Bio-Functional Analysis of Ubiquitin Ligase UBE3D

Adam William Penn

A thesis submitted in conformity with the requirements for the degree of Masters of Science Department of Medical Biophysics University of Toronto

ii Bio-Functional Analysis of Ubiquitin Ligase UBE3D

Adam William Penn

Masters of Science

Department of Medical Biophysics University of Toronto

2020 Abstract

The ubiquitin system is comprised of a reversible three step process: E1 activating enzyme, E2 conjugating enzyme and an E3 ligase, leading to ubiquitin molecules being post-translationally modified onto substrate proteins leading to a plethora of downstream effects (localization, function and half-life).

UBE3D, a HECT (homologous to E6-AP carboxylic terminus) E3 ligase, has a relatively elusive regulatory role within the cell. Here, we systematically analyze and characterize UBE3D as well as its highest confidence interactor, Dynein axonemal assembly factor (DNAAF2) through: Autoubiquitylation assay; intracellular localization with immunofluorescence; interaction network using proximity-dependent biotin identification (BioID) to better understand the relationship of these two proteins. DNAAF2 protein interaction mapping allowed for insight into PIH domain. In summary, I have used multiple approaches to gain novel knowledge and insight into the potential functional role of UBE3D within the cell, and its putative partner protein, DNAAF2.

