Identifying the Mechanism Underlying Tissue-specific Deamidation of Translation Repressor 4E-BP2

Tai Vinh Truong

Department of Biochemistry

McGill University, Montreal

May, 2017

A thesis submitted to the Faculty of Graduate studies and Research in partial

fulfilment of the requirements of the degree of Master of Science

Tai Vinh Truong, 2017

1 Abstract

Protein synthesis is a crucial process underlying cellular maintenance and cellular response towards environmental stimuli. Regulation of protein synthesis is complicate, elaborate, and involves multiple checkpoints. As the first and also rate-limiting step in mRNA translation, control of translation initiation is beneficial to preserve energy expenditure that would otherwise be lost if translation is to be halted at later stages. Amongst the governing mechanisms of translation initiation, a key regulatory module is the eIF4E-binding proteins (4E-BPs) which inhibit translation by competing with eIF4G for the same binding motif on the mRNA 5’ cap binding protein eIF4E. Through competitive inhibition, 4E-BPs prevent the formation of the tripartite eIF4F complex, consequently preventing the formation of the 48S preinitiation complex. This regulation is mainly controlled by phosphorylation of 4E-BPs.

Hypophosphorylated 4E-BPs binds with high affinity to eIF4E while fully phosphorylated 4E-

BPs undergo a conformational change and is released from its cognate. Deamidation is one of the most common post-translational modifications observed in eukaryotes, where asparagine transforms to asparatate. Each deamidated residue provides an additional negative charge.

Deamidation is commonly known to be an unwanted and unregulated event as deamidated proteins usually become unstable and are successively degraded. It was reported that 4E-BP2 exhibits a tissue disparate pattern of deamidation. Specifically, deamidated 4E-BP2 was only observed in the brain. This thesis unveils another location of 4E-BP2 deamidation and aims to delineate the mechanism underlying tissue specific deamidation of 4E-BP2. Deamidation of 4E-

BP2 is coincidental with reduction of mTOR activity. 4E-BP2 is also identified to be degraded through the proteasomal pathway. Together, it is hypothesized that specific reduction of mTOR

2 activity within specific tissues renders the proteasome less active, resulting in the accumulation of deamidated 4E-BP2.

Résumé

La synthèse protéique est un processus crucial sous-jacent l’homéostasie cellulaire et la réponse aux stimuli environnementaux. La régulation de la traduction d’ARNm en protéines est compliquée et implique de nombreux points de contrôle. Le premier et le plus important de ceux- ci est l’initiation de la traduction, qui permet de prévenir le gaspillage d’énergie que constituerait l’arrêt de la synthèse protéique à des étapes ultérieures. Parmi les acteurs impliqués dans ce mécanisme, les protéines liant le facteur d’initiation de la traduction eIF4E (4E-BPs) jouent un rôle prépondérant de par leur compétition avec eIF4G pour le même site d’interaction sur la protéine liant la coiffe des ARNm, eIF4E. Ainsi, les 4E-BPs empêchent la formation du complexe eIF4F nécessaire au recrutement du complexe ribosomal de pré-initiation 48S.

L’action des 4E-BPs est principalement contrôlée via leur phosphorylation par la kinase mTOR, ce qui diminue leur affinité pour eIF4E. L’une des 4E-BPs, 4E-BP2, peut aussi être régulée par déamidation, transformant un résidu d’asparagine en aspartate. La déamidation est l’une des modifications post-traductionnelles les plus communément observées chez les eukaryotes. La déamidation est spontanée, et est généralement considérée comme néfaste, menant à l’apparition d’une nouvelle charge négative sur un protéine, qui devient alors instable et est rapidement dégradée. Nous avons précédemment identifié un site de déamidation dans 4E-BP2 qui apparaît uniquement dans le cerveau. La présente thèse identifie un second tissu exhibant la déamidation de 4E-BP2 et vise à identifier le mécanisme conférant la spécificité de ce processus à ces deux tissus. Ainsi, nous établissons que la déamidation de 4E-BP2 coïncide avec une réduction de l’activité de mTOR et mène à sa dégradation par le protéasome. Sur ces bases, nous présentons

3 l’hypothèse que la réduction programmée de l’activité de mTOR au cours du développement de certains tissus mène à une réduction de l’activité protéasomale, et donc à l’accumulation de 4E-

BP2 déaminée.

Acknowledgement

First of all, 爸爸，妈妈，谢谢您们！

Thank you to my parents for giving me the greatest and most unique opportunity – to be alive. Thank you for working through various physical demanding jobs to support me through years of education. Evolutionarily speaking, you could have left me working my way through life after I reached 16 as you have successfully passed on your genes but you didn’t. You continue supporting me financially and most of all, emotionally. You make me who I am. And this is as much my effort as it is yours. As part of our tradition and culture, I have learned and memorized this Vietnamese idiom since elementary school and I don’t think there is a better place to phrase it other than here:

“Công cha, nghĩa mẹ, ơn thầy Nghìn năm bia đá vẫn hoài tri ân”

Thank you to Dr. Nahum Sonenberg for the precious opportunity to work in such an engaging and challenging environment. In which, I was exposed to high quality research as well as was able to challenge myself to become a better scientist.

Thank you to Dr. Arkady Khoutorsky for his thoughtful advice, guidance, and mentoring. You gave me the freedom and independence to lead my own research projects but are always available to help me when I needed.

Thank you to Dr. Edna Matta for being a wonderful lab member. You have been incredibly helpful in giving me many valuable insights and constructing experiments to shape my thesis. You have made the Master degree experience in the laboratory a very pleasant one!

Thank you to my RAC members: Dr. Sidong Huang and Dr. Wayne Sossin for your valuable inputs towards the project.

Thank you to members of the Neuro group: Dr. Agnieszka Skalecka, Dr. Argel Aguilar-Valles, Dr. Ilse Gantois, Dr. Rapita Naresh Sood, Dr. Vijendra Sharma, Dr. Yelena Popic, Shane Wiebe, and Anmol Nagpal.Thank you to other members of the Sonenberg lab especially Chadi Zakaria, Dana Pearl, Dr. Souroush Tahmasebi , and Dr. Peng Wang for making it a pleasant and almost fun experience.

Thank you Annie Sylvestre, Annick Lafrance for being the best A-team that our lab can have. Without you, all my mouse experiments would not have worked out.

4 Thank you to Eva Mignon and Sandra Perreault for making sure that the orders are always delivered as soon as possible. You guys have made it super convenient for me to plan experiments.

Thank you to Isbelle Harvey and Annamaria Kiss for your administrative assistance.

Thank you to my housemates and friends whom I have made throughout the 2 years at McGill. Life at McGill wouldn’t have been as good without you guys. Table of Contents

Abstract…………………………………………………………………………………………...2 Résumé……………………………………………………………………………………………3 Acknowledgement…………………………………………………………………………….….4 Table of Contents………………………………………………………………………………...5 Introduction………………………………………………………………………………………6 Overview of Translation……………………………………………………………………….6 Mechanism of Translation Initiation…………………………………………………………...8 Regulation of Translation Initiation ………………………………………………………….11 The eIF4E Binding Proteins (4E-BPs) and pathway…………………………………………12 mTOR………………………………………………………………………………………...15 Translational Control in Disease……………………………………………………………..16 Translational Control in Learning and Memory……………………………………………...19 Deamidation…………………………………………………………………………………..21 Results…………………………………………………………………………………………...31 Deamidation of 4E-BP2 is spontaneous……………………………………………………...31 Deamidated 4E-BP2 is also observed in skeletal muscles……………………………………32 Developmental reduction of Akt-mTOR pathway is only observed in the brain where deamidated 4E-BP2 is observed but not in the liver………………………………………….35 Single and double deamidated 4E-BP2 is less stable compared to wild type 4E-BP2 4E-BP2 is degraded through the proteasomal pathway………………………………………36 In vivo accumulation of deamidated 4E-BP2 in skeletal muscles with INK128 injection…...38 Model of tissue specific deamidation of 4E-BP2………………………………….…………40 Tissue specific turn over rate of 4E-BP2……………………………………………………..43 Discussion and Future Directions……………………………………………………………...44 Materials and Methods…………………………………………………………………………47 Supplementary Figures………………………………………………………………………...52 References……………………………………………………………………………………….54

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Introduction

1. Overview of Translation

The translation process involves three major steps: initiation, elongation, and termination

(Figure 1). In the first step, the mRNA and the initiator methionyl tRNA are recruited to the 40S ribosomal subunit in such a way that AUG on the mRNA is located at the peptidyl site and is base-paired to the methionine carrying initiator tRNA. The initiation complex is completely formed when the 60S ribosomal is recruited. In most of the cases, translation initiation is the rate limiting step. This early control checkpoint can prevent wasting of energy instead of arrest at later stages. Since the focus of my thesis involves the very first step, initiation will be discussed more in depth below.

