nassif Tabaja1 and Rhett A. Kovall1

1Department of Molecular Genetics, Biochemistry and Microbiology, University of

Cincinnati College of Medicine, Cincinnati, Ohio, USA. Correspondence should be addressed to R. A. K. Tel.: 513-558-4631 Fax: 513-558-1885 E-mail: [email protected].

Keywords: Notch pathway, isothermal titration calorimetry (ITC), protein-protein interactions

Abstract

Notch signaling is essential for tissue development and maintenance in the animal kingdom. Aberrations in the Notch signaling pathway can cause developmental defects in the skeletal, cardiovascular, and nervous systems and a variety of malignant cancers. The Notch signaling pathway controls transcription of target genes by interacting with the transcription factor CSL. CSL can bind corepressor and coactivator proteins and, therefore, functions as both a repressor and activator depending on the complexes it forms. In recent years, literature focused on the biophysical and structural characterization of CSL transcriptional complexes has expanded. Since the body of biophysical work now more fully encompasses CSL’s function as both repressor and activator, comparative biophysical analysis of known CSL binding partners can reveal insights into how CSL regulates Notch target gene transcription. Here we compare the binding of previously identified CSL binding partners to various CSL point mutants. Our data adds to previous biophysical work published in the Notch literature. Molecular level details concerning how CSL functions as a transcriptional switch of Notch target gene transcription will be seminal to the design of small molecule therapeutics that may modulate Notch target gene transcription.

Introduction

The Notch signaling pathway is a critical component to the development and maintenance of the adult animal (1). In the absence of functional Notch signaling, humans, among other animals, develop birth defects and cardiovascular disease (2).

Dysfunctional Notch signaling is also oncogenic giving rise to both solid and hematological malignancies (3). Genetic knockout of the pathway is lethal to the developing embryo further underscoring the significance of Notch signaling in higher organisms (4). Since Notch signaling is central to tissue development and maintenance and its dysfunction causes or is associated with a wide variety of diseases, it is crucial for the development of disease therapeutics to understand the molecular details of how the pathway functions to regulate gene transcription.

A form of contact based cellular communication, the Notch signaling pathway activates when a membrane-bound DSL (Delta in mammals, Serrate in flies, LAG-2 in worms) ligand presented by the signaling cell directly interacts with a membrane-bound

Notch receptor on a receiving cell (5). There are five single-pass transmembrane DSL ligands (Jagged1,2 and Delta like ligand1,3,4) and four single-pass transmembrane

Notch receptors (Notch1-4) in mammals (5). Pathway activation causes the Notch receptor to release NICD (Notch intracellular domain) from the cell membrane to allow for nuclear import (5). Upon nuclear translocation, NICD is recruited to the transcription factor CSL (CBF1/RBPJ in mammals, Su(H) in flies, LAG-1 in worms) and further recruits the transcriptional coactivator MAM (Mastermind) (6-8). The structure of CSL contains three domains that make up its core domain required for binding coregulators such as NICD: the NTD (N-terminal domain), the BTD (β-trefoil domain), and the CTD

(C-terminal domain) (9). NICD consists of a RAM (RBP-J associated molecule) and an

ANK (ankyrin repeats) domain that bind CSL and allow for MAM binding to form a ternary activation complex (10-14). The CSL-NICD-MAM ternary complex binds promoter elements of Notch target genes that contain CSL binding sites resulting in

Notch target gene transcription (15).

When Notch signaling is inactive, CSL forms corepressor complexes that actively repress transcription of Notch target genes (16-21). CSL corepressors, such as, KyoT2,

MINT/SHARP, or Hairless, function to anchor the assembly of higher order repression complexes at Notch target gene sites (20,22-24). CSL-corepressor complexes modify the local chromatin in order to repress transcription of Notch target genes (18,25,26).

Additionally, the CSL corepressor termed RITA exports CSL out of the nucleus preventing it from forming activation complexes (27). It is still unknown if assembly of

CSL complexes precedes or proceeds CSL binding to DNA (28,29). More fundamentally, the degree in similarity by which certain CSL binding proteins interact with CSL has yet to be fully addressed. Few groups have done comparative binding studies analyzing binding to CSL point mutants (30,31). The identification of new CSL binding partners further necessitates the need for research into how these new binding partners also interact with important CSL binding surfaces (27).

Because structure-function studies of CSL as a transcriptional regulator have been proliferating, it becomes increasingly helpful to compare the biophysical and functional similarities and differences between well characterized CSL binding proteins

(10,23,24,30,32-36). Using a combination of isothermal titration calorimetry (ITC) along with published CSL-complex structures and biophysical data, we confirm previously

identified features of CSL shown to be important for binding coregulators and we identify a highly conserved region of the BTD of CSL important for forming high affinity interactions. Taken together, we are able to build a more complete profile of the binding requirements for forming a high affinity interaction with CSL. The details of which will better inform the development of small-molecule modulators of Notch target gene transcription.

Methods

Cloning, expression, and protein purification-The Mus musculus CSL ortholog, residues 53-474 (CSL core domain). Expression and purification was performed as previously described in (10). Transformed bacteria were grown at 37 °C in LB medium, cooled to 20 °C, induced with 0.1 mM isopropyl β-d-thiogalactopyranoside, and grown overnight at 20 °C. The bacteria were harvested by centrifugation, resuspended in phosphate-buffered saline. The resuspended cells were lysed by sonication, cleared by centrifugation and filtration, and subsequently loaded onto a glutathione-Sepharose column. The column was washed with phosphate-buffered saline, and the GST fusion proteins were eluted using buffer containing reduced glutathione. The elutants were dialyzed, and the GST tag cleaved with Precision Protease (GE Healthcare) per the manufacturer's protocol. Protein was further purified to homogeneity using ion exchange and size exclusion chromatography.

Isothermal titration calorimetry-ITC experiments were performed as described in

(10) using a Microcal VP-ITC micocalorimeter. For all binding reactions, syringe concentrations varied between approximately 100-200 μM and cell concentrations varied between approximately 10-20 μM CSL. Titrations consisted of an initial 1µL

injection followed by 39 7µL injections. ITC binding experiments were performed in 50 mM sodium phosphate pH 6.5, 150 mM NaCl 25°C. Samples were buffer matched using size-exclusion chromatography. The collected data were analyzed using the

ORIGIN software and fit to a one site binding model.

Structural models, alignments and comparisons- PyMOL (Molecular Graphics

System, Version 1.3) was used for structural visualization and alignments

(pymol.sourceforge.net) (37). The PDBePISA server (http://www.ebi.ac.uk/pdbe) was used to analyze protein-protein interfaces (38).

Thermal Stability Shift Assays- Thermal Shift studies were performed in a

StepOne Real Time PCR system (Applied Biosystems) in MicroAmp™ 48-well reaction plate (Applied Biosytems). Melt reactions consisted of a 20µL volume containing 7µM of CSL sample and 70uM ligand sample in a buffer of 25mM MES pH 6, 500mM NaCl,

1mM EDTA, and 0.1mM TCEP. Reactions were started at 20°C and ended at 99°C over the course of an hour. Sypro Orange dye was used to track protein unfolding as indicated by increased fluorescence due to exposure of hydrophobic surfaces. Protein

Thermal Shift Software v1.2 (Applied Biosystems) was used to determine Tm values.

Results

Thermal Stability of CSL Complexes

It has been shown that intermolecular interactions, such as those that occur between a macromolecular binding a ligand, increase the thermal stability of the macromolecule (39). To begin to look at the differences of RITA, KyoT2, and RAM binding to CSL, we observed their effects on CSL stability. In order to measure the thermal stability of mammalian CSL, we used a fluorescence based thermal stability

shift assay in which peak fluorescence (Tm) indicates approximately half of the protein sample is denatured. We observed Tm of approximately 55.6°C for CSL in the absence of ligand (Figure 1). In the presence of excess RITA ligand, CSL showed a significant, yet modest, increase in peak fluorescence (Figure 1). More strikingly, in the presence of excess KyoT2 ligand, the Tm of CSL increased to ~60.6°C, a 5°C change from CSL alone (Figure 1). Similarly, in the presence of RAM ligand, the Tm of CSL increased to

~59.9°C (Figure 1). This is in agreement with binding affinities measured by isothermal titration calorimetry (ITC) demonstrating that, RAM and KyoT2 bind CSL with a dissociation constant (Kd) of ~10nM, while RITA binds with a ~500nM Kd (Table 1).

Figure 1: Thermal Stability Shift Assay data for CSL in the absence and presence of ligands. CSL undergoes a gain in stability when bound to coregulators, as shown by increasing Tm (melting temperature). CSL alone has a 55.6°C Tm. The Tm of CSL increases up to 5°C in the presence of ligand.

Table 1: Calorimetric binding data for binding to wild-type CSL.

-1 K ΔG° ΔH° -TΔS° CSL Ligand K (M ) d (uM) (kcal/mol) (kcal/mol) (kcal/mol)

WT RITA 1.9 ± 0.3 x 106 0.53 -8.5 ± 0.1 -7.6 ± 0.3 -0.9 ± 0.2

7 -17.4  WT RAM 7.1 ± 0.1 x 10 0.014 -11.3  0.2 6.1 ± 2.9 3.1 7 -15.5 ± WT KyoT2 8.5 ± 2.3 x 10 0.012 -10.8 ± 0.2 4.7 ± 0.5 0.7 WT EBNA2 1.6 ± 0.6 x 105 7.3 -7.3  0.2 -9.4  1.0 2.1  1.0

8 -10.4  WT MINT 1.1 ± 0.1 x 10 0.01 -10.9  0.1 -0.5  0.1 0.1 All experiments were performed at 25°C. Values are the mean of at least three independent experiments and errors represent the S.D. of multiple experiments. NBD represents no binding detected.

Thermodynamic Analysis of RITA, RAM, KyoT2, EBNA2, and MINT Binding CSL

Mutants

To further probe the differences in binding of CSL ligands, we utilized known CSL point mutants demonstrated to be differentially disruptive on binding subsets of CSL ligands. The body of literature on CSL contains examples of CSL point mutations that show differential binding activity. Previous work has shown that RAM and EBNA2

(Epstein-Barr virus nuclear antigen 2), a viral activator of Notch target gene transcription, differentially bind to different CSL point mutants, CSL F261L and CSL

Q333L mutants, as well as others (30,31,36,40). Initially, these results were recapitulated by ITC. While we demonstrated that the affinity of EBNA2 for CSL is only modestly affected by the CSL F261L mutant, the CSL Q333L mutant showed no detectable binding (Table 2 and 3). The opposite held true for RAM binding the F261L and Q333L CSL mutants (Table 2 and 3). Once we confirmed that our CSL mutants were trending as previously reported in the literature, we conducted the same set of experiments with CSL binding proteins, RITA, KyoT2, and MINT. Similar to RAM and as expected based on their structural similarity in complex, RITA, KyoT2, and MINT all demonstrated dramatically reduced binding to the CSL F261L mutant (Table 2). With the exception of EBNA2, the CSL Q333L mutant had a minimal effect on disrupting complex formation (Table 3). In the case of RAM, our measurements show no significant difference in binding energies between wild-type CSL and CSL Q333L mutant (Table 1 and 3).

Table 2: Calorimetric data for binding to CSL mutant F261L.

-1 K ΔG° ΔH° -TΔS° ΔΔG° CSL Ligand K (M ) d (uM) (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)

F261L RITA 2.2 ± 0.1 x 104 45.9 -5.9  0.1 -7.9  0.6 2.0  0.6 2.7 F261L RAM 4.0 ± 0.7 x 105 2.6 -6.3  2.0 -10.0  1.0 -3.7 ± 1.1 4.4 F261L KyoT2 4.6 ± 0.8 x 104 21.9 -6.3  0.1 -4.0  0.9 -2.3  1.0 4.4 F261L EBNA2 3.7 ± 1.5 x 104 32.5 -6.2  0.2 -16.0  2.2 -9.8  2.4 2.8 F261L MINT 1.0 ± 0.2 x 107 0.1 -9.6  0.1 -7.1  0.1 -2.5  0.2 1.3 All experiments were performed at 25°C. Values are the mean of at least three independent experiments and errors represent the S.D. of multiple experiments. NBD represents no binding detected.

Table 3: Calorimetric data for binding to CSL mutant Q333L.

-1 K ΔG° ΔH° -TΔS° ΔΔG° CSL Ligand K (M ) d (uM) (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)

Q333L RITA 3.5 ± 0.9 x 105 3.0 -7.5  0.1 -3.8  0.2 -3.7  0.3 1.1

8 -11.0  Q333L RAM 1.1 ± 0.3 x 10 0.01 -15.0  1.0 4.0 ± 1.3 -0.3 0.1 7 -10.0  Q333L KyoT2 2.5 ± 0.8 x 10 0.05 -14.1  0.4 4.2  0.7 0.7 0.3 Q333L EBNA2 NBD ------

7 -10.2  Q333L MINT 3.2 ± 0.6 x 10 0.03 -8.2  0.2 -2.0  0.4 0.4 0.1 All experiments were performed at 25°C. Values are the mean of at least three independent experiments and errors represent the S.D. of multiple experiments. NBD represents no binding detected.

Thermodynamic Analysis of the Hydrophobic Tetrapeptide Motif

With the exception of MINT, a common feature of the CSL binding proteins analyzed in this study is a hydrophobic tetrapeptide (φWφP) motif in which the first and third residues are any hydrophobic amino acids and the second and fourth residues are invariably a tryptophan and a proline (Figure 2). When present, the φWφP motif has been shown to be critical for binding to CSL (11,24,30). In order to better understand, the basis of EBNA2 binding differently from the other tested CSL ligands. We purchased synthetic tetrapeptide corresponding to the φWφP motif of Notch1/3,

Notch2/4, RITA, and KyoT2. Interestingly, the Notch1/3 and Notch2/4 tetrapeptides corresponding to their respective RAM domains showed no detectable binding by ITC

(Table 4). Less surprisingly, the RITA tetrapeptide also showed no binding by ITC

(Table 4). The φWφP motif of KyoT2 bound CSL with a ~12uM Kd (Table 4). The

φWφP motif of EBNA2, the weakest binding partner of CSL tested in this study, also showed binding by ITC albeit an order of magnitude weaker than KyoT2 tetrapeptide

(Table 4).

Table 4: Calorimetric data for the hydrophobic tetrapeptide motif binding to CSL.

-1 K ΔG° ΔH° -TΔS° Macromolecule Ligand K (M ) d (uM) (kcal/mol) (kcal/mol) (kcal/mol)

CSL LWTPRITA NBD ------

CSL LWFPN1/3RAM NBD ------

CSL LWLPN2/4RAM NBD ------4 CSL VWWPKyoT2 9.3 ± 2.7 x 10 11.9 -6.8  0.1 -9.2  4.2 2.4  4.3 3 CSL PWWPEBNA2 7.5 ± 0.7 x 10 134.0 -5.5  0.1 -23.3  1.7 17.8  1.7 All experiments were performed at 25°C. Values are the mean of at least three independent experiments and errors represent the S.D. of multiple experiments. NBD represents no binding detected.

Figure 2: CSL binding partners and the hydrophobic tetrapeptide motif. (Top) The surface of the BTD of CSL shown in white with Q333 and F261 surfaces highlighted in green. The hydrophobic tetrapeptides of RITA, (purple), RAM (red) and KyoT2 (blue) are shown as stick representations. (Bottom) A sequence alignment of the BTD binding regions of various CSL coregulators. The conserved hydrophobic tetrapeptide motif is boxed in yellow.

In addition to the F261L and Q333L mutants, a CSL EEF259AAA mutant has also been shown to differentially affect binding of RAM and EBNA2 (31). However, this data is nuanced by the demonstrated importance of CSL F261 for binding RAM

(12,32,36). Furthermore, recent structural data in chapter 2 has suggested the existence of a salt-bridge contact mediated by the -EE- of CSL EE259FF. This is further substantiated by the CSL-KyoT2 structure (PDB ID: 4J2X) in which KyoT2 forms a similar salt-bridge with CSL EE259 (Figure 3). In order to begin to understand differences in CSL binding, the importance of this di-glutamate feature was analyzed by generating a CSL EE259AA point mutant. Both RITA and RAM showed decreased binding affinity to CSL EE259AA when compared to wild-type CSL (Table 5). The CSL-

RITA structure suggests that R138 of RITA forms the salt-bridge contacts with EE259 of

CSL. In order to test whether binding could be rescued by a CSL-RITA charge-reversal experiment, we purchased a synthetic mutant RITA peptide with an R138E mutation and produced a CSL EE259RR mutant. Surprisingly, RITA R138E did not rescue binding to CSL EE259RR, even further reducing the strength of the interaction (Table

5).

Figure 3: The CSL-KyoT2 interaction may require salt bridge interactions. (A) The CSL-Kyot2 complex bound to DNA (PDB ID: 4J2X) showing KyoT2 binding the BTD groove of CSL (24). CSL is modeled as a ribbon diagrams, shown as the NTD in cyan, the BTD in green, and the CTD in orange. KyoT2 is shown in blue. DNA is modelled as a stick diagram and colored according to element. (B) Multiple potential salt-bridges being formed between K187 of KyoT2 and EE259 of CSL in the CSL-kyoT2 structure. (C) ITC data demonstrating dramatic loss of binding affinity for CSL upon KyoT2 truncation of K187 (24). The top panel shows CSL binding to a KyoT2 peptide containing K187 with a nanomolar affinity. The bottom panel demonstrates that loss of K187 of KyoT2 results in decreased binding affinity for CSL.

Table 5: Calorimetric data for binding to CSL mutant EE259AA and EE259RR.

-1 K ΔG° ΔH° -TΔS° ΔΔG° CSL Ligand K (M ) d (uM) (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol) 1.7 ± 0.4 x EE259AA RITA 5 6.2 -7.1  0.1 -5.0  0.5 -2.1  0.6 1.4 10 R143E 5.1 ± 1.2 x EE259RR RITA 4 21.1 -6.4  0.1 -7.7  0.1 1.3  0.1 2.1 10 4.7 ± 0.6 x EE259AA RAM 6 0.2 -9.2  0.1 -17.6  0.5 8.4  0.6 2.1 10 All experiments were performed at 25°C. Values are the mean of at least three independent experiments and errors represent the S.D. of multiple experiments. NBD represents no binding detected.

Discussion

Functioning as the nuclear effector activation of Notch target gene transcription,

CSL interacts with a wide variety of coregulators, many of which have yet to be comparatively characterized. It is apparent that between EBNA2 and RAM exists a striking contrast that has been well documented in the literature. Initial work categorized

CSL mutations that were favorable for RAM binding but detrimental for EBNA2 binding as R+/E- and vice versa R-/E+. Originally seven CSL point mutations were identified to be R+/E- while only two were confirmed to be R-/E+, F261L and Q333L (36). In parallel, our data shows that RITA, KyoT2, and MINT also segregate away from EBNA2 with regards to binding CSL mutants F261L and Q333L (Tables 2 and 3). This is in agreement with previous literature that suggests EBNA2 binds the BTD of CSL in confirmation distinct from other BTD binding proteins (33,36). When looking at a homology model of EBNA2 bound to CSL, it suffers the same loss of intermolecular contacts when F261 and Q333 are mutated to leucines (Figure 4). However, our binding data as well as previously published binding data show that CSL mutants F261L and Q333L have a distinct effect on EBNA2 not observed with RITA, KyoT2, MINT, or

RAM binding (Tables 2 and 3). This suggests that binding is not occurring along the

BTD groove like other BTD ligands, such as RAM, KyoT2, MINT, or RITA (Figure 4A-D).

Figure 4: Structural models and mutational analysis. (A) RAM (red), (B) KyoT2 (blue), (C) RITA (purple), and (D) MINT (green) binding the BTD of CSL (white). (C) Based on these structures, a homology model of EBNA2 (orange) does not support what is seen in thermodynamic studies using CSL mutants F261L and Q333L. Green BTD surface highlights indicate little to no decreased binding to leucine mutant while red and orange indicate significantly to dramatically binding to leucine mutant. CSL EE259 is denoted with black stars.

Mutations focusing on the periphery of the BTD binding surface of CSL shown in figure 2 may reveal where and how EBNA2 binding diverges from the binding path of

RITA, KyoT2, MINT, and RAM. Previous work has shown that CSL mutant

EEF259AAA is detrimental to RAM binding while not affecting EBNA2 binding to CSL

(31). Furthermore, it has been shown that CSL mutant F261A is severely reduces the affinity of RAM and RITA for CSL. In our study, we demonstrate that EE259AA in the absence of the F261A mutation is still detrimental to RAM and RITA binding (Table 5).

If EBNA2 is also dramatically effected by CSL mutant EE259AA, this suggest that the

EE259 feature may be a hinge point at which EBNA2 takes on a binding path distinct from that of RITA, KyoT2, MINT and RAM. Additionally, four RAM-binding deficient mutants have been characterized in the literature: F261R, V263R, A284R, and Q333R

(Supplementary Figure 1) (12,32,41). While all these mutants severely weaken RAM binding to CSL by at least 150-fold, only CSL mutants A284R and V263R display dramatic decreases in binding affinity for other CSL ligands, such as RITA and KyoT2

(Supplementary Figure 1) (24). Studies characterizing EBNA2 binding to these four

CSL mutants may provide clues as to the binding path of EBNA2 on CSL.

Supplementary Figure 1: Thermodynamic data for CSL cofactors binding to CSL RAM-binding deficient mutants. (Top) The BTD surface of CSL (white) shown with four surface residues (red) critical for RAM (pink) binding. (Bottom) A summary of published data demonstrating the differences among RAM, RITA, and KyoT2 binding for CSL RAM-binding deficient mutants, F261R, V263R, A284R, and Q333R (12,24).

Dissociation constants (Kd) were determined by ITC. Change in disassociation constant (ΔKd) is reported as fold-change with arrows denoting increased or decreased binding.

When analyzing the binding of the φWφP motif tetrapeptides, it is important to note that the only φWφP motifs to show binding to CSL were those of KyoT2 (-VWWP-) and EBNA2 (-PWWP-), both of which contain a di-tryptophan that fits very well to the hydrophobic pocket found on CSL (Table 4, Figure 2). It is apparent that the first position proline in the φWφP motif of EBNA2 lays next to CSL EE259 (Figure 2D). This residue is a valine in the KyoT2 φWφP motif, a less bulky residue. The bulk of the proline in the first position of the EBNA2 φWφP motif may hint at a divergence occurring at EE259 of CSL leading to the R-/E+ observed effect with F261L. The EBNA2 φWφP tetrapeptide may bind tighter to the CSL EE259AA mutant if this hypothesis is correct.

Furthermore, the φWφP motifs of Notch1-4 RAM contained either phenylalanine or leucine in place of the third position tryptophan contained in the φWφP motifs of KyoT2

(-VWWP-) and EBNA2 (-PWWP-). However, the Notch1-4 RAM tetrapeptides did not show binding by ITC. The RITA tetrapeptide contains a threonine in the third position of the φWφP motif, a less hydrophobic residue than leucine, alanine, or tryptophan.

Future studies may be able to find even more energetically favorable hydrophobic residue combinations by taking advantage of the di-tryptophan core found in the φWφP motifs of KyoT2 and EBNA2 shown to be biophysically favorable.