ii iii Table of Contents

1 Introduction 1

1.1 The Ubiquitin System 1

1.1.1 E1 Ubiquitin Activating Enzyme 3

1.1.2 E2 Conjugating Enzyme 3

1.1.3 E3 Ubiquitin Ligases 4

1.2 HECT E3 Ubiquitin Ligases 4

1.2.1 E2 conjugating enzyme interactions with HECT E3 ligase enzymes 7

1.2.2 Mechanism and Biological Impact of Ub Chain Formation 8

1.2.3 Determining Ub linkage specificity 9

1.2.4 Analysis of ubiquitin linkage by mass spectrometry 9

1.3 UBE3D, a member of the E6-AP HECT E3 ligase family 10

1.3.1 Age-Related Macular Degeneration (AMD) and UBE3D 11

1.3.2 UBE3D implicated in East-Asian AMD 12

1.4 Cilia, Dynein Motors and associated proteins 13

1.4.1 Cilia 13

1.4.2 Dynein motors 16

1.4.3 DNAAF2 and the assembly of axonemal dynein motors 17

1.5 Identifying protein:protein interactions using BioID coupled to mass spectrometry 18

1.5.1 Analysis of protein interaction data 19

1.6 Research hypothesis and thesis outline 20

2 Results 21

2.1 Identifying the Ub Chain Building Activity of UBE3D 21

iii iv 2.1.1 Conducting an E2-UBE3D HECT E3 functional analysis 21

2.1.2 Characterization of Ub chain linkages in UBE2D3 E2 – UBE3D HECT E3 reactions24

2.1.3 Summary 24

2.1.4 Experimental Details 24

2.2 Identifying potential UBE3D Protein Interactors 27

2.2.1 Using BioID to identify UBE3D protein interactors 27

2.2.2 Using IF to identify UBE3D subcellular localization 31

2.2.3 Using siRNA to evaluate UBE3D function in ciliation 37

2.2.4 Summary 41

2.2.5 Experimental Details 41

2.3 Characterization of the relationship of DNAAF2 with UBE3D 47

2.3.1 Using BioID to identify DNAAF2 protein interactors 47

2.3.2 DNAAF2 Localization in hTERT RPE1 cells 51

2.3.3 DNAAF2 N-terminal (PIH domain) BioID Results 54

2.3.4 DNAAF2 C-Terminal BioID Results 58

2.3.5 Summary 62

2.3.6 Experimental Details 69

3 Discussion 64

3.1 Novel insights on the regulation of ciliation by UBE3D 64

3.2 Characterization of DNAAF2 reveals new insights on Axonemal Dynein assembly 67

Introduction 1.1 The Ubiquitin System

Ubiquitin (Ub) is a 76 amino acid polypeptide that is only found in eukaryotes and is highly conserved. Ubiquitin can be covalently conjugated to a protein substrate through a multistep cascade (ubiquitylation), which, in turn leads to distinct downstream effects of the fate and/or function of the targeted molecule. Ubiquitylation of a substrate is a complex and highly regulated cascade involving an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligation reaction(1). In the ubiquitylation process, an isopeptide bond occurs between the carboxyl group of Ub and the amine group of a lysine residue on the protein substrate (2-4). There are many examples demonstrating that specific deregulation of the Ub system can result in many pathological effects such as cancers, inflammation, diabetes and neurodegenerative disorders (5). This is not surprising as many critical cellular processes are regulated by substrate ubiquitylation. The specific directionality of the downstream effects of ubiquitylation is dependent on the type of Ub linkage conjugated to the substrate. Ub can be conjugated either as a single Ub molecule (monoubiquitylation), single Ub molecules on multiple substrate lysine residues (multiubiquitylation), or as a chain of multiple Ubs connected through internal lysine residues found within the Ub molecule (polyubiquitylation). Each manner of Ub conjugation that occurs leads to unique downstream effect(s) on the substrate, including localization, function and half-life(2).

Monoubiquitylation on substrate proteins has experimentally been implicated in DNA damage signaling, transcriptional control, membrane-associated processes and endocytosis. Monoubiquitylation often occurs on newly synthesized proteins to be directed to the transGolgi where they are either sent for lysosomal degradation or transported to the plasma membrane(6). Multiubiquitylation has also been shown to be involved in mechanisms of membrane-protein internalization and endocytic sorting (6). Polyubiquitylation, the process of Ub conjugation, has several unique configurations which each lead to unique downstream effects to protein substrates as well as biological processes. Ub molecules contain 7 internal lysine residues (K6, K11, K27, K29, K33, K48 and K63), which all have the ability to be ubiquitylated. Upon ubiquitylation of a Ub molecule, a Ub chain is formed. The Ub lysine residue being ubiquitylated as well as length of the chain cause unique downstream effects(3, 7-9).

Several of the most biologically prevalent categories of polyubiquitylation have been elucidated but many remain to be fully understood. The best-described category of polyubiquitylation is the lysine-48 (K48)- linked Ub chains. Protein substrates conjugated with K48 linked Ub chains are targeted to the 26S

1 2 proteasome for degradation(10). In addition, lysine-6 (K6), lysine-11 (K11), lysine-27 (K27) and lysine- 29 (K29) Ub linkages upon substrate conjugation are also believed to function as targeting modifications for 26S proteasomal degradation amongst other effects(11-14). Another well-described Ub linkage is lysine-63 (K63)-linked Ub chains. K63 chains have been shown to contribute to multiple biological activities including endocytosis, aggresome formation, proteasomal degradation and DNA damage response (15). Thus, the linkage specificity of Ub chain formation can regulate protein substrate activity and/or degradation.

ATP Ub Ub SH S S E1 E1 E2

Ub Ub Ub K Ub S K S Ub E2 Cys Target Target E2 HECT E3 RING-E3

Ub Ub Ub K Ub K K K Ub Ub K Ub Ub Ub K Ub K K Ub K K K Ub K Target Target Target Target

MonoUbiquiAlaAon MulA-mono-ubiquiAnaAon Poly-ubiquiAnaAon Poly-ubiquiAnaAon (Branched)

Figure 1.1.1

A schematic representation of the general ubiquitin system and different classes of ubiquitin linkages.

1.1.1 E1 Ubiquitin Activating Enzyme

The first step in the ubiquitylation cascade is performed by the E1 activating enzyme. In mammals there are two known E1 enzymes, UBE1 (predominantly) and UBA6 (16-18). E1 activating enzymes function by first creating an adenylate-intermediate through binding of MgATP and Ub (a bond between the C- terminal carboxylate of the Ub and AMP) (3) (4) Ub is then transferred to the active cysteine site found in the catalytic domain of UBA1 to form the activated Ub-UBA1 complex via a thiol-ester bond (4, 19). The activated Ub-UBA1 complex binds a second Ub molecule to the adenylation domain and subsequently converts it to an Ub-adenylate. Upon the E1 complex being doubly bound by Ub it is then recognized by an E2 conjugating enzyme. Only the thiol-ester Ub from the E1 activating enzyme is transferred to the E2 conjugating enzyme to form a Ub-charged E2 complex.