After the formation of the initiation complex, translation proceeds by elongating the polypeptide chain. The ribosome has three sites that can be occupied by tRNA: P (peptidyl), A (aminoacyl), and E (exit). The first step of elongation is the escort of the second tRNA to the A site by a GTP- conjugated elongation factor. At the A site, the second tRNA can base pair to the second mRNA codon. The large 60S ribosomal subunit binds the aa-tRNA acceptor arm and catalyzes peptide bond formation by transferring the peptide from the P-site tRNA to the A site tRNA. The uncharged tRNA is transferred to the E site, where it will be expelled from the ribosome. The

6 translocation of mRNA and release of free tRNA allows for entry of a new amino acid-tRNA

(Jackson, Hellen, & Pestova, 2010).

Termination of translation takes place once a stop codon is recognized at the A site. Instead of tRNA, stop codon is conjugated to release factors, which allow for the dissociation of completely synthesized peptide from ribosome. The ribosome is then broken down into subunits and is

Figure 1: Pathway of mRNA translation. The simplified cartoon depicts the basic steps involved translation of mRNA including initiation, elongation, termination, and ribosomal recycling. Not shown in this picture is the specific factors involved in protein synthesis. Termination of protein synthesis occurs when a termination codon is recognized in the A-site. Hydrolysis of the peptidyl-tRNA occurs in the P-site. Picture is adapted from (Hershey, Sonenberg, & Mathews, 2012) 7 recycled for new translation(Cooper, 2000).

2. Mechanisms of Translation Initiation

Translation initiation is responsible for positioning the initiation codon at the P site of the fully assembled 80S ribosome with the help of multiple initiation factors. The very first step of translation initiation is the formation of the 43S Preinitiation Complex (PIC) (Dever, 2002). The

43S PIC is formed when eIFs 1, 1A, 3 and 5 assemble with the 40S subunit and the eIF2 ternary complex. Formation of the ternary complex is dependent on the loading of guanosine triphosphate (GTP) onto eIF2 since GTP-carrying eIF2 has much higher affinity for the initiator

Met tRNA, Met-tRNAi , compared to the GDP-loaded one (Asano, Clayton, Shalev, & Hinnebusch,

2000). The loading of GTP onto eIF2 is modulated by eIF2B, a guanine nucleotide exchange factor (GEF), which replaces GDP with GTP. eIF2 possesses 3 subunits: α, β, and γ. The loading of GTP at the γ subunit can be prevented by the phosphorylation of the α subunit, providing a mean of translational control (Maitra, 1999). After formation of the ternary complex, eIFs 1, 1A, and 3 promote the recruit of the complex to the 40S ribosome, forming the 43S PIC (Kolupaeva,

Unbehaun, Lomakin, Hellen, & Pestova, 2005).

eIFI4F complex mediates the association of the 43S PIC to the mRNA with the helps of its subunits: eIF4E, eIF4G, and eIF4A. eIF4A is an ATP-dependent helicase. eIF4E binds selectively to the mRNA 5’ cap while eIF4G bridges the mRNA to the incoming 43S PIC. eIF4G also interacts with the 13-subunit-eIF3, connecting the 43S PIC to the mRNA 5’ end(Altmann,

Schmitz, Berset, & Trachsel, 1997; Mader, Lee, Pause, & Sonenberg, 1995).

8 Once docked onto the mRNA, the 43S complex migrates through the mRNA 5’ end and scans for the start codon. The ATP-dependent eIF4A helicase unwinds the secondary structure in the 5’ end to allows for scanning (Jackson, 1991; Svitkin et al., 2001). eIF1 and eIF1A promote scanning of the 43S complex and ensure the correct recognition of the start codon by favoring the open conformation of the 40S binding cleft until the initiation codon is within the P site

Met (Maitra, 1999). Once the start codon base pairs to the anti-codon of Met-tRNAi , the 48S complex is established, promoting the dissociation of eIF1 and hydrolysis of eIF2-GTP. The 60S ribosome subunit is then recruited to the 48S complex. GDP-bound eIF2 has weak affinity for

Met Met-tRNAi , thus is released (Myasnikov et al., 2005). However, eIFs 1, 1A, and 3 still occupy the binding surfaces of the 40S subunit, preventing the joining of the 60S ribosome subunit. GTP associated eIF5B interacts with eIF1A, together with the 60S subunit remove eIF1 and 3(Acker & Lorsch, 2008; Guillon, Schmitt, Blanquet, & Mechulam, 2005). This step completes the formation of the 80S complex. eIF5B is hydrolyzed and released along with eIF1A.

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10 Figure 2: Mechanisms of translation initiation The picture depicts detailed steps involved in translation initiation. First eukaryotic Met initiation factor 2 (eEF2)-GTP-Met-tRNA -I ternary complex forms. 43S preinitiation complex (PIC) establishes, comprising a 40S subunit, eIF1, eIF1A, eIF3, eIF2-GTP-Met- Met tRNA i. mRNA attachs to the 43S PIC. 5’ UTR of mRNA is scanned from 5’ to 3’ for the initiation codon. Initiation codon recognition leads to the formation of the 48S complex, hydrolysis of eIF2-bound GTP and. Joining of the 60S ribosomal subunit to the 48S complex allows the dissociation of eIF2-GDP and other factors (eIF1, eIF3, eIF4B, eIF4F, eIF5). Formation of the 80S complex leads to GTP hydrolysis by eIF5B, release of eIF1A, GDP bound eIF5B. Picture is adapted from (Jackson et al., 2010)

3. Regulation of Translation Initiation

One key mechanism of translational control during stress is the phosphorylation of eIF2 on Ser51 of its α subunit which then competes against eIF2B, thus decreasing formation of the ternary complex. Up till now, four kinases have been documented to phosphorylate eIF2α under different conditions: PKR – viral infection, PERK – ER stress, HRI – heme deprivation, GCN2 – amino acid starvation (Dever, 2002). Second way to control rate of translation initiation is by preventing the binding of eIF4F to the cap structure of the mRNA by an eIF4E homolog, 4E-HP.

Interaction between eIF4G and eIF4E in the eIF4F complex is also inhibited by eIF4E-binding proteins (4E-BPs). 4E-BPs compete with eIF4G for eIF4E and consequently inhibits cap- dependent translation (Gingras, Raught, & Sonenberg, 1999).

MicroRNAs (miRNAs) are short (~22 nt) oligonucleotides, which are estimated to control approximately half of the human genome. There are approximately 1000 miRNAs; each of which can control up to 10 mRNAs. After processing from its primary transcript precursor, miRNA can form an RNA-induced silencing complex (RISC) with the protein complex. RISC effectively targets and inhibits translation of specific mRNAs. RISC either inhibits translation or destabilizes and leads to the breakdown of mRNAs. Currently, it is still unclear whether

11 translation initiation inhibition by this mechanism is a cap-dependent process or not. It was shown that Argonaute (Ago), a component of RISC, binds directly to the 5’ cap structure and thereby prevents binding of mRNAs with eIF4E. However, more recent results challenged this finding. Further, two other RISC-associated proteins: FXR2 and Ago 2, were shown stimulate translation of certain mRNAs when cells are under quiescent state(Chekulaeva & Filipowicz,

2009).

4. The eIF4E-Binding Proteins (4E-BPs) and mTOR pathway

Mammalian eIF4E binding proteins (4E-BPs) is a family of translation inhibiting proteins consisting of 3 mammalian paralogs. 4E-BP1 and 4E-BP2 were first identified (T. Lin et al.,

1994; Pause et al., 1994). 4E-BP3 was later identified due to structural similarity to the previous two proteins (Poulin, Gingras, Olsen, Chevalier, & Sonenberg, 1998). These proteins have been demonstrated to inhibit translation in vitro or in vivo by overexpression in cells. Their potency is based on their efficacy to bind to eIF4E and is regulated by phosphorylation (Altmann et al.,

1997; Gingras, Gygi, et al., 1999; Mader et al., 1995). The middle portion of the proteins that contains eIF4E binding sequence and regulatory phosphorylation sites are conserved within the protein family (Poulin et al., 1998).

4E-BPs compete with eIF4G by binding to a common eIF4E recognition motif (Altmann et al.,

1997; Mader et al., 1995). Structural analyses indicated that the binding site on eIF4E is located on the dorsal convex surface, opposite the concave mRNA 5’ cap binding site (Marcotrigiano,

Gingras, Sonenberg, & Burley, 1997). 4E-BP is a small polypeptide spanning between 100 and

120 amino acids. It is an inherently unstructured protein. Upon binding to eI4FE, the

12 conformation of the protein stabilize and forms an alpha helix loop structure. Due to being an inherently unstructured protein, 4E-BPs demonstrate remarkable stability to heat and acid. In fact, when the protein was first discovered, their original nomenclature is phosphorylated-heated- stable-acid-stable (PHAS)(T. Lin et al., 1994).

4E-BPs expression varies from tissues to tissues. 4E-BP1 is the most dominant form in the most tissues and is the most extensively studied protein within the family. Specifically, 4E-BP1 has been reported to be most abundant in adipose tissue and muscle while 4E-BP2 is the most prominent in the brain (Ohara et al., 2001). Their physiological importance has been demonstrated with knock out (KO) mouse models. 4E-BP1 KO or both (4E-BP1 and 4E-BP2) double KO mice display impaired metabolic function and fat regulation (Bacquer et al., 2007).