Therapeutics that affect Notch target gene transcription by acting on Notch pathway components have, until recently, focused on extracellular disruption of Notch signaling (42,43). This presents a problem when attempting to target Notch activation by viral protein such as EBNA2 which requires no active Notch signaling for activation of

Notch target gene transcription. Furthermore, by restricting therapeutic development to extracellular components of the Notch pathway, it is challenging to develop therapeutics

that provide for amplification or prolonging of Notch signaling. As comparative studies broaden and begin to include more of the growing number of identified CSL ligands, differentially important residues such as those identified by Fuchs et al. will be seminal to designing therapeutics that target specific Notch transcriptional complexes.

Acknowledgments

Project support was provided by the supported by National Institutes of Health Grants

5R01CA178974-03 and 5T32CA117846-09.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Author contributions

R.A.K. conceived of the study. R.A.K. and N.T. coordinated the research and contributed to data interpretation. Both authors edited and approved the final draft of the manuscript.

Chapter 4: Conclusions and Future Directions

Keeping the Notch Signaling Pathway in Context

In the process of gene regulation, many diverse proteins assemble together to activate or repress transcription. These complexes work together and are usually made up of downstream messengers of signaling pathways. Pathway target gene transcriptional regulation is an intricate interplay of many different types of proteins all acting in concert to repress or activate transcription.

Transcriptional dysregulation can be caused by mutations in transcriptional regulators, transcriptional cofactors, or signaling components. Dysregulation disrupts gene feedforward and feedback loops. Though homologous gene expression may sometimes rescue or partially compensate loss-of-function aberrations in transcriptional regulatory pathways, patients may be slow to develop symptoms of disease or may present differently from complete loss-of-function clinical cases. In signaling pathways, transcriptional regulators are the DNA binding effectors of upstream signaling, where the rubber meets the road or where the amino acid meets the nucleic acid. Because of the central role of transcriptional regulators in modulating gene activity, they are often targets of post-translational modifications that modulate DNA binding activity and, thus, activation and repression. A simple signaling pathway that can be designed using these principles is one that involves a ligand, receptor, and, most importantly, a transcriptional regulator.

The transcriptional regulator of the Notch signaling pathway is CSL. As a nuclear effector, CSL has DNA binding activity and is the recruitment hub for activating and repressing cofactors. Coactivators and corepressors that bind CSL recruit chromatin modifying enzymes in order to affect transactional output. Data that characterize the

structure and function of CSL transcriptional regulatory complexes is critical for refining models of pathway activation and repression in human development, maintenance, and disease.

In the final chapter, we will add context to findings presented in the previous chapters, including work from others as well as my own work. The goal of this document is to add to the body of research relating to the Notch signaling field. The goal of this chapter is to focus this work’s perspective. More specifically, this body of work adds to what is known about CSL-corepressor complexes, modes of CSL repression, and differences and similarities in various cofactors binding to CSL.

CSL in the nucleus and the cytoplasm

Initial understanding of Notch target gene transcription, hypothesized that CSL was constitutively bound to DNA (1,2). Multiple groups have demonstrated that levels of CSL bound to DNA at target sites increase after activation of Notch signaling, suggesting that not all exposed CSL binding sites are occupied and that CSL is a limiting factor in the Notch pathway (3-5). Supporting this, unpublished data has quantified the amount of CSL in cells to approximately 500 molecules using western blot analysis. With thousands of CSL binding sites in the genome, it remains unclear how the single nuclear effector of Notch signaling, CSL, is able to regulate at all of these sites. An additional question is whether CSL transcriptional complexes are preassembled before being recruited to CSL binding sites on DNA.

In parallel to DNA occupancy studies, CSL has been shown to form cytoplasmic pools that incur nuclear import after Notch pathway activation (3). This phenomenon, observed in Drosophila, supports the hypothesis that CSL may be, at least, partially

preassembling transcriptional complexes before binding DNA (3). Since the affinity of

CSL for DNA is relatively unchanged when bound to corepressors, preassembly of CSL transcriptional complexes may be necessitated by the bottleneck of CSL molecules present for binding cofactors. The leading model proposes that CSL repression complexes may not be constitutively required for Notch target gene repression and simply leave lasting structural changes on local chromatin that maintain repression.

RITA and transcriptional repression

Analogous to Drosophila, in mammals RITA can bind CSL and mediated its export out of the nucleus and into the cytoplasm (6). While RITA knockout mice display no obvious phenotype, studies have shown that RITA overexpression leads to pools of

CSL in the cytoplasm, hinting at a function but lacking a context (6). RITA is the only known CSL cofactor that utilizes nuclear export to achieve Notch target gene transcription. The ubiquitousness of Notch signaling coupled with studies of CSL and its DNA occupancy requirements in the Notch pathway have yet to be synthesized into a unified theory of Notch target gene regulation. Interestingly, in the presence of DNA,

RITA has a small, but significant, increase in affinity for CSL (chapter 2). It is hypothesized this may be due to the increased stability of the BTD, caused by CSL-

DNA contacts, provides a more energetically favorable binding surface for RITA. This may be happenstance or it may be suggestive of a preference for RITA to bind and export CSL molecules already bound to DNA.

Along with previous studies, this dissertation brings the field closer to understanding the function of RITA in Notch target gene regulation and how it diverges from previously characterized CSL corepressors. Unlike other characterized CSL

corepressors, such as KyoT2 and MINT, RITA is not known to recruit chromatin modification machinery and help anchor a multi-subunit complex to CSL. For example,

KyoT2 recruits PRC (polycomb repressive complex) to CSL (7,8). PRC contains histone methyltransferase activity that leads to silencing of the local chromatin (8).

Similarly, MINT recruits complexes containing histone modification activity, such as

HDAC, which silence local chromatin structure (9,10). MINT mediates it’s interactions with the repression machinery via the SPOC domain, interactions that are further explored in Appendix B and Appendix C. Furthermore, KyoT2 and MINT both bind CSL with low nanomolar affinity, similar to the coactivator RAM (11-13). Given the results of this dissertation, it is clear that RITA has a weaker binding affinity for CSL, approximately two-orders of magnitude weaker than RAM, KyoT2, and MINT. This seemingly presents no way for RITA to out compete other cofactors for CSL binding in the nucleus. In the cytoplasm however, RITA may exert its influence because of possible decreased competition for CSL binding.

Notch Therapeutics: A New Hope

As a focal point of Notch signaling, CSL is a mutual and exclusive binding partner of many different proteins. The most recent studies of transcription factors that interact with CSL complexes have identified a protein termed ZMIZ1 (14). Expression of ZMIZ1 has been shown to drive certain T-ALL cell lines in a Notch dependent manner with studies suggesting that ZMIZ1 directly interacts with CSL via the RAM domain of

NICD (14). Because ZMIZ1-induced transcription is a requirement of T-ALL cell survival and proliferation, this interaction may represent a specific therapeutic target with minimal functional overlap with homeostatic processes. In parallel, RITA upregulation in

HCC (hepatocellular carcinoma) cells reduces cell survival and induces apoptosis.

Notch gene targets have been implicated in HCC (15). In the context of phosphorylation and acetylation post-translational modifications that weaken the CSL-

RITA interaction, identification of the kinase(s) and acetyltransferases(s) may present attractive targets for repression in HCC, allowing for increased repression of Notch gene transcription. These two instances provide examples as to how to impart specificity to a

Notch based therapeutic. As such, a goal of this dissertation is to add to and expand on work that characterizes CSL transcription complexes in order to continue to identify binding surfaces unique to different CSL binding cofactors, further increasing the amount exclusive binding surfaces for specificity in therapeutic targeting.

Although limiting the therapeutic targeting of the Notch pathway to specific tissues is yet to be achieved, some researchers have taken a step back from the nucleus and looked at the Notch pathway “outside the box” of CSL and transcriptional regulation. The Notch signaling pathway is a fundamental pathway of animal development. The core components of the pathway consist of DSL ligand, Notch receptor, and CSL transcription factor. Because Notch receptor activation requires a mechanical pulling force, by its very nature, the pathway senses contact between adjacent cells. The fundamental function of the Notch receptor is to tether a transcription cofactor, NICD, to the cell membrane. After Notch receptor activation and subsequent S3 cleavage by the γ-secretase complex, NICD binds its target promoters via CSL. Researchers have co-opted this relatively simple pathway by taking advantage of the modular design to the Notch receptor (16). By genetically engineering chimeric Notch receptors, termed syn (synthetic) –Notch receptors, researchers have

been able to tether different ligand recognition domains to the extracellular portion of

Notch (16,17). These synNotch receptors are capable of being activated by membrane bound ligands other than the canonical DSL ligands, based on bioengineering requirements (16). Furthermore, the intracellular portion of the Notch can be substituted with any transcription factor (16). Because the transmembrane domain and NRR of mutant synNotch receptors are left unchanged from wild-type, synNotch still requires pulling force (cell-to-cell contact) for S2 activation and γ-secretase mediated S3 cleavage (16).

In work by the same group, researchers inserted a synNotch receptor gene into

T-cells containing an antibody based ligand-recognition domain targeting tumor antigen and a transcription factor that activated the transcription of T-cell CARs (Chimeric antigen receptors) (17). Current trends in cancer therapy have developed a immune therapy technique in which T-cell CARs, also using antibody based ligation-recognition motifs, are targeted to CD25 positive tumor cells, CD25 being a tumor specific cell surface marker (18). A current problem with this cancer immunotherapy is that severe off target effects, resulting in death, can sometimes occur when transgenic CAR - presenting T-cells attack host wild-type cells (19). Using the principals of logic gating, the synNotch approach allows for recognition of an additional antigen prior to CAR surface presentation (16). Therefore, doubly transgenic CAR+/synNotch+ T-cells initially present synNotch receptors targeting a tumor specific antigen 1, in the presence of antigen A. Notch receptor will be activated and drive transcription of antigen 2 (CD25)- targeting CARs (17). These CARs will present to the surface and only activate in the presence of antigen 2, leading to an immune response and tumor degradation (17). By

targeting two tumor-specific antigens, synNotch-modulated cancer immunotherapy becomes more specific and can decrease the occurrence and severity of off target effects (17).

Future Directions in Notch pathway research

Upon discovery of Notch and the elucidation the signaling pathway, initial work emphasized answering basic research questions addressing structural, biophysical and functional perspectives of Notch signaling. As more is understood about the Notch signaling pathway, research is shifting from basic research questions to clinical applications. As described in previous sections, therapeutic inhibitors and activators of

Notch target gene transcription is still a relatively underdeveloped field, but growing rapidly. Because the Notch signaling pathway offers a wide range of therapeutic targets and demonstrates adaptability for engineered use, it will become increasingly important to understand in what cellular contexts different coactivators and corepressors are required. Increased tissue specific knockout studies will help elucidate molecular pathways of cell-specific regulation.

In order to better understand the molecular details of RITA and other CSL coregulators in the Notch pathway, SPR (surface plasmon resonance) experiments should be performed across known CSL binding proteins with CSL coupled onto the chip surface. The binding kinetics of RITA, RAM, KyoT2, MINT, and EBNA2 for CSL have yet to be described. Experiments using DNA (containing a CSL binding motif) coupled to the SPR chip surface may also yield valuable insights into the nature of

CSL’s DNA occupancy mentioned earlier in the chapter. As structural studies expand into multi-transcription factor complexes that include CSL in complex with other DNA

binding proteins, new therapeutic targets with greater specificity can be developed.

Additionally, structures of viral activators of Notch target gene transcription, such as

EBNA2, may reveal novel modes of transcriptional activation further informing therapeutic research and development.

In closing, it is important to be reminded of the labors of all the scientists who faithfully labored away at uncovering, literally, the smallest details of Notch signaling. In reading and reviewing the Notch literature, it is quickly lost on the reader the years of life that go into proving any given minute detail. In performing research in the Notch field and life science in general, it becomes even more quickly apparent that chunks of lifetime have gone into the building of the Notch pathway model (Figure 4). Like any great human achievement, the road of progress is spans generations. At a cursory glance, the Notch seems like a simple pathway with a plethora of therapeutic targets.

However, as more Notch-related clinical mutations are characterized and more components of the Notch pathway are discovered, there will an abundance of opportunity to test the mettle of young researchers and employ the genius of seasoned scientists in further characterization and therapeutic modulation of the Notch signaling pathway.

Figure 1: The expansion of knowledge. Adapted from “The Illustrated guide to a Ph.D.”, this figure attempts to convey the contribution of this body work to the Notch field in the framework of the expansion of (A) total human knowledge. The colored portions represent a path of discovery (not to scale). (B) Basic core concepts that were established by the founders of modern biology are at the foundation of molecular biology (blue), (C) allowing for the elucidation of cellular models (green) and (D) signaling pathways (pink), (F) which was seminal for discovery of the Notch signaling pathway (red). (G-H) At the edge of our understanding of the Notch pathway, is the gap in knowledge (black). (I-J) The experimental results herein, ever so slightly, push the boundary of knowledge forward. (K-L) In the context of what is known about Notch signaling, this dissertation is but a dimple. And in the context of total human knowledge, Notch signaling represents but a cluster of dimples on an ever expanding human sphere of knowledge that has been growing since pen was put to paper.

Bibliography

100

Chapter 1:

1. Maury, J.-P. (1992) Newton : the father of modern astronomy, Harry N. Abrams, New York 2. Darwin, C. (1859) On the origin of species by means of natural selection, or, The preservation of favoured races in the struggle for life, John Murray, Albemarle Street, London 3. Mendel, G. (1901) Experiments in plant hybridisation, Royal Horticultural Society, London, 4. Dexter, J. S. (1914) The analysis of a case of continuous variation in Drosophila, Salem, Mass., 5. Morgan, T. H. (1910) Sex Limited Inheritance in Drosophila. Science 32, 120-122 6. Morgan, T. H. (1911) The Origin of Nine Wing Mutations in Drosophila. Science 33, 496-499 7. Morgan, T. H., and Bridges, C. B. (1916) Sex-linked inheritance in Drosophila, Carnegie Institution of Washington, Washington, 8. Morgan, T. H. (1926) The theory of the gene, Yale university press; etc., New Haven, 9. Morgan, T. H. (1919) The genetic and the operative evidence relating to secondary sexual characters, Carnegie Institution of Washington, Washington 10. Kidd, S., Kelley, M. R., and Young, M. W. (1986) Sequence of the notch locus of Drosophila melanogaster: relationship of the encoded protein to mammalian clotting and growth factors. Molecular and cellular biology 6, 3094-3108 11. Wharton, K. A., Johansen, K. M., Xu, T., and Artavanis-Tsakonas, S. (1985) Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43, 567-581 12. Yochem, J., and Greenwald, I. (1989) glp-1 and lin-12, genes implicated in distinct cell-cell interactions in C. elegans, encode similar transmembrane proteins. Cell 58, 553-563 13. Ellisen, L. W., Bird, J., West, D. C., Soreng, A. L., Reynolds, T. C., Smith, S. D., and Sklar, J. (1991) TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66, 649-661 14. Rebay, I., Fleming, R. J., Fehon, R. G., Cherbas, L., Cherbas, P., and Artavanis- Tsakonas, S. (1991) Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67, 687-699 15. Aster, J., Pear, W., Hasserjian, R., Erba, H., Davi, F., Luo, B., Scott, M., Baltimore, D., and Sklar, J. (1994) Functional analysis of the TAN-1 gene, a human homolog of Drosophila notch. Cold Spring Harbor symposia on quantitative biology 59, 125-136 16. Lieber, T., Kidd, S., Alcamo, E., Corbin, V., and Young, M. W. (1993) Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes & development 7, 1949-1965 17. Struhl, G., Fitzgerald, K., and Greenwald, I. (1993) Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 74, 331-345

101

18. Bierkamp, C., and Campos-Ortega, J. A. (1993) A zebrafish homologue of the Drosophila neurogenic gene Notch and its pattern of transcription during early embryogenesis. Mechanisms of development 43, 87-100 19. Weinmaster, G., Roberts, V. J., and Lemke, G. (1991) A homolog of Drosophila Notch expressed during mammalian development. Development 113, 199-205 20. Nye, J. S., Kopan, R., and Axel, R. (1994) An activated Notch suppresses neurogenesis and myogenesis but not gliogenesis in mammalian cells. Development 120, 2421-2430 21. Krebs, L. T., Xue, Y., Norton, C. R., Shutter, J. R., Maguire, M., Sundberg, J. P., Gallahan, D., Closson, V., Kitajewski, J., Callahan, R., Smith, G. H., Stark, K. L., and Gridley, T. (2000) Notch signaling is essential for vascular morphogenesis in mice. Genes & development 14, 1343-1352 22. Hitoshi, S., Alexson, T., Tropepe, V., Donoviel, D., Elia, A. J., Nye, J. S., Conlon, R. A., Mak, T. W., Bernstein, A., and van der Kooy, D. (2002) Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes & development 16, 846-858 23. Hilton, M. J., Tu, X., Wu, X., Bai, S., Zhao, H., Kobayashi, T., Kronenberg, H. M., Teitelbaum, S. L., Ross, F. P., Kopan, R., and Long, F. (2008) Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nature medicine 14, 306-314 24. Engin, F., Yao, Z., Yang, T., Zhou, G., Bertin, T., Jiang, M. M., Chen, Y., Wang, L., Zheng, H., Sutton, R. E., Boyce, B. F., and Lee, B. (2008) Dimorphic effects of Notch signaling in bone homeostasis. Nature medicine 14, 299-305 25. Sander, G. R., and Powell, B. C. (2004) Expression of notch receptors and ligands in the adult gut. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 52, 509-516 26. Crosnier, C., Vargesson, N., Gschmeissner, S., Ariza-McNaughton, L., Morrison, A., and Lewis, J. (2005) Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine. Development 132, 1093-1104 27. Fre, S., Huyghe, M., Mourikis, P., Robine, S., Louvard, D., and Artavanis- Tsakonas, S. (2005) Notch signals control the fate of immature progenitor cells in the intestine. Nature 435, 964-968 28. Wilson, A., Ferrero, I., MacDonald, H. R., and Radtke, F. (2000) Cutting edge: an essential role for Notch-1 in the development of both thymus-independent and - dependent T cells in the gut. Journal of immunology 165, 5397-5400 29. Wilson, A., MacDonald, H. R., and Radtke, F. (2001) Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. The Journal of experimental medicine 194, 1003-1012 30. Kuroda, K., Han, H., Tani, S., Tanigaki, K., Tun, T., Furukawa, T., Taniguchi, Y., Kurooka, H., Hamada, Y., Toyokuni, S., and Honjo, T. (2003) Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. Immunity 18, 301-312 31. Yuan, Z. R., Kohsaka, T., Ikegaya, T., Suzuki, T., Okano, S., Abe, J., Kobayashi, N., and Yamada, M. (1998) Mutational analysis of the Jagged 1 gene in Alagille syndrome families. Human molecular genetics 7, 1363-1369

102

32. Lorent, K., Yeo, S. Y., Oda, T., Chandrasekharappa, S., Chitnis, A., Matthews, R. P., and Pack, M. (2004) Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy. Development 131, 5753-5766 33. Yuan, Z. R., Kobayashi, N., and Kohsaka, T. (2006) Human Jagged 1 mutants cause liver defect in Alagille syndrome by overexpression of hepatocyte growth factor. Journal of molecular biology 356, 559-568 34. Oda, T., Elkahloun, A. G., Pike, B. L., Okajima, K., Krantz, I. D., Genin, A., Piccoli, D. A., Meltzer, P. S., Spinner, N. B., Collins, F. S., and Chandrasekharappa, S. C. (1997) Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nature genetics 16, 235-242 35. Bulman, M. P., Kusumi, K., Frayling, T. M., McKeown, C., Garrett, C., Lander, E. S., Krumlauf, R., Hattersley, A. T., Ellard, S., and Turnpenny, P. D. (2000) Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis. Nature genetics 24, 438-441 36. Joutel, A., Corpechot, C., Ducros, A., Vahedi, K., Chabriat, H., Mouton, P., Alamowitch, S., Domenga, V., Cecillion, M., Marechal, E., Maciazek, J., Vayssiere, C., Cruaud, C., Cabanis, E. A., Ruchoux, M. M., Weissenbach, J., Bach, J. F., Bousser, M. G., and Tournier-Lasserve, E. (1996) Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 383, 707-710 37. Joutel, A., Chabriat, H., Vahedi, K., Domenga, V., Vayssiere, C., Ruchoux, M. M., Lucas, C., Leys, D., Bousser, M. G., and Tournier-Lasserve, E. (2000) Splice site mutation causing a seven amino acid Notch3 in-frame deletion in CADASIL. Neurology 54, 1874-1875 38. Weng, A. P., Ferrando, A. A., Lee, W., Morris, J. P. t., Silverman, L. B., Sanchez- Irizarry, C., Blacklow, S. C., Look, A. T., and Aster, J. C. (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306, 269-271 39. Swiatek, P. J., Lindsell, C. E., del Amo, F. F., Weinmaster, G., and Gridley, T. (1994) Notch1 is essential for postimplantation development in mice. Genes & development 8, 707-719 40. Fehon, R. G., Kooh, P. J., Rebay, I., Regan, C. L., Xu, T., Muskavitch, M. A., and Artavanis-Tsakonas, S. (1990) Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila. Cell 61, 523-534 41. Zweifel, M. E., Leahy, D. J., and Barrick, D. (2005) Structure and Notch receptor binding of the tandem WWE domain of Deltex. Structure 13, 1599-1611 42. Hambleton, S., Valeyev, N. V., Muranyi, A., Knott, V., Werner, J. M., McMichael, A. J., Handford, P. A., and Downing, A. K. (2004) Structural and functional properties of the human notch-1 ligand binding region. Structure 12, 2173-2183 43. Lafkas, D., Shelton, A., Chiu, C., de Leon Boenig, G., Chen, Y., Stawicki, S. S., Siltanen, C., Reichelt, M., Zhou, M., Wu, X., Eastham-Anderson, J., Moore, H., Roose-Girma, M., Chinn, Y., Hang, J. Q., Warming, S., Egen, J., Lee, W. P., Austin, C., Wu, Y., Payandeh, J., Lowe, J. B., and Siebel, C. W. (2015)