1.1.2 E2 Conjugating Enzyme

The broad overarching function of E2 conjugating enzymes is to accept the activated Ub from E1 enzyme and then subsequently bind E3 ligases and facilitate the Ub transfer to substrates (3). There are approximately 40 E2 conjugating enzymes encoded by the human genome (both active and inactive E2 variants). These 40 E2s are responsible for conjugation of Ub to over 800 E3 ligases (each with their own specific substrate profile).(20) It has been shown that each E2 can cooperate with several E3 ligases and each E3 can cooperate with several E2 conjugating enzymes (21, 22).

Structural characteristics of the E2 conjugating family of enzymes are key to their activity have been well described for the majority of family members (20). The ubiquitin conjugation domain (UBC) is conserved amongst all E2s. The UBC consists of ~150 amino acid residues including the cysteine residue to which the active Ub molecule is accepted from the E1. The UBC domain is responsible for catalysis and Ub binding (22). The UBC is made up of 4 alpha-helices with a 310 helix extension and 4 corresponding anti- parallel beta-sheets. The main mechanism in most E2 conjugating enzymes requires a standard E2 fold of the UBC (22).

The less conserved residues in the E2 conjugating enzymes mostly occurs in the two loop regions, which confer the majority of variability in length, sequence and conformation. These two loop regions are responsible for E1 and E3 binding. They play a central role in aligning the substrate lysine toward the E2

4 catalytic cysteine (20, 23, 24). Thus increased variability in the loop regions can affect flexibility and thus E3 selection specificity (25). Specific E2-E3 interactions determine how E2 conjugating enzymes perform specific biological functions. Established specificity between E2s and E3s has yet to be determined. E2s differ in E3 interaction specificity, intracellular localization and regulatory properties based on the presence or absence of N-terminal and/or C-terminal amino acid extensions. These extensions are used to categorize E2 conjugating enzymes into different functional classes. (21).

1.1.3 E3 Ubiquitin Ligases

The final step in the Ub-substrate cascade is facilitated by Ub E3 ligases. Generally E3s are required for the transfer of Ub bound to an E2 to another Ub molecule or to a substrate protein. E3s are responsible for determining which specific substrate will be targeted for ubiquitylation. Although in majority cases substrates aren’t directly recognized by the E3. In these cases there either has to be a post-translational modification (PTM) to the E3 to become active, the substrate must be modified to be recognized or the E3 has an associated ancillary protein responsible for substrate interaction. (22).

The two major classes of E3s are RING finger E3s (RING finger domain E3s) or HECT (Homologuous to the E6-AP Carboxylic Terminus) E3s. The vast majority of identified E3s in the human genome are RING finger E3s. RING finger E3s act as scaffolding proteins, which recruit active E2-Ub complexes to substrates to allow Ub transfer (7). RING finger E3s usually require additional associated ancillary proteins and/or modifications to either the E3 or substrate to facilitate transfer of Ub. In the much smaller E3 family, HECT E3s form an intermediate thiol-ester linkage with Ub via an active cysteine residue prior to its transfer to substrates (Bernassola et al., 2008; Rotin and Kumar, 2009). A much smaller and less well-described E3 family is referred to as E4s. E4s include U-box domain containing E3s and have been shown to play a role in the elongation of Ub chains by facilitating the transfer of E2 bound Ub to an already conjugated Ub (22).