Double KO mice are also resistant to certain forms of viral infection due to translational upregulation of proteins involved in the type I interferon pathway (Colina et al., 2008).

Intriguingly, deletion of 4E-BP homologue in Drosophila, Thor, renders flies more susceptible to bacterial infection (Bernal & Kimbrell, 2000). Hyperactive eIF4E harbors oncogenic activity, suggesting that 4E-BPs can play a tumor suppressive role. In fact, overexpression of 4E-BPs can partially reverse the transformation of eIF4E overexpressed cells (Polunovsky et al., 2000).

The response of protein synthesis machineries to external stimuli is at least partially governed by the phosphorylation of 4E-BPs. 4E-BP1 was actually shown to be one of the most phosphorylated proteins following insulin stimulation of rat adipocytes (Pause et al., 1994).

Hyperphosphorylation of 4E-BPs promotes their dissociation from eIF4E, allowing the joining of eIF4G, and eventually the formation of eIF4F. 4E-BPs have 4 phosphorylation sites which are

13 conserved within the family. Considerable work has delineated the inducible, hierarchical mechanisms of phosphorylation of 4E-BPs. At the N-terminal to the eIF4E, Thr37/46 seems to be the most readily phosphorylated residues even in serum starving conditions. Phosphorylation of Thr37/46 primes the phosphorylation of Ser65, followed by Thr70, and the dissociation from eIF4E. Phosphorylation of all 4 sites is critical for detachment from eIF4E since phosphorylation at Ser65 and Thr70 alone or together cannot abolish the interaction (Gingras et al., 2001).

Mitogen-activated protein (MAP) kinase was initially thought to be responsible for phosphorylating 4E-BPs due to its role with PHAS (T. Lin et al., 1994). However, failure of

MAPK inhibitor to prevent phosphorylation of 4E-BPs suggests otherwise (T.-A. Lin et al.,

1995). The mammalian target of rapamycin (mTOR) has now been backed up with concrete evidences to be the in vivo kinase of 4E-BPs. mTOR preferentially phosphorylates Thr37/46 in vitro but seems to affect the serum-responsive stress phospho-sites Ser65/Thr70 (Beretta,

Gingras, Svitkin, Hall, & Sonenberg, 1996). Hence rapamycin treatment mimics growth factor deprivation by allowing the phosphorylation of Thr37/46 but not Ser65 and Thr70 (Manteuffel,

Gingrast, Ming, Sonenbergt, & Thomas, 1996)(Gingras et al., 2001). It is still unclear why this is the case. However, there is hypothesis that mTOR promotes hyperphosphorylation of 4E-BPs by inhibiting 4E-BP phosphatase (Peterson, Beal, Comb, & Schreiber, 2000).

The 4E-BPs contain two regulatory domains essential for proper phosphorylation. The TOR signaling motif is located at the C terminal and mediates the interaction between 4E-BPs and raptor (Schalm & Blenis, 2002). Raptor is an important mTOR-associated protein necessary for rapamycin-sensitive outputs. Mutation at this sequence prevents the dissociation of 4E-BP from

14 eIF4E since they cannot be phosphorylated at Ser65 and Thr70 (Hidayat et al., 2003; Schalm,

Fingar, Sabatini, & Blenis, 2003). The other regulatory sequence is located at the N terminal, the

RAIP motif owing its name to its amino acid sequence. It is required for phosphorylation of

Thr37 and 46. 4E-BP3 which possess CPIP instead of RAIP motif has been shown to have impaired insulin-induced phosphorylation and reduced dissociation from eIF4E (Tee & Proud,

2002).

5. mTOR

Mammalian target of rapamycin (mTOR) is a key pathway that is responsible for responding to environmental cues by phosphorylating several key players involved in translation (Ma & Blenis,

2009). mTOR is a conserved Ser/Thr kinase that belongs to the PIKK family (Jacinto & Hall,

2003). There are two mTOR complexes: mTOR complex 1 (mTORC1) and mTOR complex 2

(mTORC 2). mTORC1 consists of mTOR in complex with raptor, and LST8. mTORC2 contains mTOR, rictor, LST8, and SIN1. Due to relevance of mTORC1 to this thesis, only mTORC1 will be discussed from this point on. The specificity of mTORC1 depends almost entirely on raptor, which interacts with substrates containing TOR interacting motif, TOS (Schalm & Blenis, 2002).

Major downstream targets of mTORC1 are components of the translation machinery. Hence mTORC1 has been shown to directly regulate protein synthesis in mammals. The first major target of mTORC1 is 4E-BPs (Schalm et al., 2003). Hyperphosphorylated 4E-BPs lose affinity for eIF4E, leading to the dissociation from eIF4E allowing for the binding of eIF4G and formation of eIF4F complex. The other major target of mTORC1 is the S6 Kinases (S6K1 and

S6K2)(Guertin et al., 2006). Phosphorylation of S6K by mTORC1 can phosphorylate S6 ribosomal protein, which correlates with increased protein synthesis. Recently, a TOS-containing

15 motif, PRAS40, was identified to be the inhibitor of mTORC1. Overexpression of PRAS40 in vitro abolishes mTORC1-mediated phosphorylation of 4E-BPs and S6Ks. Depletion of PRAS40 by siRNA induces the opposite effect where phosphorylation of the aforementioned substrates is enhanced. Activity of PRAS40 is regulated by phosphorylation and association by 14-3-3

(Fonseca, Smith, Lee, Mackintosh, & Proud, 2007).

6. Translational Control in Disease

Dysregulation in translation control has been implicated in many human diseases including certain cancers and metabolic disorders. Most types of cancer are associated with aberrant signaling pathways involved in cell growth and proliferation. Increased cancer cell proliferation requires increased global protein synthesis. Hence, it is expected that translational control plays an important role in cancer. Increase in expression and activities of initiation factors, translation regulatory factors, tRNAs have been observed in cancer formation and progression (Silvera,

Formenti, & Schneider, 2010).

eIF2α can prevent or stimulate cancer development depending on the context. Overexpression of a dominant interfering form of PKR inhibits eIF2α phosphorylation and promotes tumorigenesis in mice (Donze, Jagus, Koromilas, Hershey, & Sonenberg, 1995; Koromilas, Roy, Barber, Katze,

& Sonenbergt, 1992). There are evidences that suggest increased PKR expression reduces global protein synthesis, which is required for tumor cell differentiation and proliferation. In addition, increased eIF2α level is correlated with the severity and aggressiveness of brain tumor due to the greater capacity to escape inactivation by phosphorylation (Tejada et al., 2009).

16 Mammalian eIF3 is composed of 10-13 proteins that are involved in bridging interaction between the 43S pre-initiation complex and eIF4F bound to mRNA. Overexpressions of multiple eIF3 subunits have been implicated in many types of cancer (Table 1) (Silvera et al., 2010).

Levels of eIF3α and eIF3C have been upregulated in many cancers (Dong & Zhang, 2003,

2006). Increased expression of eIF3H, which is located adjacent to the MYC proto-oncogene on chromosome region 8q, is increased in breast and prostate cancers (L. Zhang, Pan, & Hershey,

2007). On the other hand, loss of expression of eIF3 subunits also plays a role in cancer. For instance, the tumor suppressor eIF3F is downregulated in several human cancers such as melanomas. Decreased eIF3E expression is observed in some breast and lung carcinomas, while overexpression of the same subunit inhibits growth of cancer cells and increases apoptosis

(Tejada et al., 2009).

The best characterized role for translation factors in cancer development is the eIF4F complex, which consists of eIF4E, the eIF4A helicase, and RNA binding eIF4B. eIF4E is regulated by its availability but its activity is controlled by 4E-BPs and phosphorylation of Serine 209 by MNK1 and MNK2 (Sonenberg & Hinnebusch, 2009; Waskiewicz et al., 1999). In many transgenic mouse models, overexpression of eIF4E has been shown to promote B-cell lymphomas, angiocarcinomas, hepatocellular carcinomas, and lung adenocarcinomas. Increased level of eIF4E and thus the eIF4F complex preferentially augments translation of a subset of mRNA with extensive secondary structure at the 5’ UTR (Koromilas & Sonenberg, 1992; Petroulakis et al.,

2007). These mRNA usually encodes for proteins that are involved in cell cycle progression

(MYC, CCND1, and ODC1), angiogenesis (VEGFA and FGF2) (Rosenwald, Rhoads, Callanan,

Isselbacher, & Schmidt, 1993), and cell growth and survival (MIF) (Petroulakis et al., 2007).

17 Overexpression of eIF4E has also been observed to selective recruits to the ribosomes mRNAs encoding cancer-promoting genes (Larsson et al., 2007).