103

Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung. Nature 528, 127-131 44. Kershaw, N. J., Church, N. L., Griffin, M. D., Luo, C. S., Adams, T. E., and Burgess, A. W. (2015) Notch ligand delta-like1: X-ray crystal structure and binding affinity. The Biochemical journal 468, 159-166 45. Luca, V. C., Jude, K. M., Pierce, N. W., Nachury, M. V., Fischer, S., and Garcia, K. C. (2015) Structural biology. Structural basis for Notch1 engagement of Delta- like 4. Science 347, 847-853 46. Cordle, J., Johnson, S., Tay, J. Z., Roversi, P., Wilkin, M. B., de Madrid, B. H., Shimizu, H., Jensen, S., Whiteman, P., Jin, B., Redfield, C., Baron, M., Lea, S. M., and Handford, P. A. (2008) A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition. Nature structural & molecular biology 15, 849-857 47. Weisshuhn, P. C., Sheppard, D., Taylor, P., Whiteman, P., Lea, S. M., Handford, P. A., and Redfield, C. (2016) Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies. Structure 24, 555-566 48. D'Souza, B., Miyamoto, A., and Weinmaster, G. (2008) The many facets of Notch ligands. Oncogene 27, 5148-5167 49. Tax, F. E., Yeargers, J. J., and Thomas, J. H. (1994) Sequence of C. elegans lag-2 reveals a cell-signalling domain shared with Delta and Serrate of Drosophila. Nature 368, 150-154 50. Parks, A. L., Stout, J. R., Shepard, S. B., Klueg, K. M., Dos Santos, A. A., Parody, T. R., Vaskova, M., and Muskavitch, M. A. (2006) Structure-function analysis of delta trafficking, receptor binding and signaling in Drosophila. Genetics 174, 1947-1961 51. Hamel, S., Fantini, J., and Schweisguth, F. (2010) Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster. The Journal of cell biology 188, 581-594 52. Chillakuri, C. R., Sheppard, D., Ilagan, M. X., Holt, L. R., Abbott, F., Liang, S., Kopan, R., Handford, P. A., and Lea, S. M. (2013) Structural analysis uncovers lipid-binding properties of Notch ligands. Cell reports 5, 861-867 53. Henderson, S. T., Gao, D., Christensen, S., and Kimble, J. (1997) Functional domains of LAG-2, a putative signaling ligand for LIN-12 and GLP-1 receptors in Caenorhabditis elegans. Molecular biology of the cell 8, 1751-1762 54. Geffers, I., Serth, K., Chapman, G., Jaekel, R., Schuster-Gossler, K., Cordes, R., Sparrow, D. B., Kremmer, E., Dunwoodie, S. L., Klein, T., and Gossler, A. (2007) Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo. The Journal of cell biology 178, 465-476 55. Komatsu, H., Chao, M. Y., Larkins-Ford, J., Corkins, M. E., Somers, G. A., Tucey, T., Dionne, H. M., White, J. Q., Wani, K., Boxem, M., and Hart, A. C. (2008) OSM-11 facilitates LIN-12 Notch signaling during Caenorhabditis elegans vulval development. PLoS biology 6, e196 56. Shimizu, K., Chiba, S., Kumano, K., Hosoya, N., Takahashi, T., Kanda, Y., Hamada, Y., Yazaki, Y., and Hirai, H. (1999) Mouse jagged1 physically interacts

104

with notch2 and other notch receptors. Assessment by quantitative methods. The Journal of biological chemistry 274, 32961-32969 57. Fleming, R. J., Scottgale, T. N., Diederich, R. J., and Artavanis-Tsakonas, S. (1990) The gene Serrate encodes a putative EGF-like transmembrane protein essential for proper ectodermal development in Drosophila melanogaster. Genes & development 4, 2188-2201 58. Vassin, H., Bremer, K. A., Knust, E., and Campos-Ortega, J. A. (1987) The neurogenic gene Delta of Drosophila melanogaster is expressed in neurogenic territories and encodes a putative transmembrane protein with EGF-like repeats. The EMBO journal 6, 3431-3440 59. Lissemore, J. L., and Starmer, W. T. (1999) Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG-2 (DSL) proteins. Molecular phylogenetics and evolution 11, 308-319 60. Vitt, U. A., Hsu, S. Y., and Hsueh, A. J. (2001) Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules. Molecular endocrinology 15, 681-694 61. Cordle, J., Redfieldz, C., Stacey, M., van der Merwe, P. A., Willis, A. C., Champion, B. R., Hambleton, S., and Handford, P. A. (2008) Localization of the delta-like-1-binding site in human Notch-1 and its modulation by calcium affinity. The Journal of biological chemistry 283, 11785-11793 62. Xu, A., Lei, L., and Irvine, K. D. (2005) Regions of Drosophila Notch that contribute to ligand binding and the modulatory influence of Fringe. The Journal of biological chemistry 280, 30158-30165 63. Fitzgerald, K., and Greenwald, I. (1995) Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo. Development 121, 4275-4282 64. Gao, D., and Kimble, J. (1995) APX-1 can substitute for its homolog LAG-2 to direct cell interactions throughout Caenorhabditis elegans development. Proceedings of the National Academy of Sciences of the United States of America 92, 9839-9842 65. Gu, Y., Hukriede, N. A., and Fleming, R. J. (1995) Serrate expression can functionally replace Delta activity during neuroblast segregation in the Drosophila embryo. Development 121, 855-865 66. Zeng, C., Younger-Shepherd, S., Jan, L. Y., and Jan, Y. N. (1998) Delta and Serrate are redundant Notch ligands required for asymmetric cell divisions within the Drosophila sensory organ lineage. Genes & development 12, 1086-1091 67. Ladi, E., Nichols, J. T., Ge, W., Miyamoto, A., Yao, C., Yang, L. T., Boulter, J., Sun, Y. E., Kintner, C., and Weinmaster, G. (2005) The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands. The Journal of cell biology 170, 983-992 68. Pintar, A., De Biasio, A., Popovic, M., Ivanova, N., and Pongor, S. (2007) The intracellular region of Notch ligands: does the tail make the difference? Biology direct 2, 19 69. Lai, E. C., Roegiers, F., Qin, X., Jan, Y. N., and Rubin, G. M. (2005) The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta. Development 132, 2319-2332

105

70. Song, R., Koo, B. K., Yoon, K. J., Yoon, M. J., Yoo, K. W., Kim, H. T., Oh, H. J., Kim, Y. Y., Han, J. K., Kim, C. H., and Kong, Y. Y. (2006) Neuralized-2 regulates a Notch ligand in cooperation with Mind bomb-1. The Journal of biological chemistry 281, 36391-36400 71. Pavlopoulos, E., Pitsouli, C., Klueg, K. M., Muskavitch, M. A., Moschonas, N. K., and Delidakis, C. (2001) neuralized Encodes a peripheral membrane protein involved in delta signaling and endocytosis. Developmental cell 1, 807-816 72. Itoh, M., Kim, C. H., Palardy, G., Oda, T., Jiang, Y. J., Maust, D., Yeo, S. Y., Lorick, K., Wright, G. J., Ariza-McNaughton, L., Weissman, A. M., Lewis, J., Chandrasekharappa, S. C., and Chitnis, A. B. (2003) Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta. Developmental cell 4, 67-82 73. Wang, W., and Struhl, G. (2005) Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytosis and signaling in Drosophila. Development 132, 2883-2894 74. Koo, B. K., Yoon, M. J., Yoon, K. J., Im, S. K., Kim, Y. Y., Kim, C. H., Suh, P. G., Jan, Y. N., and Kong, Y. Y. (2007) An obligatory role of mind bomb-1 in notch signaling of mammalian development. PloS one 2, e1221 75. Koutelou, E., Sato, S., Tomomori-Sato, C., Florens, L., Swanson, S. K., Washburn, M. P., Kokkinaki, M., Conaway, R. C., Conaway, J. W., and Moschonas, N. K. (2008) Neuralized-like 1 (Neurl1) targeted to the plasma membrane by N-myristoylation regulates the Notch ligand Jagged1. The Journal of biological chemistry 283, 3846-3853 76. Le Borgne, R., Remaud, S., Hamel, S., and Schweisguth, F. (2005) Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila. PLoS biology 3, e96 77. Ruan, Y., Tecott, L., Jiang, M. M., Jan, L. Y., and Jan, Y. N. (2001) Ethanol hypersensitivity and olfactory discrimination defect in mice lacking a homolog of Drosophila neuralized. Proceedings of the National Academy of Sciences of the United States of America 98, 9907-9912 78. Vollrath, B., Pudney, J., Asa, S., Leder, P., and Fitzgerald, K. (2001) Isolation of a murine homologue of the Drosophila neuralized gene, a gene required for axonemal integrity in spermatozoa and terminal maturation of the mammary gland. Molecular and cellular biology 21, 7481-7494 79. Pitsouli, C., and Delidakis, C. (2005) The interplay between DSL proteins and ubiquitin ligases in Notch signaling. Development 132, 4041-4050 80. Popovic, M., De Biasio, A., Pintar, A., and Pongor, S. (2007) The intracellular region of the Notch ligand Jagged-1 gains partial structure upon binding to synthetic membranes. The FEBS journal 274, 5325-5336 81. De Biasio, A., Guarnaccia, C., Popovic, M., Uversky, V. N., Pintar, A., and Pongor, S. (2008) Prevalence of intrinsic disorder in the intracellular region of human single-pass type I proteins: the case of the notch ligand Delta-4. Journal of proteome research 7, 2496-2506 82. Mizuhara, E., Nakatani, T., Minaki, Y., Sakamoto, Y., Ono, Y., and Takai, Y. (2005) MAGI1 recruits Dll1 to cadherin-based adherens junctions and stabilizes it on the cell surface. The Journal of biological chemistry 280, 26499-26507

106

83. Morrissette, J. D., Colliton, R. P., and Spinner, N. B. (2001) Defective intracellular transport and processing of JAG1 missense mutations in Alagille syndrome. Human molecular genetics 10, 405-413 84. Warthen, D. M., Moore, E. C., Kamath, B. M., Morrissette, J. J., Sanchez-Lara, P. A., Piccoli, D. A., Krantz, I. D., and Spinner, N. B. (2006) Jagged1 (JAG1) mutations in Alagille syndrome: increasing the mutation detection rate. Human mutation 27, 436-443 85. Colliton, R. P., Bason, L., Lu, F. M., Piccoli, D. A., Krantz, I. D., and Spinner, N. B. (2001) Mutation analysis of Jagged1 (JAG1) in Alagille syndrome patients. Human mutation 17, 151-152 86. Maine, E. M., Lissemore, J. L., and Starmer, W. T. (1995) A phylogenetic analysis of vertebrate and invertebrate Notch-related genes. Molecular phylogenetics and evolution 4, 139-149 87. Logeat, F., Bessia, C., Brou, C., LeBail, O., Jarriault, S., Seidah, N. G., and Israel, A. (1998) The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proceedings of the National Academy of Sciences of the United States of America 95, 8108-8112 88. Sanchez-Irizarry, C., Carpenter, A. C., Weng, A. P., Pear, W. S., Aster, J. C., and Blacklow, S. C. (2004) Notch subunit heterodimerization and prevention of ligand-independent proteolytic activation depend, respectively, on a novel domain and the LNR repeats. Molecular and cellular biology 24, 9265-9273 89. Gordon, W. R., Vardar-Ulu, D., L'Heureux, S., Ashworth, T., Malecki, M. J., Sanchez-Irizarry, C., McArthur, D. G., Histen, G., Mitchell, J. L., Aster, J. C., and Blacklow, S. C. (2009) Effects of S1 cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2. PloS one 4, e6613 90. Lake, R. J., Grimm, L. M., Veraksa, A., Banos, A., and Artavanis-Tsakonas, S. (2009) In vivo analysis of the Notch receptor S1 cleavage. PloS one 4, e6728 91. Haltiwanger, R. S., and Stanley, P. (2002) Modulation of receptor signaling by glycosylation: fringe is an O-fucose-beta1,3-N-acetylglucosaminyltransferase. Biochimica et biophysica acta 1573, 328-335 92. Shao, L., Moloney, D. J., and Haltiwanger, R. (2003) Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand- binding site and the Abruptex region. The Journal of biological chemistry 278, 7775-7782 93. Taylor, P., Takeuchi, H., Sheppard, D., Chillakuri, C., Lea, S. M., Haltiwanger, R. S., and Handford, P. A. (2014) Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands. Proceedings of the National Academy of Sciences of the United States of America 111, 7290-7295 94. Okajima, T., Xu, A., Lei, L., and Irvine, K. D. (2005) Chaperone activity of protein O-fucosyltransferase 1 promotes notch receptor folding. Science 307, 1599-1603 95. Acar, M., Jafar-Nejad, H., Takeuchi, H., Rajan, A., Ibrani, D., Rana, N. A., Pan, H., Haltiwanger, R. S., and Bellen, H. J. (2008) Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132, 247-258

107

96. Takeuchi, H., Fernandez-Valdivia, R. C., Caswell, D. S., Nita-Lazar, A., Rana, N. A., Garner, T. P., Weldeghiorghis, T. K., Macnaughtan, M. A., Jafar-Nejad, H., and Haltiwanger, R. S. (2011) Rumi functions as both a protein O- glucosyltransferase and a protein O-xylosyltransferase. Proceedings of the National Academy of Sciences of the United States of America 108, 16600- 16605 97. Fernandez-Valdivia, R., Takeuchi, H., Samarghandi, A., Lopez, M., Leonardi, J., Haltiwanger, R. S., and Jafar-Nejad, H. (2011) Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi. Development 138, 1925-1934 98. Arboleda-Velasquez, J. F., Rampal, R., Fung, E., Darland, D. C., Liu, M., Martinez, M. C., Donahue, C. P., Navarro-Gonzalez, M. F., Libby, P., D'Amore, P. A., Aikawa, M., Haltiwanger, R. S., and Kosik, K. S. (2005) CADASIL mutations impair Notch3 glycosylation by Fringe. Human molecular genetics 14, 1631-1639 99. Okajima, T., and Irvine, K. D. (2002) Regulation of notch signaling by o-linked fucose. Cell 111, 893-904 100. Lei, L., Xu, A., Panin, V. M., and Irvine, K. D. (2003) An O-fucose site in the ligand binding domain inhibits Notch activation. Development 130, 6411-6421 101. Lee, T. V., Sethi, M. K., Leonardi, J., Rana, N. A., Buettner, F. F., Haltiwanger, R. S., Bakker, H., and Jafar-Nejad, H. (2013) Negative regulation of notch signaling by xylose. PLoS genetics 9, e1003547 102. Fleming, R. J., Gu, Y., and Hukriede, N. A. (1997) Serrate-mediated activation of Notch is specifically blocked by the product of the gene fringe in the dorsal compartment of the Drosophila wing imaginal disc. Development 124, 2973-2981 103. Panin, V. M., Papayannopoulos, V., Wilson, R., and Irvine, K. D. (1997) Fringe modulates Notch-ligand interactions. Nature 387, 908-912 104. Gordon, W. R., Vardar-Ulu, D., Histen, G., Sanchez-Irizarry, C., Aster, J. C., and Blacklow, S. C. (2007) Structural basis for autoinhibition of Notch. Nature structural & molecular biology 14, 295-300 105. Gordon, W. R., Roy, M., Vardar-Ulu, D., Garfinkel, M., Mansour, M. R., Aster, J. C., and Blacklow, S. C. (2009) Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL. Blood 113, 4381-4390 106. Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tun, T., Furukawa, T., and Honjo, T. (1995) Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-J kappa/Su(H). Current biology : CB 5, 1416-1423 107. Fryer, C. J., White, J. B., and Jones, K. A. (2004) Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Molecular cell 16, 509-520 108. Yuan, Z., Friedmann, D. R., VanderWielen, B. D., Collins, K. J., and Kovall, R. A. (2012) Characterization of CSL (CBF-1, Su(H), Lag-1) mutants reveals differences in signaling mediated by Notch1 and Notch2. The Journal of biological chemistry 287, 34904-34916

108

109. Beatus, P., Lundkvist, J., Oberg, C., and Lendahl, U. (1999) The notch 3 intracellular domain represses notch 1-mediated activation through Hairy/Enhancer of split (HES) promoters. Development 126, 3925-3935 110. James, A. C., Szot, J. O., Iyer, K., Major, J. A., Pursglove, S. E., Chapman, G., and Dunwoodie, S. L. (2014) Notch4 reveals a novel mechanism regulating Notch signal transduction. Biochimica et biophysica acta 1843, 1272-1284 111. Chu, D., Zhang, Z., Zhou, Y., Wang, W., Li, Y., Zhang, H., Dong, G., Zhao, Q., and Ji, G. (2011) Notch1 and Notch2 have opposite prognostic effects on patients with colorectal cancer. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 22, 2440-2447 112. Fan, X., Mikolaenko, I., Elhassan, I., Ni, X., Wang, Y., Ball, D., Brat, D. J., Perry, A., and Eberhart, C. G. (2004) Notch1 and notch2 have opposite effects on embryonal brain tumor growth. Cancer research 64, 7787-7793 113. Graziani, I., Eliasz, S., De Marco, M. A., Chen, Y., Pass, H. I., De May, R. M., Strack, P. R., Miele, L., and Bocchetta, M. (2008) Opposite effects of Notch-1 and Notch-2 on mesothelioma cell survival under hypoxia are exerted through the Akt pathway. Cancer research 68, 9678-9685 114. Liu, Z., Brunskill, E., Varnum-Finney, B., Zhang, C., Zhang, A., Jay, P. Y., Bernstein, I., Morimoto, M., and Kopan, R. (2015) The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis. Development 142, 2452-2463 115. Hallaq, R., Volpicelli, F., Cuchillo-Ibanez, I., Hooper, C., Mizuno, K., Uwanogho, D., Causevic, M., Asuni, A., To, A., Soriano, S., Giese, K. P., Lovestone, S., and Killick, R. (2015) The Notch intracellular domain represses CRE-dependent transcription. Cellular signalling 27, 621-629 116. Fortini, M. E., and Artavanis-Tsakonas, S. (1994) The suppressor of hairless protein participates in notch receptor signaling. Cell 79, 273-282 117. Christensen, S., Kodoyianni, V., Bosenberg, M., Friedman, L., and Kimble, J. (1996) lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis elegans, is homologous to human CBF1 and Drosophila Su(H). Development 122, 1373-1383 118. Dou, S., Zeng, X., Cortes, P., Erdjument-Bromage, H., Tempst, P., Honjo, T., and Vales, L. D. (1994) The recombination signal sequence-binding protein RBP-2N functions as a transcriptional repressor. Molecular and cellular biology 14, 3310- 3319 119. Moellering, R. E., Cornejo, M., Davis, T. N., Del Bianco, C., Aster, J. C., Blacklow, S. C., Kung, A. L., Gilliland, D. G., Verdine, G. L., and Bradner, J. E. (2009) Direct inhibition of the NOTCH transcription factor complex. Nature 462, 182-188 120. Kovall, R. A., and Hendrickson, W. A. (2004) Crystal structure of the nuclear effector of Notch signaling, CSL, bound to DNA. The EMBO journal 23, 3441- 3451 121. Nam, Y., Weng, A. P., Aster, J. C., and Blacklow, S. C. (2003) Structural requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 transcriptional activation complex. The Journal of biological chemistry 278, 21232-21239

109

122. Chung, C. N., Hamaguchi, Y., Honjo, T., and Kawaichi, M. (1994) Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-J kappa. Nucleic acids research 22, 2938-2944 123. Tun, T., Hamaguchi, Y., Matsunami, N., Furukawa, T., Honjo, T., and Kawaichi, M. (1994) Recognition sequence of a highly conserved DNA binding protein RBP-J kappa. Nucleic acids research 22, 965-971 124. Friedmann, D. R., and Kovall, R. A. (2010) Thermodynamic and structural insights into CSL-DNA complexes. Protein science : a publication of the Protein Society 19, 34-46 125. Torella, R., Li, J., Kinrade, E., Cerda-Moya, G., Contreras, A. N., Foy, R., Stojnic, R., Glen, R. C., Kovall, R. A., Adryan, B., and Bray, S. J. (2014) A combination of computational and experimental approaches identifies DNA sequence constraints associated with target site binding specificity of the transcription factor CSL. Nucleic acids research 42, 10550-10563 126. Wu, L., Aster, J. C., Blacklow, S. C., Lake, R., Artavanis-Tsakonas, S., and Griffin, J. D. (2000) MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nature genetics 26, 484-489 127. VanderWielen, B. D., Yuan, Z., Friedmann, D. R., and Kovall, R. A. (2011) Transcriptional repression in the Notch pathway: thermodynamic characterization of CSL-MINT (Msx2-interacting nuclear target protein) complexes. The Journal of biological chemistry 286, 14892-14902 128. Oswald, F., Kostezka, U., Astrahantseff, K., Bourteele, S., Dillinger, K., Zechner, U., Ludwig, L., Wilda, M., Hameister, H., Knochel, W., Liptay, S., and Schmid, R. M. (2002) SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. The EMBO journal 21, 5417-5426 129. Kao, H. Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C. R., Evans, R. M., and Kadesch, T. (1998) A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes & development 12, 2269-2277 130. Morel, V., Lecourtois, M., Massiani, O., Maier, D., Preiss, A., and Schweisguth, F. (2001) Transcriptional repression by suppressor of hairless involves the binding of a hairless-dCtBP complex in Drosophila. Current biology : CB 11, 789- 792 131. Barolo, S., Stone, T., Bang, A. G., and Posakony, J. W. (2002) Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless. Genes & development 16, 1964-1976 132. Qin, H., Du, D., Zhu, Y., Li, J., Feng, L., Liang, Y., and Han, H. (2005) The PcG protein HPC2 inhibits RBP-J-mediated transcription by interacting with LIM protein KyoT2. FEBS letters 579, 1220-1226 133. Qin, H., Wang, J., Liang, Y., Taniguchi, Y., Tanigaki, K., and Han, H. (2004) RING1 inhibits transactivation of RBP-J by Notch through interaction with LIM protein KyoT2. Nucleic acids research 32, 1492-1501 134. Wacker, S. A., Alvarado, C., von Wichert, G., Knippschild, U., Wiedenmann, J., Clauss, K., Nienhaus, G. U., Hameister, H., Baumann, B., Borggrefe, T.,

110

Knochel, W., and Oswald, F. (2011) RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J. The EMBO journal 30, 43-56 135. Collins, K. J., Yuan, Z., and Kovall, R. A. (2014) Structure and function of the CSL-KyoT2 corepressor complex: a negative regulator of Notch signaling. Structure 22, 70-81 136. Shimizu, K., Chiba, S., Hosoya, N., Kumano, K., Saito, T., Kurokawa, M., Kanda, Y., Hamada, Y., and Hirai, H. (2000) Binding of Delta1, Jagged1, and Jagged2 to Notch2 rapidly induces cleavage, nuclear translocation, and hyperphosphorylation of Notch2. Molecular and cellular biology 20, 6913-6922 137. Shimizu, K., Chiba, S., Saito, T., Kumano, K., and Hirai, H. (2000) Physical interaction of Delta1, Jagged1, and Jagged2 with Notch1 and Notch3 receptors. Biochemical and biophysical research communications 276, 385-389 138. Varnum-Finney, B., Wu, L., Yu, M., Brashem-Stein, C., Staats, S., Flowers, D., Griffin, J. D., and Bernstein, I. D. (2000) Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. Journal of cell science 113 Pt 23, 4313-4318 139. Small, D., Kovalenko, D., Kacer, D., Liaw, L., Landriscina, M., Di Serio, C., Prudovsky, I., and Maciag, T. (2001) Soluble Jagged 1 represses the function of its transmembrane form to induce the formation of the Src-dependent chord-like phenotype. The Journal of biological chemistry 276, 32022-32030 140. Sun, X., and Artavanis-Tsakonas, S. (1997) Secreted forms of DELTA and SERRATE define antagonists of Notch signaling in Drosophila. Development 124, 3439-3448 141. Gordon, W. R., Zimmerman, B., He, L., Miles, L. J., Huang, J., Tiyanont, K., McArthur, D. G., Aster, J. C., Perrimon, N., Loparo, J. J., and Blacklow, S. C. (2015) Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch. Developmental cell 33, 729-736 142. van Tetering, G., van Diest, P., Verlaan, I., van der Wall, E., Kopan, R., and Vooijs, M. (2009) Metalloprotease ADAM10 is required for Notch1 site 2 cleavage. The Journal of biological chemistry 284, 31018-31027 143. de Celis, J. F., and Bray, S. (1997) Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124, 3241-3251 144. Micchelli, C. A., Rulifson, E. J., and Blair, S. S. (1997) The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate. Development 124, 1485- 1495 145. Klein, T., Brennan, K., and Arias, A. M. (1997) An intrinsic dominant negative activity of serrate that is modulated during wing development in Drosophila. Developmental biology 189, 123-134 146. Klein, T., and Arias, A. M. (1998) Interactions among Delta, Serrate and Fringe modulate Notch activity during Drosophila wing development. Development 125, 2951-2962 147. Simpson, P. (1997) Notch signalling in development: on equivalence groups and asymmetric developmental potential. Current opinion in genetics & development 7, 537-542