1.2 HECT E3 Ubiquitin Ligases

HECT E3 ligases were the first class of E3 ligases to be identified. There are ~30 predicted HECT E3 ligases in the human genome, the first identified being UBE3A. The acronym HECT, Homologous to the E6-AP Carboxilic Terminus, was derived from the first C-terminal HECT domain identified in UBE3A (formerly known as E6-AP) (26). UBE3A has been shown to associate with human papilloma virus (HPV)

5 encoded protein E6 and effects proteasomal degradation of tumour suppressor protein p53, thus facilitating development of HPV-associated cervical cancer (27). HECT domains are conserved ~40kDa domains found on the C-terminus of the E3 ligase. The HECT domain contains an active-cysteine residue, which is used to create an intermediate thiol-ester bond with Ub (27). The family of E2 conjugating enzymes most associated with HECT E3 contain a conserved phenylalanine residue found in the loop between the 3rd and 4th beta-sheets. This phenylalanine is located at the E2-HECT interface and is required for proper binding.

The HECT domain functionality can be separated into two lobes, N-terminal lobe (N-Lobe) and C- terminal lobe (C-Lobe). For the majority of HECT E3s the N-lobe contains the E2 binding site and the C- lobe is where the active cysteine residue is located. (28). Between the N and C lobes of the HECT domain is a flexible linker sequence which is required for proper positioning of the E2 and E3 active sites. The flexible linker sequence is required for optimal orientation and thus plays a role in accessibility restriction of certain Lys residues on Ub chains or substrates.

Along with variability in the HECT domain sequence leading to unique E2 and Ub chain profiles, the N- terminal domains of HECT E3s also play a role in HECT E3 functionality. The variability in N-terminal domains of HECT E3s allows for unique functions classified into HECT E3 subfamilies. Ubiquitylation is often associated with protein degradation through the 26S proteasome but additional N-terminal domains found in different HECT E3 families have been shown to confer additional regulatory roles in the control of cellular processes, including intracellular vesicle trafficking, DNA repair, and receptor and transporter activities. (29)

1.1.2 Figure

Schematic of Ubiquitin HECT-E3 Substrate Ubiquitination

A. Transthioesterfication mechanism of ubiquitin HECT-E3 ligase (where the C- and N-terminal lobes are flexible at the hinge loop). This process involves the transfer of ubiquitin from the E2 conjugating enzyme loaded with ubiquitin to the cysteine residue located on the C-lobe of the HECT domain of the E3 ligase indicated in dark blue. B. The ubiquitin HECT-E3 ligase intermediate transfers ubiquitin through the hinge function of the HECT domain (light blue) to ubiquitinate acceptor protein substrate (via lysine residue) resulting in an isopeptide bond formation between the donor ubiquitin and the target protein.

7 The best-studied class of HECT E3s are the Nedd4 family with ~9 family members. The Nedd4 HECT E3s are classified by their common N-terminal region, composed of a C2 domain followed by 2-4 WW domains. The C2 domain has been implicated in intracellular trafficking as well as plasma membrane targeting. (30). The WW domains contribute to plasma membrane receptor turnover by binding to proline motifs found on plasma membrane channels, which subsequently leads to their ubiquitylation, followed by endocytosis into multivesicular bodies and then finishing in protein degradation via the lysosome (29). Other known Nedd4 family members confer other unique functionality. This occurs due to additional N- terminal binding sites. For example NEDD4 and ITCH E3 ligases have been shown to be implicated in cellular growth and proliferation signaling cascades as well as immune response. NEDD4 contains a TNIK (TRAF2 and NCK-interacting protein kinase) binding motif, which has been shown to play an important role in stress response and dendrite cytoskeleton rearrangement and extensions (31). ITCH contains a FYN (Tyrosine-protein kinase Fyn) binding motif which has shown to be involved in cell growth and survival as well as immune response. Thus demonstrating that even within subfamilies of HECT E3s there is still variability in specific cellular functions. (32).