18 Table 1: Translation factor and translation regulatory factor alternations in human cancer (Adopted from (Silvera et al., 2010))

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Obesity in human and mice leads to insulin resistance, which prompts pancreatic β cells to secrete more pro-insulin. The upregulation in turn promotes glucose-dependent translation of insulin and other secreted proteins (Scheuner et al., 2005). Accumulation of such proteins in β cells can ultimately lead to the failure of the pancreas, thus is hypothesized to contribute to the onset of diabetes mellitus (Scheuner et al., 2001; Skelly, Schuppin, Hisamitsu, Oka, & Rhodes,

1996). PERK-mediated eIF2α phosphorylation was shown to control the level of secreted unfolded proteins within the pancreas, and has a protective role against ER stress. Mice with heterozygous eIF2αS51A/+ are more glucose-tolerant than wild type mice, which is in part due to compromised insulin secretion suggesting impaired functioning in the pancreas β cells (Scheuner et al., 2005). In addition, these mice demonstrated other non β cell related metabolic disorders such as increased body weight, and hyperlipidemia. Mice homozygous for the eIF2αS51A/S51A are embryonic lethal due to their inability to maintain blood glucose level, leading to the the disruption of the feto-maternal circulation (Scheuner et al., 2001). These mice also demonstrate a profound impairment in pancreatic islet development due to defective glucagon production.

These evidences suggest a pivotal role for eIF2α, and translation initiation control, in metabolic disorders.

7. Translation Control in Learning and Memory

In addition to cancer and obesity, recent discovery has linked translational control to learning and memory. Alterations in synaptic strengths of neurons have been described to account for learning and memory formation. Importantly, novel protein synthesis is responsible for long- lasting forms of synaptic plasticity and memory (Costa-mattioli, Sossin, Klann, & Sonenberg,

20 2009). Translation control, in addition to governing general protein synthesis, is important in synthesis of specific proteins in response to neuronal activity. Long term potentiation (LTP) is a cellular model that aims to explain learning and memory in terms of changes in synaptic strengths in different groups of synapses in response to different stimuli. It was previously shown that the dendrites and synapses of neurons contain ribosomes, mRNAs, and components of the translation machinery. At those locations, two pathways: PI3K/Akt/mTOR and MAPK/ERK regulate local translation; hence, are important for protein synthesis dependent LTP, memory, and learning (Buffington, Huang, & Costa-mattioli, 2016).

Due to the interconnectedness of synaptic plasticity, learning, memory and translation, mouse models associated with translation deficiency have been utilized to investigate these processes.

Mice lacking 4E-BP2, which is the major isoform of 4E-BPs in the brain, require less stimulation for developing lasting LTP but exhibit impaired spatial learning, and long-term contextual fear conditioning (Banko et al., 2005, 2007). These observations suggest that excessive translation can be deleterious to synaptic plasticity and damaging to learning and memory. This notion is further supported by mice lacking GCN2, a kinase of eIF2α. GCN2-/- mice demonstrated altered synaptic plasticity, enhanced memory formation with weak training protocol (Costa-mattioli et al., 2006). They require less stimuli for activation of lasting LTP. eIF2αser51ala/+ heterozygote

“knockin” mice exhibit long-lasting LTP (Costa-mattioli et al., 2007). The molecular mechanism underlying these behaviors is reduction in GCN2-mediated eIF2α phosphorylation results in a decrease in translation of ATF4 in the brain. ATF4 inhibits cyclic AMP response element binding protein (CREB)-mediated gene expression, which is crucial for long term synaptic plasticity and memory.

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Further, 4E-BP2 KO mice demonstrate increased translation of neuroligin 2, a postsynaptic protein that has been linked to autistic spectrum disorders (ASDs). These mice have increased excitatory to inhibitory synaptic inputs (Gkogkas et al., 2012). Physical changes at the synapses are suggested to be responsible for the observed autistic-like behaviors such as social interaction deficits, altered communication, and repetitive/stereotyped behaviors.

8. Deamidation

Nonenzymatic deamidation is thought to be one of the most common post-translational modifications. However, its roles are still poorly defined. Deamidation is the process where either an asparagine or glutamine is converted to asparatate or glutamate respectively. Due to the focus of my thesis, from this point on, only asparagine deamidation will be discussed. The initial step of deamidation involves a nucleophilic attack of the peptide bond of the α amino group on the asparagine side chain carbonyl group, forming an intermediate cyclic succinimide. Water molecules can attack the succinimide intermediates giving rise to aspartyl and iso-asparatyl residues in the ratio of 1:3 (Figure 3). Since asparagine deamidation is spontaneous, the only requirements for the chemical reaction to occur are the presence of a water molecule and negligent steric hindrance. Formation of the succinimide intermediate is the rate limiting step.

Hence, asparagine residue which is preceded by a glycine or serine or histidine is more susceptible to deamidation due to their relatively small side chains(N E Robinson & Robinson,

2001; Noah E Robinson & Robinson, 2000). Nonenzymatic glutamine deamidation also occurs; however, to a much lesser extent. Modified amino acid harbors an extra negative charge.

22

Figure 3: Formation of asparatate and isoasparatate. Deamidation of asparagine occurs by the nucleophilic attack of the α amino group of the C terminal amino acid, which results in the succinimide intermediate. The cyclic imide can be hydrolyzed to give iso-Asp or Asp in a 3:1 ratio. Adapted from (Reissner & Aswad, 2003)

Deamidation of asparagine results in asparatyl and isoaspartyl. Accumulation of isoasparatyl residues are often associated with protein damage. Protein L-isoaspartyl O-methyltransferase

(PIMT) can methylate iso-Asp residues and target an isoAsp-containing protein for repair or degradation. The methyl group is added to the free alpha-carboxyl group by PIMT using

23 AdoMet as a substrate (Zhu, Doyle, Mamula, & Aswad, 2006). Enzymatic methylation followed by spontaneous ester hydrolysis results in the formation of succinimide. The cyclic imide can hydrolyze again to form IsoAsp or Asp, allowing IsoAsp a chance to convert to Asp. The role of

PIMT as a protein repair has been proven both in vitro and in vivo. In vitro protein aging is carried out by incubation of proteins at physiological pH and 37C (Bidinosti, Martineau, Frank,

& Sonenberg, 2010). This process yields significant isoAsp as seen in calmodulin, tissue plasminogen activator, tubulin, human growth hormone, angiogenin, and serine hydroxymetthyltransferase. PIMT is used to repair damaged calmodulin/calmodulin-dependent protein kinase and is able to recover its activity from 18% to 68% (Johnson, Langmack, &

Aswad, 1987). Repair of deamidated HPr phosphocarrier protein also partially recovers its function (Andersonn, Jian, & Waygoodn, 1994). In Drosophila, transgenic overexpression of

PIMT leads to a 3-7 fold increase in PIMT and increased life span (Chavous, Jackson, & Connor,

2001). PIMT KO mice show accumulation of isoAsp in various tissues with the highest accumulation in the brain. These mice demonstrate phenotypes including retarded growth and fatal epileptic seizers at 4-6 weeks of age. Interestingly, when PIMT transgene is inserted into a neuron specific enolase promoter of the KO mice, the PIMT activity is partially restored (6.5-

13% compared to wild type). They are of similar size to wild type and lived 5 times longer than the KO mice (Kim, Lowenson, MacLaren, Clarke, & Young, 1997; Yamamoto et al., 1998).

Deamidation was thought to be an artifact. In 1967, Flatmark demonstrated the existence of in vivo deamidation in cytochrome c. The different physiochemical properties of different deamidated forms of cytochrome c prove that the modification is not a purification artifact.

Subsequently, deamidation is a signal for the degradation of cytochrome c (Flatmark & Sletten,

24 1968). Since their discovery, deamidation was generally viewed as unintended and undesirable form of protein aging or damage. It is now known that deamidation widely occurs in vivo and it affects the stability and activity of the affected proteins (Flatmark & Sletten, 1968). Up until now, Bcl-XL is the only protein whose deamidation has been shown to have cellular significance.

Bcl-XL is an anti-apoptotic protein which functions to inhibit programmed cell death. It was shown that under DNA-damage inducing reagent, two asparagines (N52 and N66) undergo deamidation (Deverman et al., 2002). Deamidated Bcl-XL has less potent anti-apoptotic activity, thus cells with deamidated Bcl-XL is more susceptible to apoptosis. Intriguingly, the sequence of

Bcl-XL that is deamidated is conserved in all upper vertebrate forms. If deamidation of Bcl-XL is detrimental to its overall function, one would expect that it would be selected against throughout evolution. In fact, the 60 amino acids surrounding the asparagines are dispensable for the antiapoptotic activity of Bcl-XL. Hence, deamidation of Bcl-XL is an intended post-translational modification that governs the activity of the enzyme. Regulation of Bcl-XL activity by deamidation is further supported by the fact that deamidation is suppressed in normal cells that are resistant to apoptosis. This resistance is reliant on deamidation since these cells become susceptible when Bcl-XL demidation is mimicked by other mechanisms that inactivates the enzyme (Deverman et al., 2002; R. Zhao et al., 2007). Recently, it was shown that an important class of oncoproteins suppresses Bcl-XL deamidation, which renders the cells resistant to antineoplastic agent induced apoptosis (R. Zhao et al., 2008). This suggests that asparagine deamidation is the critical checkpoint of the apoptotic process in some cancers. In addition, deamidation of Bcl-XL is a process that occurs over a period of several days. The length of this process allows cells to repair minor DNA damage without undergoing apoptosis. Bcl-XL is an important proof that deamidation can serve important regulatory and biological roles in cells.