111

148. Pan, D., and Rubin, G. M. (1997) Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 90, 271-280 149. Simpson, P. (1990) Lateral inhibition and the development of the sensory bristles of the adult peripheral nervous system of Drosophila. Development 109, 509-519 150. Wakamatsu, Y., Maynard, T. M., and Weston, J. A. (2000) Fate determination of neural crest cells by NOTCH-mediated lateral inhibition and asymmetrical cell division during gangliogenesis. Development 127, 2811-2821 151. Sprinzak, D., Lakhanpal, A., Lebon, L., Santat, L. A., Fontes, M. E., Anderson, G. A., Garcia-Ojalvo, J., and Elowitz, M. B. (2010) Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature 465, 86-90 152. Sprinzak, D., Lakhanpal, A., LeBon, L., Garcia-Ojalvo, J., and Elowitz, M. B. (2011) Mutual inactivation of Notch receptors and ligands facilitates developmental patterning. PLoS computational biology 7, e1002069 153. Shaya, O., and Sprinzak, D. (2011) From Notch signaling to fine-grained patterning: Modeling meets experiments. Current opinion in genetics & development 21, 732-739 154. Collier, J. R., Monk, N. A., Maini, P. K., and Lewis, J. H. (1996) Pattern formation by lateral inhibition with feedback: a mathematical model of delta-notch intercellular signalling. Journal of theoretical biology 183, 429-446 155. Miller, A. C., Lyons, E. L., and Herman, T. G. (2009) cis-Inhibition of Notch by endogenous Delta biases the outcome of lateral inhibition. Current biology : CB 19, 1378-1383 156. De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J., Goate, A., and Kopan, R. (1999) A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398, 518-522 157. Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398, 513-517 158. Ye, Y., Lukinova, N., and Fortini, M. E. (1999) Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants. Nature 398, 525-529 159. Nam, Y., Sliz, P., Song, L., Aster, J. C., and Blacklow, S. C. (2006) Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes. Cell 124, 973-983 160. Hansson, M. L., Popko-Scibor, A. E., Saint Just Ribeiro, M., Dancy, B. M., Lindberg, M. J., Cole, P. A., and Wallberg, A. E. (2009) The transcriptional coactivator MAML1 regulates p300 autoacetylation and HAT activity. Nucleic acids research 37, 2996-3006 161. Saint Just Ribeiro, M., Hansson, M. L., Lindberg, M. J., Popko-Scibor, A. E., and Wallberg, A. E. (2009) GSK3beta is a negative regulator of the transcriptional coactivator MAML1. Nucleic acids research 37, 6691-6700 162. Lai, E. C. (2002) Protein degradation: four E3s for the notch pathway. Current biology : CB 12, R74-78 163. Qiu, L., Joazeiro, C., Fang, N., Wang, H. Y., Elly, C., Altman, Y., Fang, D., Hunter, T., and Liu, Y. C. (2000) Recognition and ubiquitination of Notch by Itch,

112

a hect-type E3 ubiquitin ligase. The Journal of biological chemistry 275, 35734- 35737 164. Sakata, T., Sakaguchi, H., Tsuda, L., Higashitani, A., Aigaki, T., Matsuno, K., and Hayashi, S. (2004) Drosophila Nedd4 regulates endocytosis of notch and suppresses its ligand-independent activation. Current biology : CB 14, 2228-2236 165. Liefke, R., Oswald, F., Alvarado, C., Ferres-Marco, D., Mittler, G., Rodriguez, P., Dominguez, M., and Borggrefe, T. (2010) Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex. Genes & development 24, 590-601 166. Oswald, F., Winkler, M., Cao, Y., Astrahantseff, K., Bourteele, S., Knochel, W., and Borggrefe, T. (2005) RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes. Molecular and cellular biology 25, 10379-10390 167. Friedmann, D. R., Wilson, J. J., and Kovall, R. A. (2008) RAM-induced allostery facilitates assembly of a notch pathway active transcription complex. The Journal of biological chemistry 283, 14781-14791 168. Johnson, S. E., Ilagan, M. X., Kopan, R., and Barrick, D. (2010) Thermodynamic analysis of the CSL x Notch interaction: distribution of binding energy of the Notch RAM region to the CSL beta-trefoil domain and the mode of competition with the viral transactivator EBNA2. The Journal of biological chemistry 285, 6681-6692 169. Lubman, O. Y., Ilagan, M. X., Kopan, R., and Barrick, D. (2007) Quantitative dissection of the Notch:CSL interaction: insights into the Notch-mediated transcriptional switch. Journal of molecular biology 365, 577-589 170. Ling, P. D., and Hayward, S. D. (1995) Contribution of conserved amino acids in mediating the interaction between EBNA2 and CBF1/RBPJk. Journal of virology 69, 1944-1950 171. Wilson, J. J., and Kovall, R. A. (2006) Crystal structure of the CSL-Notch- Mastermind ternary complex bound to DNA. Cell 124, 985-996 172. Choi, S. H., Wales, T. E., Nam, Y., O'Donovan, D. J., Sliz, P., Engen, J. R., and Blacklow, S. C. (2012) Conformational locking upon cooperative assembly of notch transcription complexes. Structure 20, 340-349 173. Nam, Y., Sliz, P., Pear, W. S., Aster, J. C., and Blacklow, S. C. (2007) Cooperative assembly of higher-order Notch complexes functions as a switch to induce transcription. Proceedings of the National Academy of Sciences of the United States of America 104, 2103-2108 174. Ong, C. T., Cheng, H. T., Chang, L. W., Ohtsuka, T., Kageyama, R., Stormo, G. D., and Kopan, R. (2006) Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability. The Journal of biological chemistry 281, 5106- 5119 175. Arnett, K. L., Hass, M., McArthur, D. G., Ilagan, M. X., Aster, J. C., Kopan, R., and Blacklow, S. C. (2010) Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nature structural & molecular biology 17, 1312-1317 176. Zhang, X. P., Zheng, G., Zou, L., Liu, H. L., Hou, L. H., Zhou, P., Yin, D. D., Zheng, Q. J., Liang, L., Zhang, S. Z., Feng, L., Yao, L. B., Yang, A. G., Han, H.,

113

and Chen, J. Y. (2008) Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells. Molecular and cellular biochemistry 307, 101-108 177. Fre, S., Pallavi, S. K., Huyghe, M., Lae, M., Janssen, K. P., Robine, S., Artavanis-Tsakonas, S., and Louvard, D. (2009) Notch and Wnt signals cooperatively control cell proliferation and tumorigenesis in the intestine. Proceedings of the National Academy of Sciences of the United States of America 106, 6309-6314 178. Go, M. J., Eastman, D. S., and Artavanis-Tsakonas, S. (1998) Cell proliferation control by Notch signaling in Drosophila development. Development 125, 2031- 2040 179. Dontu, G., Jackson, K. W., McNicholas, E., Kawamura, M. J., Abdallah, W. M., and Wicha, M. S. (2004) Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast cancer research : BCR 6, R605- 615 180. Ciofani, M., and Zuniga-Pflucker, J. C. (2005) Notch promotes survival of pre-T cells at the beta-selection checkpoint by regulating cellular metabolism. Nature immunology 6, 881-888 181. Giraldez, A. J., and Cohen, S. M. (2003) Wingless and Notch signaling provide cell survival cues and control cell proliferation during wing development. Development 130, 6533-6543 182. Sahlgren, C., Gustafsson, M. V., Jin, S., Poellinger, L., and Lendahl, U. (2008) Notch signaling mediates hypoxia-induced tumor cell migration and invasion. Proceedings of the National Academy of Sciences of the United States of America 105, 6392-6397 183. Wang, Z., Li, Y., Banerjee, S., Kong, D., Ahmad, A., Nogueira, V., Hay, N., and Sarkar, F. H. (2010) Down-regulation of Notch-1 and Jagged-1 inhibits prostate cancer cell growth, migration and invasion, and induces apoptosis via inactivation of Akt, mTOR, and NF-kappaB signaling pathways. Journal of cellular biochemistry 109, 726-736 184. Joshi, I., Minter, L. M., Telfer, J., Demarest, R. M., Capobianco, A. J., Aster, J. C., Sicinski, P., Fauq, A., Golde, T. E., and Osborne, B. A. (2009) Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 113, 1689-1698 185. Campa, V. M., Gutierrez-Lanza, R., Cerignoli, F., Diaz-Trelles, R., Nelson, B., Tsuji, T., Barcova, M., Jiang, W., and Mercola, M. (2008) Notch activates cell cycle reentry and progression in quiescent cardiomyocytes. The Journal of cell biology 183, 129-141 186. Noseda, M., Chang, L., McLean, G., Grim, J. E., Clurman, B. E., Smith, L. L., and Karsan, A. (2004) Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression. Molecular and cellular biology 24, 8813-8822 187. Alunni, A., Krecsmarik, M., Bosco, A., Galant, S., Pan, L., Moens, C. B., and Bally-Cuif, L. (2013) Notch3 signaling gates cell cycle entry and limits neural stem cell amplification in the adult pallium. Development 140, 3335-3347

114

188. Bray, S. J. (2006) Notch signalling: a simple pathway becomes complex. Nature reviews. Molecular cell biology 7, 678-689 189. Lai, E. C. (2004) Notch signaling: control of cell communication and cell fate. Development 131, 965-973 190. Perdigoto, C. N., and Bardin, A. J. (2013) Sending the right signal: Notch and stem cells. Biochimica et biophysica acta 1830, 2307-2322 191. Lewis, J. (1998) Notch signalling and the control of cell fate choices in vertebrates. Seminars in cell & developmental biology 9, 583-589 192. Kokubo, H., Lun, Y., and Johnson, R. L. (1999) Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split. Biochemical and biophysical research communications 260, 459-465 193. Kageyama, R., Ohtsuka, T., and Kobayashi, T. (2007) The Hes gene family: repressors and oscillators that orchestrate embryogenesis. Development 134, 1243-1251 194. Chin, M. T., Maemura, K., Fukumoto, S., Jain, M. K., Layne, M. D., Watanabe, M., Hsieh, C. M., and Lee, M. E. (2000) Cardiovascular basic helix loop helix factor 1, a novel transcriptional repressor expressed preferentially in the developing and adult cardiovascular system. The Journal of biological chemistry 275, 6381-6387 195. Ishibashi, M., Ang, S. L., Shiota, K., Nakanishi, S., Kageyama, R., and Guillemot, F. (1995) Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects. Genes & development 9, 3136-3148 196. Hirata, H., Yoshiura, S., Ohtsuka, T., Bessho, Y., Harada, T., Yoshikawa, K., and Kageyama, R. (2002) Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop. Science 298, 840-843 197. Bessho, Y., Hirata, H., Masamizu, Y., and Kageyama, R. (2003) Periodic repression by the bHLH factor Hes7 is an essential mechanism for the somite segmentation clock. Genes & development 17, 1451-1456 198. Hirata, H., Bessho, Y., Kokubu, H., Masamizu, Y., Yamada, S., Lewis, J., and Kageyama, R. (2004) Instability of Hes7 protein is crucial for the somite segmentation clock. Nature genetics 36, 750-754 199. Perdigoto, C. N., Schweisguth, F., and Bardin, A. J. (2011) Distinct levels of Notch activity for commitment and terminal differentiation of stem cells in the adult fly intestine. Development 138, 4585-4595 200. Milano, J., McKay, J., Dagenais, C., Foster-Brown, L., Pognan, F., Gadient, R., Jacobs, R. T., Zacco, A., Greenberg, B., and Ciaccio, P. J. (2004) Modulation of notch processing by gamma-secretase inhibitors causes intestinal goblet cell metaplasia and induction of genes known to specify gut secretory lineage differentiation. Toxicological sciences : an official journal of the Society of Toxicology 82, 341-358 201. van Es, J. H., van Gijn, M. E., Riccio, O., van den Born, M., Vooijs, M., Begthel, H., Cozijnsen, M., Robine, S., Winton, D. J., Radtke, F., and Clevers, H. (2005) Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435, 959-963

115

202. Wong, G. T., Manfra, D., Poulet, F. M., Zhang, Q., Josien, H., Bara, T., Engstrom, L., Pinzon-Ortiz, M., Fine, J. S., Lee, H. J., Zhang, L., Higgins, G. A., and Parker, E. M. (2004) Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. The Journal of biological chemistry 279, 12876- 12882 203. Sandy, A. R., and Maillard, I. (2009) Notch signaling in the hematopoietic system. Expert opinion on biological therapy 9, 1383-1398 204. Sandy, A. R., Jones, M., and Maillard, I. (2012) Notch signaling and development of the hematopoietic system. Advances in experimental medicine and biology 727, 71-88 205. Kumano, K., Chiba, S., Kunisato, A., Sata, M., Saito, T., Nakagami-Yamaguchi, E., Yamaguchi, T., Masuda, S., Shimizu, K., Takahashi, T., Ogawa, S., Hamada, Y., and Hirai, H. (2003) Notch1 but not Notch2 is essential for generating hematopoietic stem cells from endothelial cells. Immunity 18, 699-711 206. Han, H., Tanigaki, K., Yamamoto, N., Kuroda, K., Yoshimoto, M., Nakahata, T., Ikuta, K., and Honjo, T. (2002) Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. International immunology 14, 637-645 207. Pui, J. C., Allman, D., Xu, L., DeRocco, S., Karnell, F. G., Bakkour, S., Lee, J. Y., Kadesch, T., Hardy, R. R., Aster, J. C., and Pear, W. S. (1999) Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11, 299-308 208. Radtke, F., Wilson, A., Stark, G., Bauer, M., van Meerwijk, J., MacDonald, H. R., and Aguet, M. (1999) Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10, 547-558 209. De Smedt, M., Hoebeke, I., Reynvoet, K., Leclercq, G., and Plum, J. (2005) Different thresholds of Notch signaling bias human precursor cells toward B-, NK- , monocytic/dendritic-, or T-cell lineage in thymus microenvironment. Blood 106, 3498-3506 210. Cheng, H., and Leblond, C. P. (1974) Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. The American journal of anatomy 141, 461-479 211. VanDussen, K. L., Carulli, A. J., Keeley, T. M., Patel, S. R., Puthoff, B. J., Magness, S. T., Tran, I. T., Maillard, I., Siebel, C., Kolterud, A., Grosse, A. S., Gumucio, D. L., Ernst, S. A., Tsai, Y. H., Dempsey, P. J., and Samuelson, L. C. (2012) Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells. Development 139, 488-497 212. Lanford, P. J., Lan, Y., Jiang, R., Lindsell, C., Weinmaster, G., Gridley, T., and Kelley, M. W. (1999) Notch signalling pathway mediates hair cell development in mammalian cochlea. Nature genetics 21, 289-292 213. Lasky, J. L., and Wu, H. (2005) Notch signaling, brain development, and human disease. Pediatric research 57, 104R-109R 214. Penton, A. L., Leonard, L. D., and Spinner, N. B. (2012) Notch signaling in human development and disease. Seminars in cell & developmental biology 23, 450-457

116

215. Maliekal, T. T., Bajaj, J., Giri, V., Subramanyam, D., and Krishna, S. (2008) The role of Notch signaling in human cervical cancer: implications for solid tumors. Oncogene 27, 5110-5114 216. Koch, U., and Radtke, F. (2010) Notch signaling in solid tumors. Current topics in developmental biology 92, 411-455 217. Luxan, G., D'Amato, G., MacGrogan, D., and de la Pompa, J. L. (2016) Endocardial Notch Signaling in Cardiac Development and Disease. Circulation research 118, e1-e18 218. MacGrogan, D., Luna-Zurita, L., and de la Pompa, J. L. (2011) Notch signaling in cardiac valve development and disease. Birth defects research. Part A, Clinical and molecular teratology 91, 449-459 219. Louvi, A., and Artavanis-Tsakonas, S. (2012) Notch and disease: a growing field. Seminars in cell & developmental biology 23, 473-480 220. Hayward, S. D. (2004) Viral interactions with the Notch pathway. Seminars in cancer biology 14, 387-396 221. Veitia, R. A. (2006) The biology of genetic dominance, Landes Bioscience : Eurekah.com, Georgetown, Tex. 222. Fehon, R. G., Johansen, K., Rebay, I., and Artavanis-Tsakonas, S. (1991) Complex cellular and subcellular regulation of notch expression during embryonic and imaginal development of Drosophila: implications for notch function. The Journal of cell biology 113, 657-669 223. Fanto, M., and Mlodzik, M. (1999) Asymmetric Notch activation specifies photoreceptors R3 and R4 and planar polarity in the Drosophila eye. Nature 397, 523-526 224. Lyman, D. F., and Yedvobnick, B. (1995) Drosophila Notch receptor activity suppresses Hairless function during adult external sensory organ development. Genetics 141, 1491-1505 225. McCright, B., Lozier, J., and Gridley, T. (2002) A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Development 129, 1075-1082 226. Surendran, K., Selassie, M., Liapis, H., Krigman, H., and Kopan, R. (2010) Reduced Notch signaling leads to renal cysts and papillary microadenomas. Journal of the American Society of Nephrology : JASN 21, 819-832 227. Matsuda, T., Miyagawa, S., Fukushima, S., Kitagawa-Sakakida, S., Akimaru, H., Horii-Komatsu, M., Kawamoto, A., Saito, A., Asahara, T., and Sawa, Y. (2014) Human cardiac stem cells with reduced notch signaling show enhanced therapeutic potential in a rat acute infarction model. Circulation journal : official journal of the Japanese Circulation Society 78, 222-231 228. Guo, H., Lu, Y., Wang, J., Liu, X., Keller, E. T., Liu, Q., Zhou, Q., and Zhang, J. (2014) Targeting the Notch signaling pathway in cancer therapeutics. Thoracic cancer 5, 473-486 229. Yuan, X., Wu, H., Xu, H., Xiong, H., Chu, Q., Yu, S., Wu, G. S., and Wu, K. (2015) Notch signaling: an emerging therapeutic target for cancer treatment. Cancer letters 369, 20-27 230. Astudillo, L., da Silva, T. G., Wang, Z., Han, X., Jin, K., VanWye, J., Zhu, X., Weaver, K. L., Oashi, T., Lopes, P. E. M., Orton, D., Neitzel, L. R., Lee, E.,

117

Landgraf, R., Robbins, D. J., MacKerell, A. D., and Capobianco, A. J. (2016) The small molecule IMR-1 inhibits the Notch transcriptional activation complex to suppress tumorigenesis. Cancer research 231. Alagille, D., Estrada, A., Hadchouel, M., Gautier, M., Odievre, M., and Dommergues, J. P. (1987) Syndromic paucity of interlobular bile ducts (Alagille syndrome or arteriohepatic dysplasia): review of 80 cases. The Journal of pediatrics 110, 195-200 232. Emerick, K. M., Rand, E. B., Goldmuntz, E., Krantz, I. D., Spinner, N. B., and Piccoli, D. A. (1999) Features of Alagille syndrome in 92 patients: frequency and relation to prognosis. Hepatology 29, 822-829 233. Li, L., Krantz, I. D., Deng, Y., Genin, A., Banta, A. B., Collins, C. C., Qi, M., Trask, B. J., Kuo, W. L., Cochran, J., Costa, T., Pierpont, M. E., Rand, E. B., Piccoli, D. A., Hood, L., and Spinner, N. B. (1997) Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nature genetics 16, 243-251 234. Krantz, I. D., Colliton, R. P., Genin, A., Rand, E. B., Li, L., Piccoli, D. A., and Spinner, N. B. (1998) Spectrum and frequency of jagged1 (JAG1) mutations in Alagille syndrome patients and their families. American journal of human genetics 62, 1361-1369 235. McDaniell, R., Warthen, D. M., Sanchez-Lara, P. A., Pai, A., Krantz, I. D., Piccoli, D. A., and Spinner, N. B. (2006) NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway. American journal of human genetics 79, 169-173 236. Huppert, S. S. (2016) A faithful JAGGED1 haploinsufficiency mouse model of arteriohepatic dysplasia (Alagille syndrome) after all. Hepatology 63, 365-367 237. Chau, M. D., Tuft, R., Fogarty, K., and Bao, Z. Z. (2006) Notch signaling plays a key role in cardiac cell differentiation. Mechanisms of development 123, 626-640 238. Koyanagi, M., Bushoven, P., Iwasaki, M., Urbich, C., Zeiher, A. M., and Dimmeler, S. (2007) Notch signaling contributes to the expression of cardiac markers in human circulating progenitor cells. Circulation research 101, 1139- 1145 239. Garg, V., Muth, A. N., Ransom, J. F., Schluterman, M. K., Barnes, R., King, I. N., Grossfeld, P. D., and Srivastava, D. (2005) Mutations in NOTCH1 cause aortic valve disease. Nature 437, 270-274 240. Krantz, I. D., Smith, R., Colliton, R. P., Tinkel, H., Zackai, E. H., Piccoli, D. A., Goldmuntz, E., and Spinner, N. B. (1999) Jagged1 mutations in patients ascertained with isolated congenital heart defects. American journal of medical genetics 84, 56-60 241. Rusanescu, G., Weissleder, R., and Aikawa, E. (2008) Notch signaling in cardiovascular disease and calcification. Current cardiology reviews 4, 148-156 242. Okeda, R., Arima, K., and Kawai, M. (2002) Arterial changes in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in relation to pathogenesis of diffuse myelin loss of cerebral white matter: examination of cerebral medullary arteries by reconstruction of serial sections of an autopsy case. Stroke; a journal of cerebral circulation 33, 2565-2569