Another major class of HECT E3s is known as the HERC subfamily containing ~6 members. They are categorized together due to their common RLD domains (Regulator of Chromosome Condensation Like- Domain). HERC family members can contain multiple RLD domains based on HECT E3 sizing (100– 120 kDa contain a single RLD and >500 kDa contain multiple RLD) (29, 30). Many HERC HECT E3 ligases have been shown to be involved in membrane trafficking regardless of molecular weight category (33); (34). The larger HERC HECT E3s contain an additional WD40 domain conferring distinct functionality. For example HERC2 (~527 kDa, containing three RLD domains) has been implicated in ubiquitylation directed DNA repair in response to ionizing irradiation-induced DNA damage by recruiting DNA repair complexes (30). Furthermore, HERC1 (~532kDa, containing 2 RLD domains) has been reported to act as a guanine-nucleotide exchange factor protein found to be involved in cytoskeleton remodeling in response to stress (33). There are 5 other HECT E3 subfamilies (E6-AP, TRIP12, EDD, HACE1 and HUWE1) containing ~15 members. Each of these subfamilies contains a wide array of domains. These domains range from being implicated in mediating protein-protein interactions to domains that contribute to ubiquitylation functionality.

1.1.3 E2 conjugating enzyme interactions with HECT E3 ligase enzymes

The mechanism of action of the E2s and E3s during ubiquitylation is complex. It has been shown that the interaction is considered weak but this is crucial for proper ubiquitylation to occur (7). This is because

8 E2s are required to go through many successions of E1 and E3 binding. The binding site on E2s for E1s and E3s often superimpose each other. So binding and dissociation between E1 and E3s is required to create Ub chains, as chains contains several Ub molecules. (22); (35). In some cases E3s possess an additional E2 binding site. In these cases Ub chain formation ability is increased as constant binding and disassociation between the E2 and E3 is not required. (9). Ub chain creation specificity can be determined by both the E2 and the E3 depending on the type of E3. In RING E3s the type of E2 recruited substantially contributes to which Ub chain results. Whereas in HECT E3, it has been generally shown that the E3 regulates which Ub chain is generated, regardless of the E2 present. This is due to HECT E3s containing an additional cysteine residue in the HECT domain, which allows for Ub to be loaded directly onto the HECT domain prior to being loaded onto the protein substrate. This will be further explained below. (36); (28);

1.1.4 Mechanism and Biological Impact of Ub Chain Formation

As previously mentioned in Chapter 1.1 post translational modification of Ub motifs onto protein substrates confers many diverse downstream effects on protein functionality. Ub is bound to protein substrates in diverse structural forms such as monoubiquitylation, multi-monoubiquitylation and in a diverse range of polyUb chains. Ub itself contains 7 internal lysine residues (K6, K11, K27, K29, K33, K48 and K63), allowing for polyUb chains to have seven possible forms of homotypic Ub polymers. (37)

Ub chains are formed when the glycine residue of Ub at position 76 is conjugated with one of the 7 lysine residues in another Ub molecule. (38). Depending on which of the 7 lysine residues glycine is attached to it entails a unique downstream effect to the protein substrate upon conjugation. For these chains to effect change in functionality, localization and/or half-life of protein substrates, there are classes of proteins containing motifs or domains that interpret which type of Ub molecules have been conjugated. The ubiquitin-interaction motif (UIM) as well as ubiquitin association domains (UBA) are the best described to interpret the effects of Ub conjugation. (39); (40). Proteins containing UIM or UBA, upon binding of substrate bound Ub, also bind specific proteins of the desired outcome of each specific chain. These effector proteins thus turn the Ub code into the specific biological change. As previously stated, there are many different types of ubiquitylation so proteins containing UIM or UBA usually have a preference of Ub species present. For example the protein Rad23 contains a UBA at its C-terminus and has been shown to identify and effect change in transcription factors conjugated with K48 Ub chains. Rad23 essentially regulates the proteasome-dependent degradation of K48-conjugated transcription factors. (41)

9 1.1.5 Determining Ub linkage specificity

The ubiquitylation and selectivity for Ub chains for HECT E3s differ from RING E3s in several crucial ways. As previously mentioned, HECT E3s contain an active cysteine residue found in the HECT domain of these E3s. This is important as it allows for Ub molecules to be directly loaded onto the HECT domain to create an intermediate rather than going directly from the E2 conjugating enzyme to the substrate, as is the case with RING E3s. Due to the cysteine residue HECT E3s gain the ability to function similarly to an E2 during ubiquitylation thus causing the type of E2 present to contribute less to Ub chain type specificity. Since E2s play less of a role in the type of Ub linkage created, HECT E3s demonstrate that they have preferences in selection for Ub chain creation. For example, HECT E3 NEDD4 has been experimentally shown to demonstrate a preference for K63 Ub chains in the presence of different E2s. HECT E3 molecular size, unique domains and residues contribute to this selection.