25

Since Bcl-XL is the only example of regulated deamidation that has a specific cellular significance, it will be interesting to explore if there is any other candidate. In 2010, Bidinosti et al. reported the brain specific deamidation of 4E-BP2. Originally, tissues lysates from brains, testes, and liver were probed for 4E-BP2 expression where brain is the only tissue that exhibits a distinctive three band pattern (Figure 4A). The slower migrating bands do not resemble phosphorylated 4E-BP2 since λ phosphatase treatment of the brain lysate did not abolish this pattern (Figure 4B). To identify which post-translational modification is responsible for this slow migration, 50 mouse brain were homogenized and immunoprecipitated. Mass spectrometry analysis of purified, endogenous 4E-BP2 revealed that deamidation occurs in the asparagine rich sequence between amino acid 87-104 (Figure 5). If an amino acid is susceptible to deamidation, alkaline treatment of that polypeptide will fasten the process of deamidation. For more extensive analysis of the site of deamidation, recombinant wild type 4E-BP2 was subjected to alkaline treatment for 18 hours and the sample was run on a gel (Figure 6A). The bands visualized with dye corresponding the different forms of 4E-BP2 were excised and analyzed with MS/MS. The occurrence frequency of the immediate and slowest migrating forms is most prominent at position N99 and N102 (Figure 6B). N99A and N102A mutated 4E-BP2 did not undergo transformation seen in wild type 4E-BP2 with alkaline treatment (Figure 6C). N99D, N102D, and N99DN102D mutations resulted in the immediate and slowest migrating bands. From this analysis, it was reported that in the brain, 4E-BP2 harbors 2 other forms: single deamidated

N99D or N102D and double deamidated N99DN102D on top of the wild type protein (Figure

6D).

26 Interestingly, deamidation of 4E-BP2 occurs with age as observed in mice and cultured hippocampal neurons. There is a developmental reduction of Akt-mTOR pathway as observed in the brain and cultured neurons signified by phospho-4E-BP1 (Thr37/46), phospho-rpS6

(Ser240/244), and p-Akt (T308) (Figure 7). Co-immunoprecipitation of recombinant wild type and deamidated 4E-BP2 with eIF4E and Raptor indicated that deamidated 4E-BP2 is associated more strongly to Raptor and less efficiently to eIF4E (Figure 8). Developmental reduction of

Akt-mTOR leads to stronger association of 4E-BP2 and eIF4E thus stronger inhibition of translation initiation. Deamidated 4E-BP2 binds less strongly to eIF4E is thus thought to be a compensatory mechanism for this decrease in mTOR kinase activity.

Figure 4: Brain specific pattern of 4E-BP2 is due to deamidation A. There are three bands of 4E-BP2 in the brain but not liver or testes. There is no phospho- 4E-BP2 (Thr37/46) signal in the brain but still is observed in the other two tissues. B. λ phosphatase treatment did not abolish the three band pattern. Adapted from (Bidinosti, Ran, et al., 2010)

27

Figure 5: Mass spectrometry identifies the region of deamidation within 4E-BP2 Brain from 50 wild 2-3 week old mice were immunoprecipitated for 4E-BP2 and analysed with mass spectrometry. Region 87-104 was identified as the region containing deamidated asparagine. Adapted from (Bidinosti, Ran, et al., 2010)

28

A

B

C D

Figure 6: 4E-BP2 is deamidated at position N99 and N102. A. Recombinant 4E-BP2 is subjected to incubation for 18 hours at two different pH 7.0 and 10. Recombinant 4E-BP2 that was subjected to pH 10 after 18 hours showed a shift to slower migrating bands, which was not observed at pH 7.0. B. Recombinant 4E-BP2 that was alkaline treated was run on a gel and analyzed with MS/MS. C. 4E-BP2 was mutated at position N99 and N102 to alanine and underwent alkaline treatment. D. Position N99 and N102 were mutated to asparatatic acid (D). N99A/N102A and N96D did not show shift in migration. Adapted from Bidinosti, Ran, et al., 2010

29

B

Figure 7: Developmental pattern of 4E-BP2 A. Brain from mice at different ages show a developmental emergence of deamidated 4E- BP2 and reduction in phospho-4E-BP2 (Thr37/46). B. Developmental pattern of 4E-BP2 in hippocampus cultured neurons, resembling that of the brain. Adapted from (Bidinosti, Ran, et al., 2010)

Figure 8: Deamidated 4E-BP2 is bound less strongly to eIF4E and more strongly to raptor A. WT 4E-BP2, 4E-BP2 without TOS motif that is required to bind raptor, double deamidated 4E-BP2, and alanine mutated 4E-BP2 were co-immunoprecipitated with raptor. Levels of 4E-BP2 detected indicate the strength of the interaction between 4E-BP2 and raptor. B. Wild type 4E-BP2, double deamidated 4E-BP2 and 4E-BP2 without TOS motif were immunoprecipitated with cap (m7GDP) conjugated eIF4E. Signals of 4E-BP2 indicate the strength of the interaction between eIF4E and 4E-BP2. Adapted from (Bidinosti, Ran, et al., 2010)

30

The amino acid sequence of 4E-BP2 that undergoes deamidation is conserved in mouse, cow, dog, chimpanzee, and human (Figure 9A). However, while there are 3 isoforms in the 4E-BP family, the deamidation prone sequence is specific to 4E-BP2 (Figure 9B). Further, 4E-BP2 is only deamidated in the brain, where it is the major isoform. Up to date, histone H10 is the only example whether the ratio of deamidated to amidated protein varies in different tissues (For et al., 1998). Combined with the aforementioned details, the facts that deamidation of the major 4E-

BP2 isoform in the brain, 4E-BP2, is tissue specific and developmentally regulated make it an interesting candidate to study deamidation in general. By understanding the mechanistic regulation of tissue-specific deamidation of 4E-BP2, more insight can be gained into the

A

B

Figure 9: 4E-BP2 sequences in difference species and compared to 4E-BP1 A. 4E-BP2 sequence is conserved till mouse especially the deamidation sequence. B. Comparison between mouse 4E-BP2 and 4E-BP1. Deamidation sequence is not conserved within the family. Adapted from (Bidinosti, Ran, et al., 2010)

31 regulation of deamidation and in the process, the cellular significance of deamidated 4E-BP2 may be unveiled.

Results

1. Deamidation of 4E-BP2 is spontaneous

All asparagine deamidation up to date is spontaneous. To confirm whether 4E-BP2 follows the same trend, in vitro aging was carried out for either control (MEF lysates alone) or experimental

(MEF lysates combined with 4E-BP2 KO brain lysate) groups. The idea is that if deamidation of

4E-BP2 is carried out by a specific enzyme in the brain, in vitro aging of the experimental group would result in the accumulation of 4E-BP2 in a much faster fashion as compared to control.

During in vitro aging, lysates were extracted with Tris-HCl pH 7.0 buffer and incubated at 37C over different time points. Short term in vitro aging indicated no significant difference between the groups (Figure 10A). Long term in vitro aging indicated the emergence of the 3 bands patterns resembling deamidated 4E-BP2 at 12hr (Figure 10B). However, this was observed in both experimental and control group, suggesting that deamidation of 4E-BP2 is spontaneous.

This result is also supported by the fact that in vitro aging of recombinant 4E-BP2 resulted in the

3 band pattern over 2-day incubation (Bidinosti, Martineau, et al., 2010).

32 MEF lysate WT Brain WT BrainDKO brainMEF lysate ADKO brain B 30m 1 hr 2 hr 6 hr 1d 2d 3d 5m 12hr

MEF + DKO brain lysate MEF only

GAPDH GAPDH

MEF only MEF + DKO brain lysate

GAPDH GAPDH

Figure 10: Deamidation of 4E-BP2 from mouse embryonic fibroblast is spontaneous. In vitro aging of MEF lysate was done at 37C and pH 7.0. There are two groups: MEF and DKO brain lysates or MEF lysate alone. (A) Short term incubation ranging from 5 minutes to 6 hours. (B) Long term incubation ranging from 12 hours to 3 days.

2. Deamidated 4E-BP2 is also observed in skeletal muscles

Several tissues were isolated from adult wild type mice and probed for 4E-BP2. The distinct 3 band pattern of 4E-BP2 was also observed in the skeletal muscles, similar to the one seen in different brain regions such as cortex, hippocampus, cerebellum, trigeminal (Figure 11A).