118

243. Chabriat, H., Vahedi, K., Iba-Zizen, M. T., Joutel, A., Nibbio, A., Nagy, T. G., Krebs, M. O., Julien, J., Dubois, B., Ducrocq, X., and et al. (1995) Clinical spectrum of CADASIL: a study of 7 families. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. Lancet 346, 934- 939 244. Chabriat, H., Tournier-Lasserve, E., Vahedi, K., Leys, D., Joutel, A., Nibbio, A., Escaillas, J. P., Iba-Zizen, M. T., Bracard, S., Tehindrazanarivelo, A., and et al. (1995) Autosomal dominant migraine with MRI white-matter abnormalities mapping to the CADASIL locus. Neurology 45, 1086-1091 245. Dichgans, M., Mayer, M., Uttner, I., Bruning, R., Muller-Hocker, J., Rungger, G., Ebke, M., Klockgether, T., and Gasser, T. (1998) The phenotypic spectrum of CADASIL: clinical findings in 102 cases. Annals of neurology 44, 731-739 246. Tournier-Lasserve, E., Joutel, A., Melki, J., Weissenbach, J., Lathrop, G. M., Chabriat, H., Mas, J. L., Cabanis, E. A., Baudrimont, M., Maciazek, J., and et al. (1993) Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy maps to chromosome 19q12. Nature genetics 3, 256-259 247. Joutel, A., Vahedi, K., Corpechot, C., Troesch, A., Chabriat, H., Vayssiere, C., Cruaud, C., Maciazek, J., Weissenbach, J., Bousser, M. G., Bach, J. F., and Tournier-Lasserve, E. (1997) Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients. Lancet 350, 1511-1515 248. Ehret, F., Vogler, S., Pojar, S., Elliott, D. A., Bradke, F., Steiner, B., and Kempermann, G. (2015) Mouse model of CADASIL reveals novel insights into Notch3 function in adult hippocampal neurogenesis. Neurobiology of disease 75, 131-141 249. Joutel, A. (2011) Pathogenesis of CADASIL: transgenic and knock-out mice to probe function and dysfunction of the mutated gene, Notch3, in the cerebrovasculature. BioEssays : news and reviews in molecular, cellular and developmental biology 33, 73-80 250. Sikandar, S. S., Pate, K. T., Anderson, S., Dizon, D., Edwards, R. A., Waterman, M. L., and Lipkin, S. M. (2010) NOTCH signaling is required for formation and self-renewal of tumor-initiating cells and for repression of secretory cell differentiation in colon cancer. Cancer research 70, 1469-1478 251. Reedijk, M., Odorcic, S., Zhang, H., Chetty, R., Tennert, C., Dickson, B. C., Lockwood, G., Gallinger, S., and Egan, S. E. (2008) Activation of Notch signaling in human colon adenocarcinoma. International journal of oncology 33, 1223-1229 252. Garcia, A., and Kandel, J. J. (2012) Notch: a key regulator of tumor angiogenesis and metastasis. Histology and histopathology 27, 151-156 253. Chu, D., Li, Y., Wang, W., Zhao, Q., Li, J., Lu, Y., Li, M., Dong, G., Zhang, H., Xie, H., and Ji, G. (2010) High level of Notch1 protein is associated with poor overall survival in colorectal cancer. Annals of surgical oncology 17, 1337-1342 254. Guilmeau, S., Flandez, M., Mariadason, J. M., and Augenlicht, L. H. (2010) Heterogeneity of Jagged1 expression in human and mouse intestinal tumors: implications for targeting Notch signaling. Oncogene 29, 992-1002 255. Pellegrinet, L., Rodilla, V., Liu, Z., Chen, S., Koch, U., Espinosa, L., Kaestner, K. H., Kopan, R., Lewis, J., and Radtke, F. (2011) Dll1- and dll4-mediated notch

119

signaling are required for homeostasis of intestinal stem cells. Gastroenterology 140, 1230-1240 e1231-1237 256. Sonoshita, M., Aoki, M., Fuwa, H., Aoki, K., Hosogi, H., Sakai, Y., Hashida, H., Takabayashi, A., Sasaki, M., Robine, S., Itoh, K., Yoshioka, K., Kakizaki, F., Kitamura, T., Oshima, M., and Taketo, M. M. (2011) Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling. Cancer cell 19, 125-137 257. Noah, T. K., and Shroyer, N. F. (2013) Notch in the intestine: regulation of homeostasis and pathogenesis. Annual review of physiology 75, 263-288 258. Chiaretti, S., and Foa, R. (2009) T-cell acute lymphoblastic leukemia. Haematologica 94, 160-162 259. Hsieh, J. J., Henkel, T., Salmon, P., Robey, E., Peterson, M. G., and Hayward, S. D. (1996) Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Molecular and cellular biology 16, 952-959 260. Lee, S. Y., Kumano, K., Nakazaki, K., Sanada, M., Matsumoto, A., Yamamoto, G., Nannya, Y., Suzuki, R., Ota, S., Ota, Y., Izutsu, K., Sakata-Yanagimoto, M., Hangaishi, A., Yagita, H., Fukayama, M., Seto, M., Kurokawa, M., Ogawa, S., and Chiba, S. (2009) Gain-of-function mutations and copy number increases of Notch2 in diffuse large B-cell lymphoma. Cancer science 100, 920-926 261. Wang, N. J., Sanborn, Z., Arnett, K. L., Bayston, L. J., Liao, W., Proby, C. M., Leigh, I. M., Collisson, E. A., Gordon, P. B., Jakkula, L., Pennypacker, S., Zou, Y., Sharma, M., North, J. P., Vemula, S. S., Mauro, T. M., Neuhaus, I. M., Leboit, P. E., Hur, J. S., Park, K., Huh, N., Kwok, P. Y., Arron, S. T., Massion, P. P., Bale, A. E., Haussler, D., Cleaver, J. E., Gray, J. W., Spellman, P. T., South, A. P., Aster, J. C., Blacklow, S. C., and Cho, R. J. (2011) Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma. Proceedings of the National Academy of Sciences of the United States of America 108, 17761-17766 262. Kunnimalaiyaan, M., and Chen, H. (2007) Tumor suppressor role of Notch-1 signaling in neuroendocrine tumors. The oncologist 12, 535-542 263. Zhang, J., Chen, H., Weinmaster, G., and Hayward, S. D. (2001) Epstein-Barr virus BamHi-a rightward transcript-encoded RPMS protein interacts with the CBF1-associated corepressor CIR to negatively regulate the activity of EBNA2 and NotchIC. Journal of virology 75, 2946-2956 264. Liang, Y., Chang, J., Lynch, S. J., Lukac, D. M., and Ganem, D. (2002) The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jkappa (CSL), the target of the Notch signaling pathway. Genes & development 16, 1977-1989 265. Henkel, T., Ling, P. D., Hayward, S. D., and Peterson, M. G. (1994) Mediation of Epstein-Barr virus EBNA2 transactivation by recombination signal-binding protein J kappa. Science 265, 92-95 266. Liang, Y., and Ganem, D. (2003) Lytic but not latent infection by Kaposi's sarcoma-associated herpesvirus requires host CSL protein, the mediator of Notch signaling. Proceedings of the National Academy of Sciences of the United States of America 100, 8490-8495

120

267. Aster, J. C., and Blacklow, S. C. (2012) Targeting the Notch pathway: twists and turns on the road to rational therapeutics. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 2418-2420 268. Wu, Y., Cain-Hom, C., Choy, L., Hagenbeek, T. J., de Leon, G. P., Chen, Y., Finkle, D., Venook, R., Wu, X., Ridgway, J., Schahin-Reed, D., Dow, G. J., Shelton, A., Stawicki, S., Watts, R. J., Zhang, J., Choy, R., Howard, P., Kadyk, L., Yan, M., Zha, J., Callahan, C. A., Hymowitz, S. G., and Siebel, C. W. (2010) Therapeutic antibody targeting of individual Notch receptors. Nature 464, 1052- 1057 269. Ridgway, J., Zhang, G., Wu, Y., Stawicki, S., Liang, W. C., Chanthery, Y., Kowalski, J., Watts, R. J., Callahan, C., Kasman, I., Singh, M., Chien, M., Tan, C., Hongo, J. A., de Sauvage, F., Plowman, G., and Yan, M. (2006) Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444, 1083-1087 270. Olsauskas-Kuprys, R., Zlobin, A., and Osipo, C. (2013) Gamma secretase inhibitors of Notch signaling. OncoTargets and therapy 6, 943-955 271. Bielskiene, K., Bagdoniene, L., Mozuraitiene, J., Kazbariene, B., and Janulionis, E. (2015) E3 ubiquitin ligases as drug targets and prognostic biomarkers in melanoma. Medicina 51, 1-9 272. Wang, H., Chen, G., Wang, H., and Liu, C. (2013) RITA inhibits growth of human hepatocellular carcinoma through induction of apoptosis. Oncology research 20, 437-445

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Chapter 2:

1. Radtke, F., Wilson, A., Stark, G., Bauer, M., van Meerwijk, J., MacDonald, H. R., and Aguet, M. (1999) Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10, 547-558 2. Swiatek, P. J., Lindsell, C. E., del Amo, F. F., Weinmaster, G., and Gridley, T. (1994) Notch1 is essential for postimplantation development in mice. Genes & development 8, 707-719 3. Gridley, T. (2003) Notch signaling and inherited disease syndromes. Human molecular genetics 12 Spec No 1, R9-13 4. Koch, U., and Radtke, F. (2010) Notch signaling in solid tumors. Current topics in developmental biology 92, 411-455 5. Tolcher, A. W., Messersmith, W. A., Mikulski, S. M., Papadopoulos, K. P., Kwak, E. L., Gibbon, D. G., Patnaik, A., Falchook, G. S., Dasari, A., Shapiro, G. I., Boylan, J. F., Xu, Z. X., Wang, K., Koehler, A., Song, J., Middleton, S. A., Deutsch, J., Demario, M., Kurzrock, R., and Wheler, J. J. (2012) Phase I study of RO4929097, a gamma secretase inhibitor of Notch signaling, in patients with refractory metastatic or locally advanced solid tumors. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 2348- 2353 6. Aster, J. C., and Blacklow, S. C. (2012) Targeting the Notch pathway: twists and turns on the road to rational therapeutics. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 2418-2420 7. Kopan, R., and Ilagan, M. X. (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216-233 8. Kovall, R. A., and Blacklow, S. C. (2010) Mechanistic insights into Notch receptor signaling from structural and biochemical studies. Current topics in developmental biology 92, 31-71 9. Wallberg, A. E., Pedersen, K., Lendahl, U., and Roeder, R. G. (2002) p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro. Molecular and cellular biology 22, 7812-7819 10. Fryer, C. J., Lamar, E., Turbachova, I., Kintner, C., and Jones, K. A. (2002) Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes & development 16, 1397-1411 11. Kovall, R. A., and Hendrickson, W. A. (2004) Crystal structure of the nuclear effector of Notch signaling, CSL, bound to DNA. The EMBO journal 23, 3441- 3451 12. Friedmann, D. R., and Kovall, R. A. (2010) Thermodynamic and structural insights into CSL-DNA complexes. Protein science : a publication of the Protein Society 19, 34-46 13. Friedmann, D. R., Wilson, J. J., and Kovall, R. A. (2008) RAM-induced allostery facilitates assembly of a notch pathway active transcription complex. The Journal of biological chemistry 283, 14781-14791 14. Kovall, R. A. (2007) Structures of CSL, Notch and Mastermind proteins: piecing together an active transcription complex. Curr Opin Struct Biol 17, 117-127

122

15. Nam, Y., Weng, A. P., Aster, J. C., and Blacklow, S. C. (2003) Structural requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 transcriptional activation complex. The Journal of biological chemistry 278, 21232-21239 16. Nam, Y., Sliz, P., Song, L., Aster, J. C., and Blacklow, S. C. (2006) Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes. Cell 124, 973-983 17. Borggrefe, T., and Oswald, F. (2009) The Notch signaling pathway: transcriptional regulation at Notch target genes. Cellular and molecular life sciences : CMLS 66, 1631-1646 18. Kuroda, K., Han, H., Tani, S., Tanigaki, K., Tun, T., Furukawa, T., Taniguchi, Y., Kurooka, H., Hamada, Y., Toyokuni, S., and Honjo, T. (2003) Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. Immunity 18, 301-312 19. Oswald, F., Kostezka, U., Astrahantseff, K., Bourteele, S., Dillinger, K., Zechner, U., Ludwig, L., Wilda, M., Hameister, H., Knochel, W., Liptay, S., and Schmid, R. M. (2002) SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. The EMBO journal 21, 5417-5426 20. Oswald, F., Winkler, M., Cao, Y., Astrahantseff, K., Bourteele, S., Knochel, W., and Borggrefe, T. (2005) RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes. Molecular and cellular biology 25, 10379-10390 21. Taniguchi, Y., Furukawa, T., Tun, T., Han, H., and Honjo, T. (1998) LIM protein KyoT2 negatively regulates transcription by association with the RBP-J DNA- binding protein. Molecular and cellular biology 18, 644-654 22. Brou, C., Logeat, F., Lecourtois, M., Vandekerckhove, J., Kourilsky, P., Schweisguth, F., and Israel, A. (1994) Inhibition of the DNA-binding activity of Drosophila suppressor of hairless and of its human homolog, KBF2/RBP-J kappa, by direct protein-protein interaction with Drosophila hairless. Genes & development 8, 2491-2503 23. Barolo, S., Stone, T., Bang, A. G., and Posakony, J. W. (2002) Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless. Genes & development 16, 1964-1976 24. Kurth, P., Preiss, A., Kovall, R. A., and Maier, D. (2011) Molecular analysis of the notch repressor-complex in Drosophila: characterization of potential hairless binding sites on suppressor of hairless. PloS one 6, e27986 25. VanderWielen, B. D., Yuan, Z., Friedmann, D. R., and Kovall, R. A. (2011) Transcriptional repression in the Notch pathway: thermodynamic characterization of CSL-MINT (Msx2-interacting nuclear target protein) complexes. The Journal of biological chemistry 286, 14892-14902 26. Hsieh, J. J., Zhou, S., Chen, L., Young, D. B., and Hayward, S. D. (1999) CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proceedings of the National Academy of Sciences of the United States of America 96, 23-28

123

27. Shi, Y., Sawada, J., Sui, G., Affar el, B., Whetstine, J. R., Lan, F., Ogawa, H., Luke, M. P., Nakatani, Y., and Shi, Y. (2003) Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature 422, 735-738 28. Hsieh, J. J., and Hayward, S. D. (1995) Masking of the CBF1/RBPJ kappa transcriptional repression domain by Epstein-Barr virus EBNA2. Science 268, 560-563 29. Krejci, A., and Bray, S. (2007) Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers. Genes & development 21, 1322-1327 30. Castel, D., Mourikis, P., Bartels, S. J., Brinkman, A. B., Tajbakhsh, S., and Stunnenberg, H. G. (2013) Dynamic binding of RBPJ is determined by Notch signaling status. Genes & development 27, 1059-1071 31. Wacker, S. A., Alvarado, C., von Wichert, G., Knippschild, U., Wiedenmann, J., Clauss, K., Nienhaus, G. U., Hameister, H., Baumann, B., Borggrefe, T., Knochel, W., and Oswald, F. (2011) RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J. The EMBO journal 30, 43-56 32. Ji, Z., and Sharrocks, A. D. (2015) Changing partners: transcription factors form different complexes on and off chromatin. Molecular systems biology 11, 782 33. Kettenbach, A. N., Schweppe, D. K., Faherty, B. K., Pechenick, D., Pletnev, A. A., and Gerber, S. A. (2011) Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Science signaling 4, rs5 34. Wang, H., Chen, G., Wang, H., and Liu, C. (2013) RITA inhibits growth of human hepatocellular carcinoma through induction of apoptosis. Oncology research 20, 437-445 35. Ling, P. D., and Hayward, S. D. (1995) Contribution of conserved amino acids in mediating the interaction between EBNA2 and CBF1/RBPJk. Journal of virology 69, 1944-1950 36. Collins, K. J., Yuan, Z., and Kovall, R. A. (2014) Structure and Function of the CSL-KyoT2 Corepressor Complex: A Negative Regulator of Notch Signaling. Structure 22, 70-81 37. Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tun, T., Furukawa, T., and Honjo, T. (1995) Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-J kappa/Su(H). Current biology : CB 5, 1416-1423 38. Johnson, S. E., Ilagan, M. X., Kopan, R., and Barrick, D. (2010) Thermodynamic analysis of the CSL x Notch interaction: distribution of binding energy of the Notch RAM region to the CSL beta-trefoil domain and the mode of competition with the viral transactivator EBNA2. The Journal of biological chemistry 285, 6681-6692 39. Mossessova, E., and Lima, C. D. (2000) Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Molecular cell 5, 865-876 40. Otwinowski, Z., and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol 276, 307-326

124

41. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software. Journal of applied crystallography 40, 658-674 42. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta crystallographica. Section D, Biological crystallography 60, 2126- 2132 43. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python- based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography 66, 213-221 44. Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J., Wang, X., Murray, L. W., Arendall, W. B., 3rd, Snoeyink, J., Richardson, J. S., and Richardson, D. C. (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic acids research 35, W375-383 45. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) The Protein Data Bank. Nucleic acids research 28, 235-242 46. Schrodinger, LLC. (2010) The PyMOL Molecular Graphics System, Version 1.3r1. 47. Krissinel, E., and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state. Journal of molecular biology 372, 774-797 48. Yuan, Z., Friedmann, D. R., Vanderwielen, B. D., Collins, K. J., and Kovall, R. A. (2012) Characterization of CSL (CBF-1, Su(H), Lag-1) Mutants Reveals Differences in Signaling Mediated by Notch1 and Notch2. The Journal of biological chemistry 287, 34904-34916 49. Whitmore, L., and Wallace, B. A. (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic acids research 32, W668-673 50. Whitmore, L., and Wallace, B. A. (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89, 392-400 51. Choi, S. H., Wales, T. E., Nam, Y., O'Donovan, D. J., Sliz, P., Engen, J. R., and Blacklow, S. C. (2012) Conformational locking upon cooperative assembly of notch transcription complexes. Structure 20, 340-349 52. Sturtevant, J. M. (1977) Heat capacity and entropy changes in processes involving proteins. Proceedings of the National Academy of Sciences of the United States of America 74, 2236-2240 53. Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R., and Israel, A. (1995) Signalling downstream of activated mammalian Notch. Nature 377, 355- 358 54. Ong, C. T., Cheng, H. T., Chang, L. W., Ohtsuka, T., Kageyama, R., Stormo, G. D., and Kopan, R. (2006) Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture

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determines activation probability. The Journal of biological chemistry 281, 5106- 5119

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Chapter 3:

1. Radtke, F., Wilson, A., Stark, G., Bauer, M., van Meerwijk, J., MacDonald, H. R., and Aguet, M. (1999) Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10, 547-558 2. Gridley, T. (2003) Notch signaling and inherited disease syndromes. Human molecular genetics 12 Spec No 1, R9-13 3. Koch, U., and Radtke, F. (2010) Notch signaling in solid tumors. Current topics in developmental biology 92, 411-455 4. Swiatek, P. J., Lindsell, C. E., del Amo, F. F., Weinmaster, G., and Gridley, T. (1994) Notch1 is essential for postimplantation development in mice. Genes & development 8, 707-719 5. Kopan, R., and Ilagan, M. X. (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216-233 6. Kovall, R. A., and Blacklow, S. C. (2010) Mechanistic insights into Notch receptor signaling from structural and biochemical studies. Current topics in developmental biology 92, 31-71 7. Wallberg, A. E., Pedersen, K., Lendahl, U., and Roeder, R. G. (2002) p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro. Molecular and cellular biology 22, 7812-7819 8. Fryer, C. J., Lamar, E., Turbachova, I., Kintner, C., and Jones, K. A. (2002) Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes & development 16, 1397-1411 9. Kovall, R. A., and Hendrickson, W. A. (2004) Crystal structure of the nuclear effector of Notch signaling, CSL, bound to DNA. The EMBO journal 23, 3441- 3451 10. Friedmann, D. R., and Kovall, R. A. (2010) Thermodynamic and structural insights into CSL-DNA complexes. Protein science : a publication of the Protein Society 19, 34-46 11. Kovall, R. A. (2007) Structures of CSL, Notch and Mastermind proteins: piecing together an active transcription complex. Current opinion in structural biology 17, 117-127 12. Friedmann, D. R., Wilson, J. J., and Kovall, R. A. (2008) RAM-induced allostery facilitates assembly of a notch pathway active transcription complex. The Journal of biological chemistry 283, 14781-14791 13. Nam, Y., Weng, A. P., Aster, J. C., and Blacklow, S. C. (2003) Structural requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 transcriptional activation complex. The Journal of biological chemistry 278, 21232-21239 14. Nam, Y., Sliz, P., Song, L., Aster, J. C., and Blacklow, S. C. (2006) Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes. Cell 124, 973-983 15. Borggrefe, T., and Oswald, F. (2009) The Notch signaling pathway: transcriptional regulation at Notch target genes. Cellular and molecular life sciences : CMLS 66, 1631-1646

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16. Kuroda, K., Han, H., Tani, S., Tanigaki, K., Tun, T., Furukawa, T., Taniguchi, Y., Kurooka, H., Hamada, Y., Toyokuni, S., and Honjo, T. (2003) Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. Immunity 18, 301-312 17. Oswald, F., Kostezka, U., Astrahantseff, K., Bourteele, S., Dillinger, K., Zechner, U., Ludwig, L., Wilda, M., Hameister, H., Knochel, W., Liptay, S., and Schmid, R. M. (2002) SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. The EMBO journal 21, 5417-5426 18. Oswald, F., Winkler, M., Cao, Y., Astrahantseff, K., Bourteele, S., Knochel, W., and Borggrefe, T. (2005) RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes. Molecular and cellular biology 25, 10379-10390 19. Brou, C., Logeat, F., Lecourtois, M., Vandekerckhove, J., Kourilsky, P., Schweisguth, F., and Israel, A. (1994) Inhibition of the DNA-binding activity of Drosophila suppressor of hairless and of its human homolog, KBF2/RBP-J kappa, by direct protein-protein interaction with Drosophila hairless. Genes & development 8, 2491-2503 20. Barolo, S., Stone, T., Bang, A. G., and Posakony, J. W. (2002) Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless. Genes & development 16, 1964-1976 21. Taniguchi, Y., Furukawa, T., Tun, T., Han, H., and Honjo, T. (1998) LIM protein KyoT2 negatively regulates transcription by association with the RBP-J DNA- binding protein. Molecular and cellular biology 18, 644-654 22. Kurth, P., Preiss, A., Kovall, R. A., and Maier, D. (2011) Molecular analysis of the notch repressor-complex in Drosophila: characterization of potential hairless binding sites on suppressor of hairless. PloS one 6, e27986 23. VanderWielen, B. D., Yuan, Z., Friedmann, D. R., and Kovall, R. A. (2011) Transcriptional repression in the Notch pathway: thermodynamic characterization of CSL-MINT (Msx2-interacting nuclear target protein) complexes. The Journal of biological chemistry 286, 14892-14902 24. Collins, K. J., Yuan, Z., and Kovall, R. A. (2014) Structure and function of the CSL-KyoT2 corepressor complex: a negative regulator of Notch signaling. Structure 22, 70-81 25. Hsieh, J. J., Zhou, S., Chen, L., Young, D. B., and Hayward, S. D. (1999) CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proceedings of the National Academy of Sciences of the United States of America 96, 23-28 26. Shi, Y., Sawada, J., Sui, G., Affar el, B., Whetstine, J. R., Lan, F., Ogawa, H., Luke, M. P., Nakatani, Y., and Shi, Y. (2003) Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature 422, 735-738 27. Wacker, S. A., Alvarado, C., von Wichert, G., Knippschild, U., Wiedenmann, J., Clauss, K., Nienhaus, G. U., Hameister, H., Baumann, B., Borggrefe, T., Knochel, W., and Oswald, F. (2011) RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J. The EMBO journal 30, 43-56 28. Krejci, A., and Bray, S. (2007) Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers. Genes & development 21, 1322-1327

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29. Castel, D., Mourikis, P., Bartels, S. J., Brinkman, A. B., Tajbakhsh, S., and Stunnenberg, H. G. (2013) Dynamic binding of RBPJ is determined by Notch signaling status. Genes & development 27, 1059-1071 30. Johnson, S. E., Ilagan, M. X., Kopan, R., and Barrick, D. (2010) Thermodynamic analysis of the CSL x Notch interaction: distribution of binding energy of the Notch RAM region to the CSL beta-trefoil domain and the mode of competition with the viral transactivator EBNA2. The Journal of biological chemistry 285, 6681-6692 31. Ling, P. D., and Hayward, S. D. (1995) Contribution of conserved amino acids in mediating the interaction between EBNA2 and CBF1/RBPJk. Journal of virology 69, 1944-1950 32. Yuan, Z., Friedmann, D. R., VanderWielen, B. D., Collins, K. J., and Kovall, R. A. (2012) Characterization of CSL (CBF-1, Su(H), Lag-1) mutants reveals differences in signaling mediated by Notch1 and Notch2. The Journal of biological chemistry 287, 34904-34916 33. Johnson, S. E., and Barrick, D. (2012) Dissecting and circumventing the requirement for RAM in CSL-dependent Notch signaling. PloS one 7, e39093 34. Calderwood, M. A., Lee, S., Holthaus, A. M., Blacklow, S. C., Kieff, E., and Johannsen, E. (2011) Epstein-Barr virus nuclear protein 3C binds to the N- terminal (NTD) and beta trefoil domains (BTD) of RBP/CSL; only the NTD interaction is essential for lymphoblastoid cell growth. Virology 414, 19-25 35. Del Bianco, C., Vedenko, A., Choi, S. H., Berger, M. F., Shokri, L., Bulyk, M. L., and Blacklow, S. C. (2010) Notch and MAML-1 complexation do not detectably alter the DNA binding specificity of the transcription factor CSL. PloS one 5, e15034 36. Fuchs, K. P., Bommer, G., Dumont, E., Christoph, B., Vidal, M., Kremmer, E., and Kempkes, B. (2001) Mutational analysis of the J recombination signal sequence binding protein (RBP-J)/Epstein-Barr virus nuclear antigen 2 (EBNA2) and RBP-J/Notch interaction. European journal of biochemistry / FEBS 268, 4639-4646 37. Schrodinger, LLC. (2010) The PyMOL Molecular Graphics System, Version 1.3r1. 38. Krissinel, E., and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state. Journal of molecular biology 372, 774-797 39. Lo, M. C., Aulabaugh, A., Jin, G., Cowling, R., Bard, J., Malamas, M., and Ellestad, G. (2004) Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Analytical biochemistry 332, 153-159 40. Hsieh, J. J., and Hayward, S. D. (1995) Masking of the CBF1/RBPJ kappa transcriptional repression domain by Epstein-Barr virus EBNA2. Science 268, 560-563 41. Wilson, J. J., and Kovall, R. A. (2006) Crystal structure of the CSL-Notch- Mastermind ternary complex bound to DNA. Cell 124, 985-996 42. Tolcher, A. W., Messersmith, W. A., Mikulski, S. M., Papadopoulos, K. P., Kwak, E. L., Gibbon, D. G., Patnaik, A., Falchook, G. S., Dasari, A., Shapiro, G. I., Boylan, J. F., Xu, Z. X., Wang, K., Koehler, A., Song, J., Middleton, S. A., Deutsch, J., Demario, M., Kurzrock, R., and Wheler, J. J. (2012) Phase I study of

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RO4929097, a gamma secretase inhibitor of Notch signaling, in patients with refractory metastatic or locally advanced solid tumors. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 2348- 2353 43. Aster, J. C., and Blacklow, S. C. (2012) Targeting the Notch pathway: twists and turns on the road to rational therapeutics. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 2418-2420

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Chapter 4:

1. Ong, C. T., Cheng, H. T., Chang, L. W., Ohtsuka, T., Kageyama, R., Stormo, G. D., and Kopan, R. (2006) Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability. The Journal of biological chemistry 281, 5106- 5119 2. Kopan, R., and Ilagan, M. X. (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216-233 3. Krejci, A., and Bray, S. (2007) Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers. Genes & development 21, 1322-1327 4. Skalska, L., Stojnic, R., Li, J., Fischer, B., Cerda-Moya, G., Sakai, H., Tajbakhsh, S., Russell, S., Adryan, B., and Bray, S. J. (2015) Chromatin signatures at Notch- regulated enhancers reveal large-scale changes in H3K56ac upon activation. The EMBO journal 34, 1889-1904 5. Castel, D., Mourikis, P., Bartels, S. J., Brinkman, A. B., Tajbakhsh, S., and Stunnenberg, H. G. (2013) Dynamic binding of RBPJ is determined by Notch signaling status. Genes & development 27, 1059-1071 6. Wacker, S. A., Alvarado, C., von Wichert, G., Knippschild, U., Wiedenmann, J., Clauss, K., Nienhaus, G. U., Hameister, H., Baumann, B., Borggrefe, T., Knochel, W., and Oswald, F. (2011) RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J. The EMBO journal 30, 43-56 7. Qin, H., Wang, J., Liang, Y., Taniguchi, Y., Tanigaki, K., and Han, H. (2004) RING1 inhibits transactivation of RBP-J by Notch through interaction with LIM protein KyoT2. Nucleic acids research 32, 1492-1501 8. Qin, H., Du, D., Zhu, Y., Li, J., Feng, L., Liang, Y., and Han, H. (2005) The PcG protein HPC2 inhibits RBP-J-mediated transcription by interacting with LIM protein KyoT2. FEBS letters 579, 1220-1226 9. Ariyoshi, M., and Schwabe, J. W. (2003) A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling. Genes & development 17, 1909-1920 10. Oswald, F., Winkler, M., Cao, Y., Astrahantseff, K., Bourteele, S., Knochel, W., and Borggrefe, T. (2005) RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes. Molecular and cellular biology 25, 10379-10390 11. Collins, K. J., Yuan, Z., and Kovall, R. A. (2014) Structure and function of the CSL-KyoT2 corepressor complex: a negative regulator of Notch signaling. Structure 22, 70-81 12. VanderWielen, B. D., Yuan, Z., Friedmann, D. R., and Kovall, R. A. (2011) Transcriptional repression in the Notch pathway: thermodynamic characterization of CSL-MINT (Msx2-interacting nuclear target protein) complexes. The Journal of biological chemistry 286, 14892-14902 13. Friedmann, D. R., Wilson, J. J., and Kovall, R. A. (2008) RAM-induced allostery facilitates assembly of a notch pathway active transcription complex. The Journal of biological chemistry 283, 14781-14791 14. Pinnell, N., Yan, R., Cho, H. J., Keeley, T., Murai, M. J., Liu, Y., Alarcon, A. S., Qin, J., Wang, Q., Kuick, R., Elenitoba-Johnson, K. S., Maillard, I., Samuelson, L. C., Cierpicki, T., and Chiang, M. Y. (2015) The PIAS-like Coactivator Zmiz1 Is a

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Direct and Selective Cofactor of Notch1 in T Cell Development and Leukemia. Immunity 43, 870-883 15. Wang, H., Chen, G., Wang, H., and Liu, C. (2013) RITA inhibits growth of human hepatocellular carcinoma through induction of apoptosis. Oncology research 20, 437-445 16. Morsut, L., Roybal, K. T., Xiong, X., Gordley, R. M., Coyle, S. M., Thomson, M., and Lim, W. A. (2016) Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780-791 17. Roybal, K. T., Rupp, L. J., Morsut, L., Walker, W. J., McNally, K. A., Park, J. S., and Lim, W. A. (2016) Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits. Cell 164, 770-779 18. Barrett, D. M., Singh, N., Porter, D. L., Grupp, S. A., and June, C. H. (2014) Chimeric antigen receptor therapy for cancer. Annual review of medicine 65, 333- 347 19. Magee, M. S., and Snook, A. E. (2014) Challenges to chimeric antigen receptor (CAR)-T cell therapy for cancer. Discovery medicine 18, 265-271

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Appendix A: A phospho-dependent mechanism involving NCoR and KMT2D controls a committed chromatin state at Notch target genes

I contributed to this work by performing the SPOC-pNCoR mutant binging studies (Figure 4).

Published as:

Oswald, F., Rodriguez, P., Giaimo, B. D., Antonello, Z. A., Mira, L., Mittler, G., Thiel, V. N., Collins, K. J., Tabaja, N., Cizelsky, W., Rothe, M., Kuhl, S. J., Kuhl, M., Ferrante, F., Hein, K., Kovall, R. A., Dominguez, M., and Borggrefe, T. (2016) A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes. Nucleic acids research

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Appendix B: Unanticipated Structural Plasticity of CSL as Revealed by the X-ray structure of the Su(H)-Hairless Repressor Complex

Zhenyu Yuan1,4, Heiko Praxenthaler2,4, Nassif Tabaja1, Rubben Torella3, Anette Preiss2 Dieter Maier2, & Rhett A. Kovall1

1Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA. 2Universität Hohenheim, Institut für Genetik, Stuttgart 70593, Germany. 3Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom. 4These authors contributed equally to the work.

I contributed to this work by performing the circular dichroism and thermal shift assay experiments from purification to data collection (Figure S4).

To be published in PLOS Biology.

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Appendix C: A re-engineered humanized anti-cocaine monoclonal antibody: analysis of its temperature- and pH-stability and energetics of ligand binding

W. James Ball, Jr.1, Nichola C. Garbett2, Michael R. Tabet1, Nassif Tabaja3 and Andrew B. Norman1

1Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA, 2James Graham Brown Cancer Center, Department of Medicine, University of Louisville, Louisville, Kentucky, USA, 3Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

I contributed to this work by performing the antibody binging studies (Figure 8,9, and Table III).

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Abstract

Vaccines for active immunization and human or humanized anti-cocaine monoclonal antibodies (mAbs) administered as a passive immunotherapy are under development as interventions for countering the re-instatement of cocaine abuse in treatment seeking patients. In this study, we have compared the affinity and ligand specificity of the further humanized, recombinant anti-cocaine mAb h2E2 to that of its parent high-affinity chimeric (human γ1 heavy/murine λ light chain) mAb 2E2 and determined their thermal and pH stabilities relative to that of two murine anti-cocaine mAbs (3P1A6 and 1BB835).

These studies indicated that the re-engineered h2E2 retains the same ligand binding properties with a thermal stability modestly improved over that of 2E2 and that is similar to that of typical murine sequence mAbs. Capillary differential scanning calorimetry

(DSC) was then used to obtain thermograms of the unfolding of h2E2, its Fab fragment and 1BB835 and their enthalpies of unfolding. These results further confirmed h2E2’s suitability for continued development as a therapeutic. Interestingly, the DSC studies also indicated that the thermal stability of the Fab domain is significantly increased upon ligand binding. Isothermal calorimetry (ITC) titration studies were then used to determine the thermodynamic parameters of h2E2’s binding interactions with cocaine, cocaethylene and the analogue compound RTI-113. The Gibbs free energy (ΔG) of binding for the three ligands were similar but the enthalpy (ΔH) and entropy (TΔS) values indicated potential differences in the mechanism of RTI-113 binding versus that for cocaine.

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1. Introduction

A 2012 National Survey on Drug Use and Health (NSDUH) has estimated that 23.9 million Americans, aged 12 or older, were current illicit drug users and of these 1.6 million used cocaine with 1.1 million deemed dependent upon or abusing cocaine (1).

The challenges to addressing the health and societal problems generated by substance abuse are long-standing and unfortunately often appear to be nearly intractable. Never the less, there are many varied, continuing efforts to develop new treatment strategies.

The traditional pharmacological approach towards developing a treatment for cocaine abuse has been to attempt to moderate or antagonize cocaine’s inhibition of the dopamine transporter or alternatively dopamine’s effects on specific dopamine receptor subtypes (D2-R), by utilizing low molecular weight drugs. Unfortunately, unlike the case with heroin where there are several Food and Drug Administration (FDA)-approved medications for the treatment of opiate addiction/abuse (2), this not the case in regards to cocaine. Thus, while considerable research has been directed towards identifying and developing pharmacological agents none have proven suitable nor been approved for use by the FDA as a treatment for cocaine abuse.

The adoption of an immunotherapeutic approach to the treatment of cocaine addiction represents a particularly attractive alternative approach to therapeutic intervention. The concept is certainly not new as studies reported by Bonese et al., (3) in 1974 demonstrated the effectiveness of both a vaccine designed to stimulate the production of anti-heroin antibodies in rhesus monkeys as well as the passive transfer of antidrug sera to non-immunized monkeys (4) to moderate their self-administration responses to

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heroin. Analogously, more recently, we and others have focused on the use of cocaine-directed antibodies that bind plasma cocaine and prevent its distribution from the peripheral circulation to the CNS thereby blunting the drug’s neurological effects (5-

7). This represents a particularly attractive approach to cocaine abuse treatment because an immunological intervention that only targets an exogenous compound should have no direct psychoactive effects, neither moderating the actions of an intended target receptor nor an unintended CNS receptor.

Towards developing a safe anti-cocaine immunotherapeutic, the complementary use of recombinant DNA technologies and murine hybridoma methodologies employing normal or “humanized” transgenic mice has enabled the production of bio-manufactured humanized and human sequence monoclonal antibodies (mAbs) that can be administered as an immediately active agent. Alternatively, vaccines comprised of cocaine-derived hapten-protein conjugates can be used that upon repeated immunization elicit endogenously produced polyclonal anti-cocaine antibodies. Both approaches have been shown in animal model studies to have considerable potential as therapeutics (6, 8-10). Importantly, initial clinical trials in humans with a vaccine (11-13) have shown that some patients generate sufficient anti-cocaine antibody levels to result in decreased drug usage.

In our laboratory, we have focused on the development of a “humanized” mAb that would be administered as a passive immunotherapeutic (14). The passive immunization approach ensures the administration of a known dosage of single species of mAb with a defined high-affinity and specificity for cocaine. This approach while more costly to administer than a vaccine avoids the problems of the wide patient

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variability in the levels, affinity and specificity of the anti-cocaine immunological responses that have been observed for the vaccines currently under development (11,

12). The utility of utilizing human sequence mAbs as therapeutic drugs seems widely appreciated. While only about 25 have thus far been FDA approved in the US, this number rises to more than 40 when including those approved or under review in the US and EU and many more are in current development (15, 16).

In this report, we have compared the physical properties of our mixed-chain or chimeric mAb 2E2 that has a fully human sequence γ1 heavy (H) chain and a murine λ light (L) chain (17) with that of a re-engineered, more humanized version designated as h2E2

(18). The original mAb 2E2 having a high affinity (~4nM) and specificity for cocaine was obtained through immunization of the HumAb® transgenic mouse (19) and use of standard hybridoma cell technology (20). The transgenic mouse line was designed to produce fully human sequence mAbs but in this case produced a mixed-chain mAb.

Recombinant DNA technology has then been used to modify 2E2 so that the murine λc chain constant region domain has been replaced with a human λc region and its production established in stably transfected Chinese Hamster Ovary (CHO) cells.

This increased humanization results in h2E2 being approximately 95% identical or homologous to a human sequence IgG1 antibody. This re-engineering should then reduce its potential for immunogenicity in patients and the murine λc – human λc domain switching would generally not be expected to alter the mAb’s ligand binding properties. However, several studies have detailed instances in which H chain constant region changes have altered mAb ligand binding (21, 22). In addition, and more relevant to these studies the switching of the human κc to human λc domain has been

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shown to alter the binding site structure and kinetic properties of the organophosphate hydrolyzing human mAb A17 (23). Hence the importance of the studies reported here to determine whether h2E2’s cocaine/cocaethylene binding properties are altered from those of 2E2. To be successfully used as a therapeutic the re-engineered h2E2 needs to meet several criteria. It must not only retain the same high affinity and specificity for cocaine as 2E2 it also must be capable of high level, large-scale production and purification. Further, it should have physical and stability properties allowing for high concentration dose formulation with a long-storage or shelf-life.

As a brief introduction for these studies, mAb 2E2, as secreted by the hybridoma cell line was produced at very low levels, approximately, one-tenth (~0.1mg/ml, 17) that normally observed for murine mAbs. Further, 2E2 production levels obtained from transiently transfected CHO and human embryonic kidney (HEK) cells expressing the cloned mAb were similarly low as compared to the humanized therapeutic mAb rituximab produced as a control using the same expression methodologies (unpublished results). These results raised the possibility that the mixed-chain mAb 2E2 might be inherently unstable or there were abnormalities in the human H and mouse L chain folding and/or their hetero-dimer IgG1 assembly. During studies characterizing 2E2’s binding properties no apparent issues with its stability were observed but limitations in the quantity/purity of 2E2 obtainable precluded the use of techniques such as differential scanning calorimetry to investigate its conformational stability. Recently achieved high level production of h2E2 in serum-free media now allows us to obtain highly purified mAb at levels sufficient to do detailed studies of its physical properties and ligand binding thermodynamics.

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We have recently reported (18) initial affinity and specificity studies that demonstrated that the re-engineered recombinant h2E2 and 2E2 have essentially identical affinities for cocaine (~4nM) and similar specificities for several cocaine metabolites. In this report, we present additional studies comparing the binding specificities of h2E2 and 2E2 and investigate their pH and temperature-dependent stabilities in order to identify any potential issues of h2E2 stability that might hinder its development as a therapeutic.

The pH and temperature inactivation studies suggest a modest improvement in the stability of the re-engineered h2E2 versus 2E2. These studies also indicated that were no significant differences in the stability of the humanized mAbs as compared to two commercially available murine anti-cocaine mAbs serving as controls. In addition, differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) studies were conducted to elucidate the thermodynamics of h2E2 unfolding and ligand binding further characterizing h2E2’s physical properties and how it binds ligands.

The DSC studies enabled us to study the conformational transitions of mAb unfolding.

They demonstrated that h2E2’s overall unfolding profile was somewhat more complex than that of a control murine mAb 1BB835 but their transition-midpoint (Tm) temperatures under varying pH conditions were not significantly different. Interestingly, the presence of a binding ligand, in this case the cocaine analog RTI-113, was found to significantly stabilize h2E2’s Fab domain.

Finally, isothermal titration calorimetry (ITC) was used to determine the underlying thermodynamic parameters of ligand and mAb complex formation. The binding titration curves of the three ligands, cocaethylene, cocaine and RTI-113 to h2E2 generated similar Gibbs free energy (ΔG) values consistent with those typically found for antibody

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binding of low-molecular weight molecules. However, the ΔH and TΔS energy components for the three drugs suggested significant difference in the molecular nature of the RTI-113/mAb interactions versus that of cocaine and cocaethylene. These results provide new information about cocaine/antibody binding interactions complementing that developed in our earlier 3-dimensional quantitative structure-activity relationship (3-D CoMFA and CoMSIA) modeling studies (17, 24) and drug/mAb docking models based on homology modeling the 2E2 Fab fragment (25).

2. Methods and Materials

Murine hybridoma-derived mAb 2E2: its production and purification. The hybridoma cell line secreting anti-cocaine mAb 2E2 was generated, identified and isolated using standard hybridoma technology. This was accomplished, as previously described (17), by fusing splenocytes obtained from a human antibody producing transgenic mouse strain designated HCo7/Ko5 (19), that had been immunized with a hapten-carrier conjugate with cells of the murine myeloma cell line P3X63-Ag8.653. The immunogen was benzoylecgonine (hapten) coupled to 1,4-butanediamine-derivatized keyhole limpet hemocyanin (KLH). In these studies, mAb 2E2 used was purified as previously described (26) from endogenous murine IgG and serum proteins of the ascites fluid collected from SCID (severe combined immunodeficiency disease) mice inoculated i.p. with 2E2 producing hybridomas.

Establishing CHO-cell production of the recombinant mAb h2E2. The complete heavy (H) and light ( L) chain sequences mAb 2E2 were obtained via DNA sequencing of full-length H and L chain cDNA constructs generated from cloning the hybridoma cell

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line mRNA for 2E2’s human γ1H and murine λ L chains. Each gene was sequenced and matched to the previous H and L chain protein sequencing results. Confirmation of mAb 2E2 cloning was achieved by transiently expressing 2E2 in human embryonic kidney cells (HEK293T) and as previously reported (25) obtaining similar ligand binding properties for the hybridoma and HEK293T-cell expressed recombinant 2E2 mAb.

DNA plasmids encoding the 2E2 H and L chains as well as the electronic DNA and protein sequences were supplied to Catalent Pharma Solutions (Madison,WI). These plasmids as well as a construct encoding the human λc L chain constant region were used to construct H and modified L chain RNA plasmids that were inserted separately into proprietory GPEx® gene expression, replication-defective retroviral vectors derived from the Moloney murine leukemia virus (27, 28). High level mAb h2E2 production cell lines were generated by performing multiple rounds of transductions of the H chain (5 rounds) and L chain (4 rounds) containing vectors into the GPEx-rChinese Hamster

Ovary (CHO) cell line. Clonal cell lines were isolated and each screened by ELISA for their human mAb titers in order to identify cells expressing high levels of h2E2. The h2E2 production for these studies was about 4.7 mg/ml, achieved in 250 ml shake flasks seeded with selected clonal cells into 60 ml volumes of serum-free media. Upon completion of the cell growth period the secreted mAb was purified from spent medium using protein-A HPLC chromatography. h2E2 purity was confirmed by 1% sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE) under reduced

(β-mercaptoethanol) and non-reduced conditions. h2E2 Fab generation, purification and SDS-page gel monitoring. Fab fragments of h2E2 were generated by dialyzing a 5.2 mg/ml solution of h2E2 in 50mM phosphate

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buffered saline (PBS, pH 7.2) against 20 mM MOPS buffer, pH 8.0 then incubating 10-

30 mg mAb h2E2 at 5 mg/ml with a 1/500th mg/mg ratio of endoproteinase-lys-C for 1hr at room temperature. The protease-treated mAb sample was then adjusted to pH 7.0 and loaded onto a cation exchange column (Sulfo-propyl strong cation resin CX). The

Fc fragment and endoproteinase-lys-C were separated from the Fab as they are uncharged and elute in the void volume wash of the exchange column while the Fab fragment remained bound and was eluted from the column with 100 mM NaCl in MOPS buffer. To confirm the identity of the two fragments and the extent of Fab/Fc separation, microgram amounts of the column-bound and unbound fractions of protein, both reduced (β-mercaptoethanol) and unreduced samples were identified via Coomassie blue staining after 1%SDS-10% PAGE.

Radioligand binding and competition enzyme-linked immunosorbent assays

(ELISAs). The affinity, or dissociation constants (Kd) of mAbs 2E2 and h2E2 for cocaine were determined using a saturation radioligand binding procedure employing a double antibody precipitation and membrane filtration method as previously described

3 (24). Kd values were determined by fitting measured counts [ H]-cocaine per minute recovered as a function of cocaine concentration to a binding isotherm. The inhibition constants (IC50) for benzoylecgonine binding to 2E2 and h2E2 were determined by radioligand competition. The experimental conditions were similar to the radioligand binding assay except that the all samples had 15 nM cocaine (including 1nM [3H]- cocaine) with varying concentrations of non-radioactive, cocaine or BE added as competitor. Results presented represent the averages obtained from two independent experiments done in duplicate.

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The relative binding affinities (RBAs) of the two mAbs for cocaine, several in vivo metabolites and the analog compound RTI-113 were obtained using a competition

ELISA procedure previously detailed (17, 24). In the ELISAs the IC50 values were determined by fitting the data to a three-parameter logistic equation. The reported RBA values were obtained by dividing the IC50 values of competitors by the IC50 value for cocaine. Results are reported as the average of two-three experiments done in duplicate.