However, in other tissues/organs such as liver, heart, colon, spleen, lung, pancreas, and testes,

4E-BP2 harbors a single band corresponding to amidated protein. Treatment with protein λ phosphatase effectively abolish the signal of phospho-rpS6 (Ser240/244) but retains the 3 band pattern (Figure 11B). All phospho-4E-BP2 was stained with anti-phospho-4E-BP1 (Thr37/46) because the phosphorylation sites are conserved as mentioned above. Phospho-4E-BP2 was not shown since it is known that in adult brain, the level of phosphorylation of 4E-BPs are very weak and thus is not a very good positive control for the effectiveness of the phosphatase treatment. In addition, to make sure that deamidated 4E-BP2 is solely from skeletal muscle cells and not

33 contaminated by motor neurons, several markers of neurons were probed for such as PSD95, synaptophysin (Figure 11C), and MAP2 (Figure 11D). PSD95 is a post-synaptic protein.

Synaptophysin stains for pre-synaptic vesicles, and MAP2 stains for microtubules of neurons. In all cases, muscle samples are clear from neuronal contamination providing strong evidences that

A HippocampusCerebellum Pancreas TrigeminalHeart Spleen Testes Liver Cortex MuscleColon Lung s

4E-BP2

B C cerebellummusclecerebellum muscle cerebellum muscle - + - + λ λ λ λ P-rpS6 Synaptophysin

Total PSD95 rpS6 GAPDH GAPDH

4E-BP2 Figure 11: 4E-BP2 in different tissues. 4E- BP2 is deamidated in skeletal muscles. (A) Various tissues were isolated from 6-

D week old mice and probed for 4E-BP2. cerebellum cerebellum Skeletal muscles demonstrated the same 3 muscle muscle band pattern as seen in different parts of the brain. (B) Cerebellum and skeletal muscles lysates were treated with λ phosphatase for 1 MAP2 hour at 37C. P-rps6 (Ser240/244) was probed to indicate the efficiency of the phosphatase treatment. (C)&(D) Skeletal GAPDH muscle lysates were not contaminated with motor neurons as indicated by neuronal markers synaptophysin, PSD95, and MAP2.

34 4E-BP2 is also deamidated in skeletal muscles.

3. Developmental reduction of Akt-mTOR pathway is only observed in brain where

deamidated 4E-BP2 is observed but not liver

Mice at different developmental ages were analyzed for mTOR activity using its target such as

phospho-rpS6 (Ser240/244) and phospho-4E-BP2 (Thr37/46). The emergence of deamidated 4E-

BP2 coincided with the decrease in mTOR activity in whole brain lysate (Figure 12A,B). This

was unique to samples where deamidation of 4E-BP2 takes place since analysis of liver samples

from post-natal mice at different ages indicated consistent phosphorylation of 4E-BP1,

A B Brain

3d 10d 21d 90d E18 P0 P2 P7 P10 P13 P16 P21 P60 P90 P2y AKT T308 4E-BP2 AKT S473 AKT S6 (S240/244) p4E-BP2

S6 4E-BP2 (T37/46) GAPDH

4E-BP2 C Liver

Raptor 3d 10d 21d 90d mTOR 4E-BP2 ERK1/2 (T202/220) ERK1/2 PRAS40 (T246) p4E-BP2 PRAS40 HSP70 GAPDH

Figure 12: Developmental pattern of activity of mTOR pathway in brain and liver. (A)&(B) Whole brain tissues from mice at different ages as indicated were extracted. Phospho-4E-BP1 (Thr37/46), 4E-BP2, p-rpS6 (Ser240/244) were probed for mTOR activity. (A) was done by Dr. Arkady Khoutorsky. (B) was done to confirm the finding of (A). (C) Liver from mice at different ages were collected and analyzed for 4E-BP2, p-4E-BP1 (Thr37/46). GAPDH was used loading control.35 suggesting consistent mTOR activity (Figure 12C). From this piece of information, I hypothesized that mTOR inhibition is at least partially responsible for the accumulation of deamidated 4E-BP2 in the brain.

4. Single and double deamidated 4E-BP2 is less stable compared to wild type 4E-BP2

In most other proteins, deamidation acts as a signal that targets them for degradation. I want to investigate whether deamidation of 4E-BP2 affects its stability. Wild type 4E-BP2 plasmid compatible for mammalian transfection was generated previously. Site-directed mutagenesis kit was employed with specific primers to create plasmid encoding single (N99D and N102D) and double (N99DN102D) deamidated 4E-BP2. DNA was extracted from bacterial colonies containing mutated sequence and sent for sequencing to confirm successful generation of new plasmids (Table S1). HEK293 cells were transformed with plasmids encoding either wild type or single or double deamidated 4E-BP2. N99D and N102D 4E-BP2 migrated slower compared to the wild type 4E-BP2. N99DN102D 4E-BP2 migrated the slowest in SDS-PAGE due to 2 extra negative charges (Figure 13A). Protein stability was assessed with cyclohexamide chase assay.

Cyclohexamide is a compound that inhibits protein synthesis. By culturing cells in cyclohexamide containing media, the level of protein will decrease over time, which will help determine the stability of 4E-BP2. Plasmids encoding human wild type and double deamidated

4E-BP2 and HA tag were overexpressed in HEK293 cells. Overexpressed 4E-BP2 are detected by probing with anti-HA antibody. Wild type 4E-BP2 is more stable compared to single and especially double deamidated 4E-BP2 (Figure 13B). This result suggested that deamidation destabilizes 4E-BP2, of which the mechanism will be discussed in the following sections.

36 B A

Time (mins) 0 15 30 45 60 90 unM N99D N102D N99D/N102D

3HA-4E-BP2 WT Anti-HA

GAPDH GAPDH

3HA-4E-BP2 N99D Figure 13: WT-4E-BP2 is GAPDH more stable compared to deamidated 4E-BP2 3HA-4E-BP2 N102D (A) Unmodified (unM), single and double deamidated 4E-BP2 were transfected to cells and run GAPDH on SDS-PAGE gel to confirm the expression of proteins. (B) 3HA-4E-BP2 N99DN102D Cyclohexamide chase assay were used to treat untransfected and transfected cells. Levels of GAPDH 4E-BP2 over time is used to assess its stability

To assess whether this instability arose due to a structural change of 4E-BP2 due to deamidation,

purified recombinant wild type and double deamidated 4E-BP2 protein were generated. 4E-BP2

undergoes a disordered to ordered conformation transformation upon phosphorylation

(Muhandiram, Zhao, Forman-kay, Sonenberg, & Kay, 2015). Since, deamidation of 4E-BP2

generates an extra negative charge, it mimics phosphorylation. We utilized 1D-NMR and circular

dichroism to gain insights into the structure of deamidated 4E-BP2 compared to wild type. In the

1D-NMR experiment, two small differences in peaks were noted with red arrows. This

difference most likely arose due to chemical environmental changes around N99D and N102D

rather than structural changes (Figure 14A). Signals from circular dichroism indicated the same

result (Figure 14B). Deamidation of 4E-BP2 did not confer any significant structural changes

from the wild type protein. WT and DD-4E-BP2 were also subjected to Sypro Orange thermo

shift assay. Sypro Orange binds nonselective to all hydrophobic regions on the proteins. As

37 temperature increases, absorbance values will give insight into whether there is a transition from unfolded to folded state or vice versa (Figure 14C). Absorbances of both WT and DD 4E-BP2 increased linearly with rise in temperature indicating the lack of conformational change. This is

A

4EBP2 DD 4EBP2 wt Thermostablity Circular Dichrosim 130 4EBP2 (wt) B C 4EBP2 (DD)

120

110

100

90

80

70 0 20 40 60 80 100 Temperature (C) Figure 14: Wild type and deamidated 4E-BP2 don’t have significant difference in structure or thermodynamic stability. (A) 1D-NMR of WT and DD 4E-BP2. Red arrows indicate the difference between the two signals. (B) Circular dichroism of WT and DD 4E-BP2. (C) Thermoshift assay using Sypro Orange dye. Y-axis indicates the absorbance values. Thermostability indicates no unfolded to folded or vice versa change in structure. not unexpected for WT 4E-BP2 since the protein is an intrinsically disordered protein.

5. 4E-BP2 is degraded through the proteasomal pathway

Akiko et al. showed that hypophosphorylated 4E-BP1 is degraded through the proteasomal pathway (Yanagiya et al., 2012). In short E3 ubiquitin ligase ubiquitinates the protein at a certain lysine residue targeting it to the proteasome for degradation. Since the sequence surrounding the

38 lysine residue at which 4E-BP1 is ubiquitinated is conserved in all three members of the 4E-BP family, it is reasonable to expect that 4E-BP2 is degraded through the proteasomal pathway as well. MG132 is an effective proteasome inhibitor. HEK293 cells were treated with 10μM or

20μM MG132 for 6 hours (Figure 15A). The increase in level of 4E-BP2 and interestingly the emergence of slower migrating bands of 4E-BP2 indicated that 4E-BP2 is degraded through the proteasomal pathway. While the three band pattern in the treatment group resemble that of deamidated 4E-BP2, further experiments need to be done to definitively characterize whether these bands are deamidated 4E-BP2 or mono-ubiquitinated 4E-BP2.