The compounds, cocaine, cocaethylene, norcocaine, benzoylecgonine, ecgoninemethylester, ecgonine and RTI-113 (synthesized by Dr. Ivy Carroll of the

Research Triangle Institute) were generously provided by the drug supply program of the National Institute on Drug Abuse. mAb thermal and pH stability determinations. Antibodies, 2E2, h2E2, 3P1A6

(Biodesign Internatl., Kennebunk, ME) and 1BB835 (US Biological, Swampscott, MA) in stock solutions were diluted to 10 nM (1.5 μg/ml) into test tubes containing 200 mM glycine-HCl at pH 3.0 or 1mM EGTA-Tris, pH 7.0 solutions at room temperature (~

21oC) or in heat blocks at 50o or 65oC. At selected times aliquots were removed and diluted 1:1 into tubes containing an equal volume of a standard ELISA binding buffer (5 mg/ml BSA, 10 mM PBS, pH 7.2) pre-chilled on ice. Quantification of the 5nM mAbs’ ability to bind to ELISA plates coated with 0.2 μg/ml of the benzoylecgonine- ethylenediamine–bovine serum albumin (BSA) conjugate was determined subsequent to the timed exposure to the inactivation conditions. mAb binding was monitored as previously described (17) using biotinylated affinity-purified goat anti-human IgG antibody or biotinylated goat anti-mouse IgG antibody to detect the bound anti-cocaine

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mAb, followed by addition of the streptavidin-alkaline phosphatase conjugate and the colorimetric assay substrate p-nitrophenolphosphate. All results reported for the

ELISAs are the averages of experiments done in triplicate.

Differential scanning calorimetry (DSC). The capillary differential scanning calorimetry (DSC) measurements were performed at the University of Louisville (KY) by

Dr. Nichola Garbett (29). DSC data were collected with a Nano DSC Autosampler

System (TA Instruments, New Castle, DE). The mAb/Fab samples and matched dialysates to load the instrument sample and reference chambers, respectively, were transferred to 96-well plates and loaded into the instrument autosampler and maintained at 4oC until analysis. Sample volumes of 950 μL were required to provide sufficient volume to ensure proper rinsing and filling of the 300 μL thermal sensing area. DSC scans were recorded from 20oC to 110oC at a scan rate of 1oC/min with a pre-scan equilibration period of 900 seconds. The instrument was cycled overnight by running at least seven water-water scans followed the next morning by at least three buffer-buffer scans to condition the instrument chambers. This was followed by alternating sample- buffer and buffer-buffer scans to ensure there was no sample carryover between scans and to provide a rigorous assessment of buffer reference thermograms. Duplicate scans were obtained for each of the experimental thermograms shown in the results but most experiments were repeated additional times. mAb protein concentrations were

~1mg/ml and Fab at 0.67 mg/ml as determined using a Lowry protein assay and calculated using molar extinction coefficient (ε) values at 280nm (h2E2, ε 1% 280 =

14.63, Fab, ε1% 280 = 16.1 and 1BB835, ε1% 280 = 13.5). Samples were in 10mM PBS, pH 7.2 or 10mM acetate-saline, pH 5.5 as described in the results. For experiments

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determining the effects of mAb bound ligand, RTI-113, both ligand containing samples and mAb only samples were scanned in 10mM PBS, pH 7.2 with 1% dimethylsulfoxide

(DMSO).

DSC data were analyzed using the manufacturer supplied software, NanoAnalyze version 2.1.13. Raw DSC thermograms were corrected for instrument baseline by subtraction of a suitable buffer reference scan, normalized for sample molar concentrations and corrected for non-zero sample baselines by application of a sigmoidal baseline function. Final data were plotted as excess molar heat capacity

(kJ/mol.K) versus temperature (oC). Unfolding enthalpy and temperature at the heat capacity maximum (Tmax) were obtained by direct integration of the final data and without recourse to model fitting. Tmax values are approximated to Tm values of the major transition dominated by Fab domain unfolding. As the transitions presented in this study are irreversible, the transition midpoint temperature (Tm), unfolding enthalpy

(ΔHu) and entropy (ΔSu) values reported are “apparent” values.

Isothermal titration calorimetry (ITC). ITC experiments were performed using a

Microcal VP-ITC microcalorimeter. For cocaethylene and cocaine binding experiments 5

μl aliquots of 50 μM drug in 10 mM PBS buffer, pH 7.2 were titrated into the 1.42 ml reaction cell containing 10 μM (binding sites) mAb h2E2 in 10mM PBS that was equilibrated and maintained at 20oC. The RTI-113 stock concentration was also 50 μM but because of the drug’s poor solubility both drug and mAb were in 10 mM PBS and

1% DMSO. Heats of ligand dilution control experiments were performed by titrating each ligand into buffer and these results were subtracted from those of the ligand/mAb binding titrations. The presence of 1% DMSO was found to have no significant effect on

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the ligand only background controls. Each experiment was repeated three times and the data collected were analyzed using the manufacturer’s MicroCal ORIGIN 7.0 software and fit to a one set of sites binding model. The data analysis enables the calculation of the association constant (Ka), interaction stoichiometry and the thermodynamic parameters, Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (TΔS).

3. Results:

Determination of 2E2 and h2E2’s cocaine binding affinities and relative specificities for cocaine and metabolites. A saturation radioligand ([3H]-cocaine) binding assay was employed for initial determinations as to whether the modification of

2E2’s L chain structure and expression of h2E2 as a recombinant protein had altered the mAb’s ligand binding properties. The Kd values of the hybridoma-produced mAb

2E2 and the CHO cell-produced h2E2 for cocaine were determined to be 4.4 and 3.9 nM, respectively (18). Next, radioligand competition binding assays were used to compare the specificities of the two mAbs for cocaine versus that of the important in vivo metabolite benzyolecgonine. These experiments showed that 2E2 and h2E2 had

IC50 values for BE that were, respectively, 12- and 10-fold lower than that for cocaine

(data not shown). The Kd and IC50 values for the two mAbs were, within experimental error, essentially identical. A broader scan comparison of the two mAbs’ specificities was obtained by using a competition ELISA assay to determine their relative binding affinities (RBA values) for the series of compounds; cocaine, cocaethylene, RTI-113, norcocaine, benzoylecgonine, ecgoninemethylester and ecgonine. Figures 1a and 1b show that the two mAbs had essentially identical patterns to their sets of RBA curves.

Both mAbs bind cocaethylene with a somewhat higher affinity than cocaine, with RTI-

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113 about 2-3-fold lower than cocaine, followed by benzoylecgonine and norcocaine at about 7-10-fold lower affinities, while ecgoninemethylester and ecgonine had very low affinities. In Table 1 these RBA values are summarized and presented for comparison to the distinctly different pattern of RBA values obtained for the murine anti-cocaine mAb 1BB835 (Kd for cocaine ~ 0.1 nM) used for comparison in the subsequent stability experiments . h2E2 Stability: determinations of temperature and pH–induced inactivation of anti-cocaine mAbs, 2E2, h2E2, 3P1A6 and 1BB835. Next, the stabilities of 2E2, h2E2, as well as that of two commercially available murine anti-cocaine mAbs, 3P1A6 and 1BB835, selected as control murine mAbs, were compared under various temperature and pH conditions. An ELISA was used to monitor mAb (5 nM) binding to the microtiter plate-absorbed hapten-carrier conjugate after timed incubations in Tris-

1mM EGTA, pH 7 or glycine-HCl, pH 3 at three temperatures (~22o, 50o, 65oC). Figure

2a presents the inactivation curves of mAbs 2E2, h2E2 and 3P1A6 in PBS, pH 7 and incubated at 65oC before transfer to the ELISA plates. Also shown is h2E2’s stability at room temperature (RT) and its rapid inactivation at pH 3 and 65oC. Figure 2b, shows the inactivation curves obtained for the same three mAbs in PBS, pH 7 at 50oC.

Additionally, the stability of 3P1A6 at RT versus its rapid inactivation at pH 3 and 65oC is shown. Clearly the rates of and extent of the mAb inactivation over the 1 hour test period were substantially greater for 65o versus 50oC. In addition, the data show that at pH 7, h2E2 and 3P1A6 were both stable at RT while at pH 3 and 65oC both were inactivated rapidly, nearly completely in ~5 minutes. While the differences in stability

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between the three mAbs were modest in both sets of experiments the recombinant h2E2 was somewhat more stable than 2E2 and 3P1A6.

In a similar set of experiments, Figure 3a presents the inactivation curves for 2E2, h2E2 and Figure 3b for murine mAb 1BB835 in PBS, pH 7 at temperatures of 22o, 50o and

65oC. In this set of experiments h2E2 was more stable than 2E2 but the murine mAb

1BB835 was somewhat more stable than h2E2.

In a final set of experiments the stabilities of the four mAbs at pH 3, were determined at

22o, 50o and 65oC for 1hour (data not shown). At 22oC (RT) their binding activity decreased about 45% over the course of one hour. At 50o and 65oC, half-maximal inactivation occurred within about 5 and 2 minutes, respectively, with all four mAbs undergoing similar rates of inactivation. Overall these data showed no significant differences in stability between the mixed-chain 2E2 versus that of two “normal” murine mAb controls. The results did suggest that the re-engineered h2E2 may have a modestly improved stability over 2E2. mAb thermostability analysis of mAbs h2E2 and 1BB835 by capillary differential scanning calorimetry (DSC). Having established that neither 2E2 nor h2E2 displayed any undue sensitivity to temperature and pH inactivation, more detailed thermostability studies of h2E2 were accomplished using capillary differential scanning calorimetry

(DSC). DSC is a technique that monitors thermally-induced conformational transitions or the unfolding of macromolecule structures (30). It does this by measuring the changes in the excess heat capacity (Cp) of the protein solution as a function of temperature that occur during thermally-induced processes of macromolecule unfolding.

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The Cp changes result from restructuring of protein-solvent, solvent-solvent interactions and intra- and inter-molecular protein interactions. The protein unfolding transitions monitored by DSC yields an endothermic peak centered at the transition midpoint temperature (Tm) with an integrated area providing a direct measure of the enthalpy of the unfolding transition (ΔHu). Clearly in regards to mammalian IgG immunogobulins despite the virtually limitless variety in their binding specificities there are significant similarities to their structures hence also their unfolding patterns. IgGs are composed of identical heavy (H) and light (L) chain pairs linked by disulfide bonds, each chain with characteristic intra-chain disulfides and with a common beta-sheet “immunoglobulin fold” architecture. Further, there are a limited number of constant region sequences for these chains amongst the IgG subtypes. The characteristic or idealized DSC model of an IgG1 antibody’s unfolding transitions consists of three phases. An initial lower temperature unfolding event attributed to the CH2 regions of the H chains’ interactions followed by the higher temperature (Tm) endothermic peak for the Fab domain unfolding and then the highest temperature but lower enthalpy CH3 region unfolding transition

(31, 32).

The major variations in DSC curves for antibodies of the same isotype are then largely attributed to sequence variations in their Fab hypervariable or complementarity- determining regions (CDRs). For h2E2, we were therefore alert for potential thermal instabilities in the Fab region, perhaps attributable to atypical murine L and human H sequence interactions or specific features of the CDR regions.

Figure 4a shows a set of representative curves (each done in duplicate) for mAbs h2E2 and 1BB835 at pH 5.5 and 7.2 as well as the h2E2 Fab fragment (pH 7.2). These

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studies, in contrast to the dilute solutions used in the earlier studies, required mAb concentrations in the 1mg/ml and Fab in the 0.7 mg/ml range in order to generate high

o o quality DSC data over the 50 -100 C transition range. At pH 7.2 in PBS buffer, the Tm values of the major unfolding transition which largely represents Fab domain unfolding for h2E2, its Fab and 1BB835 were 71.9, 72.7 and 72.9oC, respectively, with h2E2 and

1BB835 having similar total unfolding enthalpies. For h2E2, at pH 5.5, in acetate-saline

o buffer, the Tm of the major transition was decreased slightly to 71.7 C and there was some broadening of the unfolding curve in the 60-70oC range that is likely attributable to a lowered stability of the CH2 region. This makes the CH2 region’s unfolding somewhat more distinct from that of the Fab domain unfolding. For 1BB835 at pH 5.5 the Tm of the major transition decreased about three degrees from that observed at pH 7.2 with a reduced unfolding enthapy but little change in the overall curve profile. While 1BB835 had a higher, more distinct major transition than h2E2, the total unfolding enthalpies of h2E2 and 1BB835 were essentially the same at both pHs.

As regards an unfolding transition in the 75-85oC range region that would commonly be attributed to CH3 unfolding 1BB835 showed little if any and while h2E2 showed evidence of further unfolding its Fab fragment also generated a shoulder in this temperature range. Thus, neither mAb gave scans with three clearly delineated component curves. However, as shown in Figure 5, the difference thermogram of h2E2 and its Fab did enable the generation of a calculated thermogram for the CH2 and CH3 domains in agreement with literature reports (32, 33). The DSC data while demonstrating the complex nature of mAb unfolding indicated that there were only subtle differences in the relative thermal stabilities of the Fab, CH2 and CH3 domains of

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h2E2 and 1BB835. Thus, h2E2’s mix of murine and human sequences has not resulted in any significant loss in stability as compared to the control murine mAb.

Ligand (RTI-113)-induced stabilization of mAb h2E2. As the stabilization of enzyme structure upon substrate binding is a common phenomenon we investigated the ability of ligand binding to increase h2E2’s thermal stability. We selected the cocaine analogue RTI-113 (Fig. 2) as the binding ligand since DEA restrictions prevented our testing cocaine or cocaethylene. Our current (Figs. 1a, 1b) and previous studies indicated that while it has a lower binding affinity than cocaine it appears to bind

2E2/h2E2 similarly (17, 25). However, as RTI-113 has poor water solubility it was dissolved in dimethyl sulfoxide (DMSO) and then added to the mAb solution or buffer control in a volume bringing these solutions to 1% DMSO.

The results presented in Figure 6 show that the presence of the organic solvent

o modestly altered h2E2’s thermogram, increasing the Tm (+ 1.7 C) of the major transition but not the total unfolding enthalpy. Figure 6 then shows a significant shift in the thermogram profiles obtained in the presence of non-saturating and saturating levels of

RTI-113. Bound RTI-113 increased the Tm of the major unfolding transition that is largely ascribed to the Fab domain. Under excess ligand conditions (as determined in

o ITC experiments) the Tm increases from 73.6 to 79.1 C. This is a rather dramatic change. The ligand-induced thermal shift of the DSC profiles now reveals a lower

o temperature unfolding transition with a Tm at about 71 C that may be attributed to the

CH2 region but which in the absence of ligand (at pH 7.2) was not distinguished from the

Fab domain unfolding. Clearly RTI-113 binding generates more stable Hv and Lv chain, domain interactions. However, any differences in total mAb unfolding enthalpy are all

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within the standard deviations of the measurements (Table II). This was expected given the small binding enthalpy of h2E2-RTI-113 interaction. Currently, we do not know but given their higher binding affinities we would expect cocaine and cocaethylene binding to have the same or greater Fab stabilizing effect.

Determination of the thermodynamic parameters of cocaethylene, cocaine and

RTI-113 binding to h2E2. In addition, to the DSC studies of h2E2 and 1BB835 unfolding the thermodynamics of cocaethylene, cocaine and RTI-113 binding to h2E2 were determined using isothermal titration calorimetry (ITC). Current ITC instrumentation enables the use of relatively modest protein concentrations for determinations of the enthalpy (ΔH) of ligand binding, the stoichiometry (N) of binding and the association constant (Ka) or affinity of ligand binding. The Gibbs free energy

(ΔG) of binding is calculated using ΔG = -RT lnKa and the entropy (TΔS) obtained as the difference of ΔH-ΔG.

These data provide considerable information about the mechanisms of the ligand/mAb interactions. Figure 7 presents representative single experiment raw data sets (upper plots) and the non-linear least squares fit of the peak areas curves (lower plots) resulting from the ligand titrations of h2E2 with cocaine (7a, b) cocaethylene (7c, d) and

RTI-113 (7e,f), respectively. Both the exothermic heats of binding from each addition of ligand and the binding isotherms are presented as a function of the ligand/protein complex formation. The solid lines represent the data fitted to a single set of sites model to give the association constant (Ka), binding stoichiometry and ΔH. Table III presents the compiled values of the thermodynamic parameters obtained from three titrations each of cocaethylene, cocaine, and RTI-113. The ligand titration curves

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showed good fits to a single binding site model for all ligands and the association constants obtained (and calculated Kd values = 2nM, 7nM and 30nM, respectively) are consistent with our previous radioligand and ELISA relative binding affinity results.

Calculations of the concentrations of ligand needed to saturate mAb binding also confirmed that the h2E2 preparation is highly pure and active.

The calculated ΔG values, for cocaethylene, cocaine and RTI-113 ranked by decreasing affinities were -11.6, -10.9 and -10.1 kcal/mol, respectively. Interestingly, both the enthalpy (ΔH) and entropy (ΔS) values for RTI-113 were quite distinct from that of cocaine and cocaethylene. The enthalpy value for RTI-113 of -2.6 kcal/mol was less than one fourth that of cocaethylene (-11.3kcal/mol) or cocaine (-14.6 kcal/mol). Also, the TΔS contribution to RTI-113 binding was a positive value (+7.5 kcal/mol) as compared with a near zero value for cocaethylene (0.3kcal/mol) and a negative value for cocaine (-3.7kcal/mol). Thus, indicating RTI-113 binding has a larger entropy driven interaction.

The difference in the binding energetics of RTI-113 likely results from altered ligand/mAb interactions due to the absence of the C-4 position carboxy group that for cocaine and cocaethylene links the phenyl and tropane rings and replacement of the methylester or ethylester group on the C-2-tropane ring with the much larger aromatic phenyl group. Structurally, RTI-113 is less polar with about half the polar surface area as cocaine or cocaethylene. It has two fewer potential hydrogen bond acceptors and has significantly more hydrophobic surface area than the other two ligands. Therefore, a larger proportion of the binding energy is likely due to hydrophobic rather than hydrophilic drug/protein interactions.

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Thus, the calorimetry studies were able to identify and quantify differences in h2E2’s binding interactions with RTI-113 as compared to cocaine and cocaethylene. This contrasts from our previous drug docking modeling studies (25) that indicated RTI-113 had a similar mode of binding as cocaine and cocaethylene.

Interestingly, the ΔG values that we report here for ligand binding to h2E2 are similar to those obtained by Ramakrishnan, et al. (34) with their murine anti-cocaine mAb, designated mAb08. They used ligand titration ITC and obtained ΔG binding values of -

11.7, -10.5 and -10.2 kcal/mol for cocaine, benzoylecgonine and cocaethylene, respectively, in order of their decreasing binding affinities (2nM, 19nM and 43 nM) to mAb08. However, as discussed below the enthalpy and entropy values differed considerably.

4. Discussion

Here we have further confirmed that the re-engineered, CHO cell-expressed recombinant anti-cocaine mAb h2E2 retains essentially the same affinity and specificity for cocaine and cocaethylene versus various metabolites and analogues as the original hybridoma cell-derived human/mouse mixed-chain mAb 2E2 (18). The re-engineering of 2E2’s L chain constant domain to a human sequence rather than murine has the potential to reduce the probability of mAb antigenicity when used as a therapy with repeat dosings. However, each mAb species has unique sequences and each patient’s immunological response is unique. Thus, it is not possible to a priori predict how any individual will react upon infusion of a therapeutic mAb. The fact that h2E2’s γ1 H chain is fully-human and the mAb binds an exogenous low-molecular weight drug rather than

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targeting an endogenous human protein or antigen should significantly reduce the likelihood of any untoward antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) immune responses.

The thermal and pH inactivation studies presented here demonstrate that 2E2 and h2E2 have similar thermal and pH stabilities that compared favorably with that of two typical murine anti-cocaine mAbs 3P1A6 and 1BB835. Therefore, it seems unlikely that poor protein folding or mAb stability is the explanation for 2E2’s low levels of production in the hybridoma cell and the transiently transfected HEK and CHO cells. These inactivation studies did indicate that under the tested conditions h2E2 showed a modest improvement in stability as compared to 2E2.

The DSC studies then provided a means to directly monitor the thermal unfolding profiles of h2E2 and the murine mAb 1BB835 and obtain more detailed stability information. The thermograms showed that the two mAbs have nearly identical total unfolding enthalpies and Tm values for the Fab domain unfolding transition. h2E2 did generate a more complex profile, especially at temperatures above the Tm but there was no evidence of h2E2’s three domain structures being problematic in regards to its stability.

DSC and differential scanning fluorimetry have been used to investigate the thermostability of therapeutic chimeric, humanized and human mAbs of differing subtypes (i.e., IgGγ1, γ2, γ3 & γ4 (32, 33, 35) and in some cases also their Fab and Fc fragments (31, 36). Overall these studies indicate a considerable variability in the thermogram profiles and the Tm values of the Fab domain unfolding reported vary over

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o o a broad range, from 57 C to 82 C (32, 33). This range in the reported Tm values may not though reflect specific differences in these antibodies’ intrinsic stabilities because mAb unfolding is dependent upon the pH, ionic strength, excipients and buffers used in the studies and these have varied widely. Amongst a number of mAbs studied, the humanized mAb trastuzumab (Herceptin®, Genentech), stands out in that it has undergone considerable re-engineering in a transition from its initial murine sequence to a humanized sequence. This has been accomplished with a concomitant engineered increase in its thermal stability. The humanization of the Fab domain has been found to

o o increase the Tm value of the Fab from 72 C to 82 C (36). A substantial increase. Our

o studies indicate that h2E2’s stability (Tm~72 C) stands about mid-point of the observed range while the work of Garber and Demarest (32) suggests that in general it is mAbs

o with Tm values below 60 C that are most likely to be unstable upon storage and prone to aggregation.

The extent of the observed stabilization of the h2E2 Fab domain upon RTI-113 binding is impressive but some degree of ligand-induced stabilization was expected as it seems intuitive that the free energy of ligand binding to a protein should contribute to stabilizing its structure. Certainly, the concept that a substrate or regulatory ligand that preferentially binds an enzyme’s native state should induce or stabilize enzyme conformational changes that increase its thermal stability has a long-standing history

(37, 38). In recent work on this topic, the computational simulations of Waldron &

Murphy (39) as well as DSC studies reviewed by Bruylants, et al. (30) provide support for this phenomenon. However, DSC studies have also shown possible exceptions to

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this assumption such as the ATP-dependent destabilization of the human erythrocyte cell membrane-bound glucose transporter GLUT-1 (40).

In h2E2’s case, as based on our models of cocaine’s interactions with the H and L chain

CDR regions likely comprising the mAb binding site pocket, it seems that ligands should provide connecting links that increase chain-chain interactions (25). These should result in an increased stability in the interactions of the mAb’s Fv domains.

Interestingly, unlike the case with enzymes, ligand-induced thermal stabilization of antibodies does not seem to have received much study.

Thus far, we have found few literature reports of ligand-dependent increases in mAb stability. Perhaps this is because x-ray crystal structure studies of antibodies currently provide few examples of significant ligand-induced conformational changes in mAb structure. Alternatively, ligand-dependent antibody stabilization may not be common because antigen binding often is achieved through a ligand’s fitting complementarily onto CDR regions’ surfaces rather than mediating H and L chain interactions.