Hypo-phopshorylated 4E-BP1 is dissociated from eIF4E, making 4E-BP1 less stable and prone to degradation. To investigate whether this is similar for 4E-BP2, eIF4E KO HeLa cells were generated by transducing regular HeLa cell line with either shRNA targeting eIF4E genes or scrambled shRNA control. There was an increase in level of 4E-BP2 when the cells were treated with 1μM PP242 for 1 and 6 hours and 20μM MG132 for 1 hours (Figure 15B). This increase was not observed in control shRNA transduced cells (Figure 15C). In sheIF4E KD HeLa, level of 4E-BP2 seemed to decrease with 6-hour treatment with MG132 (Figure 15B). This was possibly due to the high concentration of MG132 and the prolonged treatment time. It has been previously documented that high level of MG132 can cause apoptosis within cells.

39 DMSO MG132 10μM MG132 20μM A -λP +λP -λP +λP -λP +λP

4E-BP2 (short exposure) HEK293

4E-BP2 (long exposure)

GAPDH

sheIF4E HeLa shRNA HeLa B PP242 1μM MG132 20μM C PP242 1μM MG132 20μM

shRNA DMSO 1 6 1 6 Time (hrs) DMSO sh4E 1 6 1 6 Time (hrs)

p-4E-BP1 (Thr37/46) p-4E-BP1 (Thr37/46)

4E-BP2 4E-BP2

GAPDH GAPDH

eIF4E eIF4E

Figure 15: Proteasome inhibition leads to accumulation of 4E-BP2 in HEK293 and HeLa cells. PP242 leads to increase of 4E-BP2 in sheIF4E KO HeLa cells. (A) HEK293 cells were treated with MG132 at different concentrations. Lysates were treated with or without lambda phosphatase. (B) sheIF4E HeLa cells were treated with PP242 1μM and MG132 20μM at 1 or 6 hours. (C) shRNA control HeLa were treated with PP242 1μM PP242 and MG132 20μM at 1 and 6 hours

6. In vivo accumulation of deamidated 4E-BP2 in skeletal muscle with INK128 injection

Wild type C57Bl/c mice at 10-day old were injected with either .5mg/kg of INK128 or DMSO vehicle control everyday for 8 days. Tissues were collected at 2d, 4d, and 8d. The reasoning behind injecting animals at 10d old was because there was no 4E-BP2 observed in the skeletal muscle at 21d (Figure 16A). Hence, if mTOR inhibition affects the accumulation of deamidated

4E-BP2, we would observe a change that is not due to developmentral contribution. Two mTOR targets phospho-rpS6 (Ser240/244) and phospho-4E-BP1/2 (Thr37/46) were probed to show that

40 INK128, an active site mTOR inhibitor successfully impeded mTOR activity at 2 days after injection (Figure 16B). Accumulation and emergence of the slower migrating bands started to appear 2 days after INK128 injection but did not with vehicle control. The result is in accordance to the observation that deamidated 4E-BP2 increases as mTOR activity decreases in brain and neuronal cultures. However, similar treatment did not accumulate deamidated 4E-BP2 in liver

(Figre 16C). Developmental probing of 4E-BP2 in muscle tissues indicated deamidated 4E-BP2

41 does not appear until later on in development. Hence, inhibition of mTOR activity accelerates the accumulation of deamidated 4E-BP2 in muscles.

A Muscle

3d 10d 21d 90d

4E-BP2

GAPDH

DMSO INK128 (.5mg/kg) DMSO INK128 (.5mg/kg)

8d 2d 4d 8d Muscles B 8d 2d 4d 8d Liver C p-rpS6 (Ser240/244) p-rpS6 (Ser240/244)

p-4EBP1 (Thr37/46) p-4EBP1 (Thr37/46)

4E-BP2 4E-BP2

GAPDH

Figure 16: In vivo chronic injection of INK128 leads to accumulation of 4E-BP2 in muscles but not liver (A) Skeletal muscles from mice at different developmental ages were collected and probe for 4E- BP2. 0.5mg/kg of INK128 was injected daily to wild type mice. Tissues were collected at day 2, 4, and 8 and probed for p-rpS6 (Ser240/244), p-4E-BP1 (Thr37/46), and 4E-BP2. GAPDH is the loading control. (B) Accumulation of deamidated 4E-BP2 was observed in skeletal muscles starting day 2 of INK128 treatment. (C) Accumulation of deamidated 4E-BP2 was not observed in the liver.

42

7. Model of tissue-specific deamidation of 4E-BP2

In summary, spontaneous deamidation of 4E-BP2 targets the protein for degradation by the proteasomal pathway. mTOR inhibition prevents the degradation of 4E-BP2, leading to the accumulation of deamidated 4E-BP2 in the skeletal muscles and cell models. From the results above, I hypothesized that following model to explain for tissue-specific deamidation of 4E-BP2.

Since deamidation of 4E-BP2 is spontaneous, we expect to see deamidated 4E-BP2 in all tissues.

However, deamidated 4E-BP2 is targeted for degradation immediately. In tissues such as the liver, where mTOR activity is consistent throughout development, proteasome works efficiently to degrade deamidated 4E-BP2. In tissues such as the brain and skeletal muscle, mTOR activity decreases with age leading to decreased proteasomal activity. The proteasome doesn’t degrade deamidated mTOR effectively anymore leading to the accumulation of deamidated 4E-BP2 in such tissues (Figure 17). There have been multiple publications by researchers from Boston who took conflicting stances on the effect of mTORC1 on the proteasome. Zhang et al suggested that inactivation of mTORC1 reduces general proteolysis by downregulation of the proteasomal subunits through NRF1 (Yinan Zhang & Manning, 2017; Yining Zhang et al., 2014). In the following year, Zhao et al refuted this hypothesis and proposed that inactivation of mTORC1 coordinately activates proteasomal and lysosomal degradation (Y Zhang, 2014; J. Zhao, Zhai,

Gygi, & Goldberg, 2015). Zhao et al argued that the rate of degradation experiment using radioactive tracer by Zhang et al is not validated due to substantial amount of residual 35S-Met

43 radiolabels. Zhang et al later defended his hypothesis by utilizing the 3H-Phe in the pulse chase experiment, and displayed that the new result is in accordance to his previous data(Yinan Zhang,

Manning, & Diseases, 2016). The difference outcomes from two researchers might have arisen from the different concentration of mTORC1 inhibitors, rampamycin and Torin1 used in the experiments. Zhao et al stated that rampamycin and Torin1 are mTORC1 selective inhibitors, which is not entirely accurate. While the two suppresors effectively inhibits mTORC1 at low concentration, higher concentration has been demonstrated to prevent the formation of mTORC2. In their experiments, Zhao et al utilized 300nM rampamycin and 100nM of Torin1.

These concentrations have been prescribed to affect both mTOR complexes. Hence, the differences observed between the experiments might have arise due to the different mTORC targeted.

In the context of mTORC1 and 4E-BP2, reduction in the activity of the kinase complex leads to diminishing phosphorylation of 4E-BP2, in turn impedes translation initiation. As deamidated proteins bind more strongly to Raptor and less efficiently to eIF4E (Bidinosti, Ran, et al., 2010), deamidation was suspected as a compensatory mechanism to reduced mTORC1 activity. If deamidated 4E-BP2 is degraded through the proteasomal pathway, it is more likely that deamidated 4E-BP2 is sustained rather than broken down in the aged brain. Our hypothesis is in accordance with the notion that was proposed by Zhang et al: reduced MTORC1 activity down- regulates proteasomal subunits and its proteolytic capacity.

44 99 102 N N Brain 4E-BP2 Muscles Liver

Spontaneous mTOR mTOR

99 102 N D d4E-BP2 Proteasome Proteasome

99 102 Activity Activity D N d4E-BP2 99 102 99 102 (D) (D) N N 99 102 D D d4E-BP2 4E-BP2 d4E-BP2

Figure 17: Model of tissue-specific deamidation of 4E-BP2

8. Tissue-specific turn over rate of 4E-BP2

According to the proposed model described above, one would expect that the turn over rate of

4E-BP2 in the brain and muscles are much slower that those in other tissues. Turn over rate of specific protein can be measured employing mass spectrometry and pulse chasing with heavy stable isotope such as 15N in food. In fact, there are protocols that feed rats over a period of a few months with 15N algae and chase with regular 14N algae. After, mass spectrometry can be used to calculate the decrease in 15N signal to determine the half-lives of proteins. Despite being the prominent 4E-BP in the brain, the level of 4E-BP2 is still very small, making it difficult to be detected by mass spectrometry. As mentioned before, brains from 50 mice 2-3 week old were

45 needed to detect deamidated 4E-BP2. Therefore, to employ this method to measure 4E-BP2 half- life, protocol is being optimized to effectively immunoprecipitate 4E-BP2 from brain lysates using the optimal amount of antibody. 20μL of 4E-BP2 antibody was the most optimal for the use of immunopreciptation of 4E-BP2 based on the amount of recovery of proteins (Figure

18A,B). Elution protocol was optimized to make sure that the sample to be analyzed are not contaminated with heavy and light chains from IgG. Sequential elution was done with .25M,

.5M, and 1M Glycine. At .5M concentration, glycine elution buffer was effective in disrupting the interaction between 4E-BP2 and the antibody but the the interaction between the antibody and protein A beads (Figure 18C). Hence, this elution buffer can effective recover 4E-BP2 with minimal traces of contamination of IgG chains.