Munnert and Voss (41) have published DSC thermograms for the low affinity (Kd

~10μM) antifluorescein murine mAb 9-40 and find fluorescein binding modestly reduced the total enthalpy of unfolding without changing the overall thermogram profile while the

o o Fab domain Tm value increased from 75 to 77 C. They also reported that binding of a monofluoresceinated polyarginine derivative that has both ligand-binding site and non- binding site interactions with the mAb effected no change in the Tm but decreased the enthalpy of the Fab domain unfolding transition. It also modified the thermogram such that there was a broad lower temperature shoulder not present in the absence of ligand.

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These ligand-dependent DSC results for mAb 9-40 were modest and distinct from those obtained for the RTI-113-h2E2 interactions.

Inspection of our model (25) of cocaine docked to 2E2 shows hydrophobic, ionic and hydrogen bond interactions between cocaine and the H chain that include residues

Trp145 (CDR1), Glu211 and Leu212 (CDR3) as well as L chain interaction with residues Ser33, Tyr35 (CDR1) and Trp94 (CDR3) that suggest cocaine could serve as a bridge linking the two chains. As the three ligands used in these ITC studies computationally docked similarly into the modeled mAb binding site it seems likely that cocaine and cocaethylene with their higher affinities would generate corresponding larger increases in mAb stability than RTI-113. But RTI-113 does have a larger hydrophobic surface area and fewer hydrogen bonding capabilities than cocaine or cocaethylene. As with our modeling of 2E2 inspection of the crystal structures of the

Fabs of three murine mAbs, GNC92H2 (42), 15A10 (43) and M82G2 (44) with bound cocaine suggests that for all these mAbs cocaine seems capable of serving as a mediator for increased H and L chain interactions.

In reviewing the titration calorimetry results it is conceptually intriguing how high-affinity, selective binding of low-molecular weight compounds by antibodies is achieved given that the ΔG values of their binding are usually rather modest. Generally they are in the range of -5 to -10 kcal/mol as composed of varying combinations of the enthalpy and entropy components (45). In our studies, the Gibbs free energy values for cocaethylene, cocaine and RTI-113 were similar, ranging from -11.6 to -10.1 kcal/mol while the enthalpy and entropy values for RTI-113 varied significantly from those of the other two ligands.

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The ITC technique therefore appears more sensitive in detecting differences in the binding of different ligands than achieved in our 3D-QSAR and docking structure modeling. The thermodynamic differences between RTI-113 and cocaine/cocaethylene may arise from the distinct structural features of its binding to a site better designed to fit cocaethylene and cocaine, or from its having an altered mode of binding. While our modeled dockings showed RTI-113 to bind similarly as cocaine there is precedence for related ligands to bind differently. For example, Tars, et al. (46) using x-ray crystallography found the human anti-nicotine mAb Nic12 binds nicotine and the nicotine- succinimide (linker) derivative that served as the immunizing hapten (linked to the protein carrier) in distinctly different modes.

In a related comparison, discussed previously, the ΔG values reported by

Ramakrishnan, et al. (34) for cocaine, benzoylecgonine and cocaethylene binding to anti-cocaine mAb mAb08 are similar to our results. However, the ΔH (-28.1, -21.7 and -

21.1 kcal/mol) and TΔS (-16.3, -11.1 and -11.0 kcal/mol) values determined for the ligands’ binding were quite distinct from those obtained for h2E2. The ΔH values for ligand binding to h2E2 were about half or less than that for mAb08 and the TΔS values were also significantly different. These sets of results potentially further illustrate the variability and flexibility in how antibodies achieve high affinity binding specificities.

In summary, in this study we have further confirmed that the affinity and specificity of the anti-cocaine chimeric mAb 2E2 for cocaine and cocaethylene versus that for inactive metabolites has not been compromised by reengineering a more humanized h2E2 and employing stably-transfected CHO cells for its production. Further, these studies of the temperature and pH sensitivity of mAb stability have indicated that h2E2’s

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stability is modestly improved over that of 2E2 and compares favorably with that of conventional murine mAbs. Also, DSC studies done at pH 5.5 and 7.0 showed that while h2E2 has a more complex pattern of unfolding than murine mAb 1BB835 with subtle differences in the thermal stabilities of the Fab, CH2 and CH3 domains their overall unfolding energetics do not differ significantly. These stability studies further support the continued development of h2E2 as an immunotherapeutic.

The extent to which h2E2’s Fab domain stability increased upon binding RTI-113 was impressive. Additional, future studies designed towards determining whether this stabilization is a common phenomenon for mAbs or unique for this drug:mAb interaction should prove interesting and informative.

Finally, the ITC studies provided detailed characterizations of the thermodynamics of drug binding to h2E2 and an alternative to radioligand binding methodology for determination of their affinities of binding. Interestingly, these studies indicated that the thermodynamics of RTI-113 binding h2E2 were quite distinct from those of cocaine and cocaethylene with the entropy component playing the dominant role in its binding.

Thus, the ITC results provided information about ligand binding not apparent from our earlier structure-activity relationship modeling studies. Clearly though a more comprehensive understanding of the molecular nature of the drug/2hE2 Fab interactions will await our obtaining high-resolution structures through x-ray crystallography.

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References:

1. www.drugabuse.gov/drugs-abuse/cocaine; Substance Abuse and Mental Health Services Administration’s (SAMSHA’s) National Survey on Drug Use and Health 2012.

2. Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, Chapter 18: Opioids, Analgesia and Pain Management, au: T. L. Yash and M. S. Wallace, pp 481- 525, Ed. 12, pub. McGraw-Hill, New York, USA (2011).

3. Bonese, K. F., Wainer, B. H., Fitch, F.W., Rothberg, R. M. and Schuster, C. R. Changes in heroin self-administration by a rhesus monkey after morphine immunization. Nature 252: 708-710 (1974).

4. Killian, A., Bonese, K. F., Rothberg, R. M., Wainer, B. H. and Schuster, C. R. Effects of passive immunization against morphine on heroin self-administration. Pharmacol. Biochem. Behav. 9: 347-352 (1978).

5. Norman, A.B., Tabet M.R., Norman M.K., Buesing, W.R., Pesce, A.J., Ball, W.J., A chimeric human/murine anticocaine monoclonal antibody inhibits the distribution of cocaine to the brain in mice. J. Pharmacol. Exp. Ther. 320: 145-153 (2007).

6. Norman, A.B., Norman M.K., Buesing W.R., Tabet, M.R., Tsibulsky, V.L., Ball, W.J., The effect of a chimeric human/murine anti-cocaine monoclonal antibody on cocaine self-administration in rats. J. Pharmacol. Exp. Ther. 328: 873-881 (2009).

7. Kosten TR, Owens SM. Immunotherapy for the treatment of drug abuse. Pharmacol Ther 108: 76-85 (2005).

8. Fox, B.S., Kantak, K. M., Black, K. M., Bollinger, B. K., Botka, A.J., French, T.L., Thompson, T.L., Schad, V.C., Greenstein, J.L., et al., Efficacy of a therapeutic cocaine vaccine in rodent models. Nature Medicine 2 (10): 1129-1132 (1996).

9. Carrera, M.R., Ashley, J. A., Zhou, B., Wirsching, P., Koob, G. F. and Janda, K.D., Cocaine vaccines: antibody protection against relapse in a rat model. Proc. Natl. Acad. Sci. USA 97: 6202-6206 (2000)

10. Carrera, M.R., Ashley, J. A., Zhou, B., Wirsching, P., Koob, G. F. and Janda, K.D., A second-generation cocaine vaccine protects against the psychoactive effects of cocaine. Proc. Natl. Acad. Sci. USA 98: 1988-1992 (2001).

11. Martell, B.A., Mitchell, E., Poling, J., Gonsai, K. and Kosten, T.R., Vaccine pharmacotherapy for the treatment of cocaine dependence. Biol. Psychiatry 58: 158- 164, (2005).

233

12. Martell BA, Orson FM, Poling J, et al., Cocaine vaccine for the treatment of cocaine dependence in methadone maintained patients. Arch. Gen. Psychiatry 66 (10): 1116- 1123 (2009).

13. Haney M, Gunderson EW, Jiang H, Collins ED, Foltin RW. Cocaine-specific antibodies blunt the subjective effects of smoked cocaine in humans. Biol. Psychiatry 67: 59-65, (2010).

14. Norman, A. B. and Ball, W. J., Predicting the clinical efficacy and potential adverse effects of a humanized anticocaine monoclonal antibody. Immunotherapy 4 (3): 1-9, 2012.

15. Lonberg, N., Fully human antibodies from transgenic mouse and phage display platforms. Current Opinion in Immun. 20: 450-459 (2008).

16. Roopenian, D. C., Christianson, G. J. and Sproule, T. J., Chapter 6, Human FcRn transgenic mice for pharmacokinetic evaluation of therapeutic antibodies. Mouse Models for Drug Discovery, Protetzel,G and Wiles M.V. (eds.) Methods in Molecular Biology 602, 93-104, 2010.

17. Paula, S., Tabet, M.R., Farr, C.D., Norman, A.B., Ball, W.J., Three-dimensional quantitative structure-activity relationship modeling of cocaine binding by a novel human monoclonal antibody. J. Med. Chem. 47: 133-142 (2004).

18. Norman, A. B., Gooden, F. C.T, Tabet, M. R. and Ball, W. J., A recombinant humanized anticocaine monoclonal antibody inhibits the distribution of cocaine to the brain in rats. Drug Metabolism and Disposition 42 (7): 1125-1131 (2014).

19. Fishwild, D.M., O’Donnell, S.L., Bengoechea, T., Hudson, D.V., Harding, F., Bernhard, S.L., Jones, D., Kay, R.M., Higgins, K.M., Schramm, S.R., Lonberg, N., High- avidity human IgG kappa monoclonal antibodies from a novel strain of minilocustransgenic mice. Nature Biotech.14: 845-851 (1996).

20. Kohler, G, Milstein, C., Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6 (7): 511-519 (1976).

21. Torres, M., Casadevall, A., The immunoglobulin constant region contributes to affinity and specificity. Trends in Immunology 29: 91-97 (2008).

234

22. Dan, T.K., Torres, M. Brewer, C.F., Casadevall, A., Isothermal titration calorimetry reveals differential binding thermodynamics of variable region-identical antibodies differing in constant region for a univalent ligand. J. Biol. Chem. 283: 31366-31370 (2008).

23. Ponomarenko, N., Chatziefthimiou, S. D., Kurkova, I., Mokrushina, Y., Stepanova, A., Smirnov, I. et al. (Gabibov, A.), Role of κ – λ light-chain constant-domain switch in the structure and functionality of A17 reactibody. Acta Crystallographica (Section D) 70: 708-719 (2014)

24. Paula, S., Tabet, M.R., Keenan, S.M., Welsh, W.J., Ball, W.J., Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis. J. Mol. Biol. 325: 515-530 (2003)

25. Lape, M., Paula, S., Ball, W.J., A molecular model for cocaine binding by the immunotherapeutic human/mouse chimeric monoclonal antibody 2E2. Eur. J. Med. Chem. 45: 2291-2298 (2010).

26. Ball, W.J., Kasturi, R., Dey, P., Tabet, M.R., O’Donnell, S., Hudson, D. and Fishwild, D., Isolation and Characterization of human monoclonal antibodies to digoxin. J. Immunol. 163: 2291-2298 (1999).

27. Bleck, G. T., An alternative method for the rapid generation of stable high- expressing mammalian cell lines. Bioprocessing Journal, Sept./Oct.: 1-7, (2005).

28. Bleck, G.T., Consistent production of genetically stable mammalian cell lines. BioPharm. International 25:56-59, (2012).

29. Garbett, N.C. and Chaires, J.B. Thermodynamic studies for drug design and screening. Expert Opin. Drug Discov. 7 (4): 299-314 (2012).

30. Bruylants, G., Wouters, J. and Michaux, C., Differential scanning calorimetry in Life Sciences: Thermodynamics, stability, molecular recognition and application in drugs design. Current Medicinal Chem.12: 2011-2020, (2005).

31. Ionescu, R.M., Valasak, J., Price, C., Kirchmeier, M., Contribution of variable domains to the stability of humanized IgG1 monoclonal antibodies. J. Pharmaceut. Sci. vol. 97: 1414-1426 (2008).

32. Garber, E. and Demarest, S. J., A broad range of Fab stabilities with a host of therapeutic IgGs. Biochem. Biophys. Res. Commun. 355: 751-7, 2007.

235

33. Sahin, E., Grillo, A. O., Perkins, M. D. and Roberts, C. J., Comparative effects of pH and ionic strength on protein-protein interactions, unfolding and aggregation for IgG1 antibodies. J. Pharmaceut. Sci. 99 (12): 4830-4848, (2010).

34. Ramakrishnan, M., De Melo, F. A., Kinsey, B. M., Ladbury, J. E., Kosten, T. R. and Orson, F. M., Probing Cocaine-Antibody interactions in buffer and human serum. PLos ONE: Vol. 7 (Issue 7) e40518, pp 1-12, (2012).

35. Harn, N., Allan, C., Oliver, C., Middaugh, C.R., Highly concentrated monoclonal antibody solutions: direct analysis of physical structure and thermal stability. J. Pharmaceut. Sci. vol. 96: 532-546, (2007).

36. Kelley, R. F., O’Connell, M. P., Carter, P., Presta, L., Eigenbrot, C., Covarrubias, M., Snedecor, B., Bourell, J. H. and Vetterlein, D. Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185 HER2 antibody Fab fragments., Biochemistry 31: 5434-5441, (1992).

37. Schellman, J. A., Macromolecular binding. Biopolymers 14: 999-1018, (1975).

38. Layton, C. J. and Hellinga, H. W., Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes. Biochemistry 49: 10831-10841, (2010).

39. Waldron, T.T. and Murphy, K.P., Stabilization of proteins by ligand binding: application to drug screening and determination of unfolding energetics. Biochemistry 42: 5058-5064 (2003).

40. Epand, R.F., Epand, R.M. and Jung, C.Y., Ligand-modification of the stability of the glucose transporter GLUT 1. Protein Sci.10: 1363-1369 (2001).

41. Mummert, M.E. and Voss, E.W., Effects of secondary forces on the ligand binding and conformational state of antifluorescein monoclonal antibody 9-40. Biochemistry 36: 11918-11922, (1997).

42. Larsen, N.A., Zhou, B., Heine, A.K., Wirsching, P., Janda, K. D. and Wilson, I.A., Crystal structure of a cocaine-binding antibody. J. Molecular Biology 311: 9-15, (2001).

43. Larsen, N. A., de Prada, P., Deng, S.-X., Mittal, A., Braskett, M., Zhu, X., Wilson, I.A. and Landry, D.W., Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10. Biochemistry 43: 8076-8076, (2004).

236

44. Pozharski, E., Moulin, A., Hewagama, A., Shanafelt, A. B., Petsko, G. A. and Ringe, D., Diversity in hapten recognition: structural study of an anti-cocaine antibody M82G2. J. Molecular Biology 349: 570-582, (2005).

45. Perozzo, R., Folkers, G. and Scapozza, L., Thermodynamics of protein-ligand interactions: history, presence and future aspects. J. Receptors and Signal transduction 24 (1 & 2): 1-52, (2004).

46. Tars, K., Kotelovica, S., Lipowsky, G., Bauer, M., Beerli, R. R., Bachmann, M. F. and Maurer, P., Different binding modes of free and carrier-protein-coupled nicotine in a human monoclonal antibody. J. Mol. Biol. 415: 118-127 (2011).

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Acknowledgements

This work was supported by National Institutes of Health, National Institute on Drug Abuse [grant DP1DA031386, P. I. A.B Norman]. The authors would like to thank Dr. Terence L. Kirley for preparation of the Fab fragments of h2E2 and insightful comments on the manuscript.

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Figure Legends:

Figure 1 a, b: Determination of the relative binding affinities (RBAs) of mAb h2E2 for cocaine and analogues versus that of 2E2. The curves in panels a (h2E2) and b (2E2) show representative competition ELISA results. Decreasing optical density values indicate mAb binding to plate-adsorbed hapten-conjugate as reduced by increasing concentrations of ligand present in mAb binding buffer. The ligand competition curves are as indicated: cocaethylene (closed circles), cocaine (open circles), RTI-113 (closed stars), benzoylecgonine (closed squares), norcocaine (open sqsquares), ecgonine methylester (open diamonds, panel a, open triangles, panel b) and ecgonine (closed triangles). Symbols represent the average of duplicate samples and regression lines were obtained from Sigma Plot, non-linear regression-global curve fitting.

Figure 2: Chemical structures. The three panels, as identified, illustrate the structures of: a) (-)cocaine, b) (-)cocaethylene and c) (-)RTI-113. Oxygen atoms are colored in red, nitrogen in blue and chlorine green.

Figure 3 a, b: Determinations of temperature and pH-dependent inactivation of anti- cocaine mAbs 2E2, h12E2, 3P1A6 and 1BB835. Panels show normalized results of quantification of mAb binding to plate absorbed hapten-carrier conjugate upon varying treatment conditions over 0-60min. Representative sets of experiments are shown: Panel a, shows h2E2 (open squares) at room temperature (RT, pH 7), 65oC, pH 7, and 65oC, pH 3, 2E2 (closed circles) at 65oC, pH 7 and 3P1A6 (xs). Panel b, shows mAb 3P1A6 (xs) at room temperature (RT, pH 7), 50oC, pH 7 and 65oC, pH 3; h2E2 (open squares) at 50oC, pH 7 and 2E2, 50oC, pH 7. Symbols represent the averages of determinations made at the indicated times and regression lines were obtained using Sigma plot, non-linear regression, exponential decay, double, 4 parameter least squares fitting. Error bars show standard errors of the mean of determinations performed in triplicate.

Figure 4 a, b: Determination of temperature dependent inactivation of anticocaine mAbs 2E2, h2E2 and 1BB835. Panels show normalized results of quantification of mAb- binding to plate-absorbed hapten carrier conjugate upon varying treatment conditions. Representative sets of results are shown: Panel a, shows h2E2 (closed squares) at room

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temperature (RT, pH 7), (closed diamonds) 50oC, pH 7, and (closed triangles) 65oC; and 2E2 (open squares) at room temperature (RT), pH 7, (open diamonds) at 50oC and (open triangles) at 65oC, pH7. Panel b, shows mAb 1BB835 (open squares) at room temperature (RT, pH 7), (open circles) 50oC, pH 7 and (closed diamonds) 65oC, pH 7. Symbols represent the averages of determinations made at the indicated times and regression lines were obtained using Sigma plot, non-linear regression, exponential decay, double, 4 parameter least squares fitting. Error bars show standard errors of the mean of determinations performed in triplicate.

Figure 5: Thermal unfolding profiles of anticocaine mAbs h2E2, Fab h2E2 and 1BB835 via capillary DSC. Representative thermal unfolding profiles of anti-cocaine mAbs h2E2 (1mg/ml) at pH 7.2 (black line) and pH 5.5 (blue line), Fab h2E2 (0.76mg/ml) at pH 7.2 (green line) and 1BB835 (1mg/ml) at pH 7.2(red line) and pH 5.5(cyan line) recorded by DSC.

Figure 6: Thermal unfolding profiles of anti-cocaine mAb h2E2 and Fab h2E2 and deconvolution of the Fc calormetric domain. The thermal unfolding profile of h2E2 at pH 7.2 (black line) and a calculated profile of the Fab (green line) represented by twice the experimental Fab thermogram (see Figure X). The Fc CH2 and CH3 domains’ unfolding (red lines) were calculated by subtraction of the Fab profile from that of h2E2.

Figure 7: Determination of the effects of 1%DMSO and RTI-113 binding on the thermal unfolding profile of h2E2. Representative thermal unfolding profiles of mAb h2E2 (13.4 μM) in PBS, pH 7.2 (black line), in PBS plus 1%DMSO (magenta line), in buffer, 1%DMSO and 6.7μM RTI-113 (mustard line), in buffer, 1%DMSO and 39μM RTI- 113 (orange line).

Figure 8 a,b,c: Calorimetric titrations of mAb h2E2 with a) cocaine, b) cocaethylene and c) RTI-113. The top panels illustrate the raw data of ITC titrations of 5μl injections of 50μM drug to 10μM (binding sites) mAb h2E2. The bottom panels illustrate the non-linear least squares fit of the peak area from the titrations shown above to a one set of sites model.

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Figure 9 a, b, c, d: Comparison of thermodynamic binding parameters of antibody h2E2 binding of cocaine, cocaethylene and RTI-113 obtained from ITC titrations at 20oC. In each of the panels the thermodynamic parameters for cocaine are shown in blue, cocaethylene in red and RTI-113 in green. Data represent averages of three titration experiments for each ligand and error bars indicate standard errors of the mean.

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Table1: Determinations of relative binding affinities of mAbs h2E2, 2E2 and 1BB835 for cocaine and metabolites.

Competition ELISA: Relative Binding Affinity (RBA) Determinations ______RBA values (relative to cocaine)

compound mAb: h2E2 2E2 1BB835

cocaine 1 1 1

(IC50, 0.17μm) (0.17μm) (0.03μm)

Cocaethylene 0.6 0.5 2

RTI-113 3 2 1700

Norcocaine 8 7 40

Benzoylecgonine 7 11 60

Ecgoninemethylester 1900 1800 900

Ecgonine 18000 23000 n.b.

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Figure 1:

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Figure 2: Chemical structures

a) (-)cocaine b) (-)cocaethylene c) (-) RTI-113

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Figure 3:

Panels show normalized results of quantification of mAb binding to plate-adsorbed hapten- carrier conjugate upon varying treatment conditions over 0-60 minutes. Representative results

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Figure 4:

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Table II. Summary of thermodynamic parameters of h2E2, Fab h2E2 and 1BB835 unfolding obtained by DSC.

o mAb or Fab Tmax ( C) ΔHo (kJ/mol) ΔSo (kJ/mol.K) h2E2, pH 7.2 71.9 ± 0.37 3076 ± 305 8.9 ± 0.89 Fab h2E2, pH 7.2 72.7 ± 0.18 1204 ± 38 3.5 ± 0.11 h2E2, pH 5.5 71.7 ± 0.04 2915 ± 61 8.6 ± 0.18 1BB835, pH 7.2 72.8 ± 0.05 2973 ± 321 8.6 ± 0.67 1BB835, pH 5.5 69.7 ± 0.07 261 ± 69 7.6 ± 0.21 h2E2, pH 7.2, 1% DMSO 73.6 ± 0.22 3260 ± 242 9.4 ± 0.70 h2E, pH 7.2, 1% DMSO + RTI-113 (1:0.5) 77.6 ± 0.24 3267 ± 27 9.3 ± 0.05 h2E2, pH 7.2, 1%DMSO + RTI-113 (1:3) 79.1 ± 0.04 3087 ± 8 8.8 ± 0.03

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Figure 5:

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Figure 6:

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Figure 7:

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Figure 8:

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Figure 9:

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Table III. The thermodynamic binding parameters of h2E2 binding to cocaethylene, cocaine and RTI-113 obtained by ITC titration.

-1 Drug ΔG kcal/mol ΔHobs kcal/mol TΔS kcal/mol Ka (M )

Cocaethylene -11.6 ± 0.10 -11.3 ± 0.22 0.30 ± 0.2 5.0 ± 0.28 .108

Cocaine - 10.9 ± 0.09 - 14.6 ± 0.12 -3.72 ± 0.10 1.55 ± 0.2 .108

RTI-113 - 10.1 ± 0.16 - 2.62 ± 0.30 7.5 ± 0.46 3.63 ± 0.8 .107

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