46

A B Pre-immunized rb beads Ab control beads 5uL 4E10uL-BP2 20uL4E IP-BP2 4E34uL IP-BP2 4E IP-BP2 +ve control IP +ve control 70.0 60.0 57.4

50.0 48 kDa 50.1 40.0

25kDa 30.0 24.7 Recovery Recovery % 20.0

10.0 10.0 4E-BP2 0.0 -5 5 15 25 35 4E-BP2 antibody volume (uL) C +ve control.25M Glycine .5M Glycine 1M Glycine Rb IP Rb IP Rb IP

48 kDa

25kDa

Figure 18: Optimization for immunoprecipitation of 4E-BP2 from mouse brains. (A) Different volumes of antibody were used to pull down 4E-BP2 from brain samples. Result was probed with 4E-BP2 antibody. (B) Signal was quantified with ImageJ and graph was created with Microsoft Excel. (C) Sequential elution of 4E-BP2 bound beads with glycine gradient buffers. Rb stands for beads bound with non-specific rabbit IgG.

47 Discussion and Future Directions

The current model of tissue-specific deamidation of 4E-BP2 is still being developed. Assay to sensitively measure proteasome activity are being optimized. With that assay, we can investigate the effect of mTOR inhibition on the proteasome, proteasome activity within different tissues, as well as how proteasome activity changes during development. It still needs to be shown that mTOR inhibition decreases proteasome activity. Currently in vivo chronic administration of

INK128 experiment are being repeated to confirm previous finding and investigate other tissues.

INK128 treatment did not induce the accumulation of deamidated 4E-BP2 in the liver. This result is not too surprising since the liver has been long known as a tissue with very high turn over rate. Hence, it is possible that there might be other mechanisms to degrade deamidated 4E-

BP2 in the liver or that INK128 treatment at the dose used was not sufficient.

In order to strengthen the hypothesis that deamidation acts as a signal that targets deamidated

4E-BP2 to degradation by the proteasome. We are currently over-expressing WT and DD 4E-

BP2 with histidine tagged ubiquitin. Cells with treated with MG132 over a short period of time.

With this co-immunoprecipitation experiment, we will assess whether there is a difference in the amount of ubiquitin associated with WT and DD 4E-BP2. Higher amount of ubiquitin associated with DD-4E-BP2 will suggest that deamidated 4E-BP2 is more readily targeted for degradation through the proteasomal pathway.

It has been observed in multiple occasions that aged brain has decreased activity of Akt-mTOR pathway. Inhibition of mTOR activity has resulted in extended life span in mice (Harrison et al.,

2009). However, not much explanation has been given to this phenomenom. If the proposed

48 hypothesis is correct, this suggests that the decrease in mTOR activity regulates post- translational modifications such as deamidations. This suggests that the profile of proteins that undergo PTM will differ between tissues and with development. 4E-BP2 in the brain has been suggested to have functions in memory formation and learning capacity. In addition, 4E-BP2 KO mice demonstrated characteristics of autism spectrum disorders. Understanding the mechanisms of tissue-specific deamidation of 4E-BP2 will not provide another example where deamidation can be regulated at tissue specific level but also might give insight into the functions of deamidated 4E-BP2 within the tissues that they were observed.

Materials and Methods

Protein extraction from tissues

Mice were anesthetized with CO2 and Isofluorane mixture. Under anesthetization, mice were euthanized by cervical dislocation. Tissues such as brains, liver, muscles, heart, lungs, spleen, kidney, and fat were excised, collected, snap-frozen with liquid N2, and stored at -80C. RIPA buffer (Sigma#R0278) with Protease Cocktail inhibitors (Roche#11697498001) and phosphatase inhibitor cocktails 2&3 (Sigma #P5726, #P0044), were used to homogenize tissues at a ratio of

8:1 volume:weight. Homogenates were sonicated in a water bath at high frequency for 2x 5 seconds. Homogenates were centrifuged at 16,000rpm for 30 minutes at 4C. Supernatant was collected and protein concentration was detected by comparing absorbance signal at 595nm with a BSA standard curve.

49 Expression Vectors

Human 4E-BP2 cDNA was subcloned into pCDNA3-3HA (Imataka et al. 1997) using two restriction enzymes EcoRI and XhoI. Site-directed mutagenesis kit (Agilent #200521) was used to generate mutated forms of 4E-BP2 including N99D, N102D, and N99D/N102D 4E-BP2.

Generated clones were sent for sequencing at McGill University and Génome Québec Innovation

Centre. Wild type and mutated 4E-BP2 were subcloned into with EcoRI/XhoI into pGEX-6P-1

(Pharmacia) for production of recombinant proteins with GST tags. pTER-sheIF4E encoding shRNA against human eIF4E and scrambled pTER-shSceIF4E were constructed as followed:

Oligos with sequences against human eIF4E and scrambled shRNA described in Table S2 were annealed, followed by insertion into pTER vector using BglII and HindII restriction enzymes. pMV7-HA-eIF4E was described (Lazaris-Karatzas et al., 1990)

Generation of transient eIF4E-KD cell lines

HEK293H cells were transfected with pLP1 (Invitrogen), pLP2 (Invitrogen), pLP/VSVG

(Invitrogen), pTER-sheIF4E or pLP1, pLP2, pLP/VSVG, pTER-shSceIF4E using

Lipofectamine2000 (Invitrogen). Media was changed to complete DMEM (cDMEM) 6 hours after transfection. At 48 hours, supernatant was collected, filtered, and snap-frozen with liquid

N2, and stored at -80C. HeLa cells were plated at 80% confluency and allowed to settle in a

10cm plate. HeLa media was changed to new media containing virus supernatant at a dilution of

1:2. 48 hours after transduction, selection media is changed to cDMEM with 10% Puromycin

(Sigma #P8833).

50 4E-BP2 Immunoprecipitation and Mass Spectrometry Analysis

Proteins were extracted from tissues as described above. Proteins from 10 mouse’s brains were immunoprecipitated with 20μL of 4E-BP2 antibody/brain. Protein lysates were incubated with

4E-BP2 antibodies O/N on a rotator at 4C. Protein A beads were washed 3X with RIPA lysis buffer, and added to the antibody-protein lysate mixture, rotated at 4C for 3 hours. Mixture was centrifuged at 3,000rpm for 5 minutes and the beads were collected. Protein A beads were washed 5X with lysis buffer. 4E-BP2 were eluted with .5M Glycine buffer, precipitated with trichloroacetic acid, and analyzed with MS/MS.

Antibody and Reagents

The following antibodies were utilized in this project: 4E-BP2 (CST#2845), p-4E-BP1

(Thr37/46) (CST#236B4), which recognizes both phospho-4E-BP1 and phospho-4E-BP2 isoforms. p-S6 ribosomal proteins (Ser240/244) (CST#2215), S6 ribosomal protein (CST#2217),

4E-BP1 (CST#9644), eIF4E (BioScience#610270), GAPDH (SantaCruz#47724), Synaptophysin

(Millipore#MAB5258), PSD95 (CST#3450), MAP2 (Abcam#5392), HA (Biolegend#901501)

In Vitro Deamidation Assay

Mouse embryonic fibroblast (MEF) cells and brains from eIF4EBP1/eIF4EBP2 double knock out mice were lysed with 0.15M Tris/HCl pH 7.0. Mixture of MEF and brain lysates was incubated at 37C, pH 7.0 over different time points. Mixtures were collected, run on a 15%

SDS-PAGE gel and analyzed with Western Blot.

51 Protein Stability Assay

Different cell lines were treated with 100μg/mL cyclohexamide (Bioshop#CYC003) for the indicated times. Cell were lysed and analyzed with Western Blot.

In vivo mTOR inactivation

Wild type C57BL/6 mice (The Jackson Laboratory) at P21 were chronically injected with either

.5mg/kg INK128 (Selleckchem#MLN0128) or DMSO vehicle for 8 days. Mice were euthanized at 2d, 4d, 6d, and 8d. Proteins were extracted as described above and analyzed with Western

Blot.

In vitro phosphorylation assay

GST-tagged recombinant proteins were produced by chemically transfect E. Coli Rosetta 2 DE3 cells with pGEX plasmids containing either wild type 4E-BP2 or N99D/N102D 4E-BP2, purified through a sepharose column, then high liquid chromatography column, and concentrated with a filtered column. WT and N99D/N102D 4E-BP2 were incubated with 5mM ATP, and purified recombinant mTOR for indicated times. Protein were run on an SDS-PAGE gel and analyzed with Western Blot.

52 Supplementary Figures

53 Table S1: Sequencing of vectors containing mutated 4E-BP2 Vectors containing single and double mutated 4E-BP2 at position 99 and 102. GTC codes for D while GTT codes of N

Table S2: Oligos for Construction of shRNAs

54

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