The characteristics of the E2 conjugating enzyme that determine Ub linkage specificity are the similar to those that determine the Ub linkage preference of the HECT E3 ligases. The major determining characteristic is proximity and positioning of Ub donor and Ub acceptor. Recall that the HECT domain is separated into an N-terminal lobe and a C-terminal lobe connected through a flexible hinge. The N-lobe contains the substrate-binding site (36). The positioning of the substrate-binding site within the N-lobe allows or restricts accessibility of certain lysine residues of Ub. This affects specificity of Ub linkages because it restricts the ability of HECT domains to form Ub polymers of that lysine type. The C-lobe, which contains the active cysteine residue, which forms an intermediate with Ub, also has been shown to play a role in controlling Ub linkage specificity. (28) This has been experimentally demonstrated by interchanging the N-lobe and C-lobe between two distinct HECT E3s, UBE3A and ITCH. It is known that UBE3A and ITCH create K48 and K63 chains, respectively. Upon swapping the C-lobe of ITCH with that of UBE3A it was demonstrated that Ub chain creation switched from K63 to K48. Although this was done in-vitro they were able to demonstrate that the C-lobe plays a role in Ub chain specificity. They proposed that the C-lobe controls the positioning of the bound Ub and that altering its relative position to the substrate can restrict lysine residue accessibility (28).

1.1.6 Analysis of ubiquitin linkage by mass spectrometry

Depending on which Ub linkage form is conjugated to protein substrates it can lead to very different downstream outcomes. Therefore determining the type of Ub conjugated (e.g. monoUb, polyUb linkage type) to a substrate can be just as important as the substrate profile itself. Through the use of mass

10 spectrometry there are approaches to identifying which exact Ub form is preferentially conjugated by a specific E3 ligase (42); (43); (12, 44).

Prior to entering a sample into the liquid chromatography-tandem mass spectrometer (LC-MS/MS) it must first be digested using trypsin (or other proteases) into tryptic peptides before being fractionated by liquid chromatography. Once digested and separated by LC the mass spectrometer then ionizes the peptides based on their mass-to-charge (m/z) ratio. Using a second round MS in tandem mass spectrometry will further fragment the peptides to create a set of fragmented peptides. To analyze the data collected through MS/MS it is compared to theoretical spectral databases to identify the full sequence of the peptide fragments which in turn allowed for the identification of the original protein the peptides came from (45).

Identifying which type Ub conjugate was present in the sample is made easy because upon tryptic digestion of a Ub linkage a di-glycine tag on the specific modified lysine residue remains. (22, 44). The di-glycine tag adds an additional mass of 114.0429 Da to the peptide. This molecular weight is added to the search parameters to more efficiently identify ubiquitylation sites (46)(47) (44) (42).

The results found by this in vitro ubiquitin linkage assay only identify ubiquitin linkages that a HECT domain can produce under these specific in vitro conditions. These results aren’t necessarily what occurs in vivo but demonstrates that the HECT domain has the ability to form that type of ubiquitin linkages under these specific conditions.

1.2 UBE3D, a member of the E6-AP HECT E3 ligase family

Although the E6-AP HECT E3 ligases family was the first identified E3 family it is relatively understudied. E3 ligases are categorized into the E6-AP family based on lack of domains associated with other HECT E3 families. Thus members of the E6-AP family have very little sequence homology other than that found in the C-terminal HECT domain. The E6-AP HECT E3 ligase family consists of ~ 4 different HECT E3s; UBE3A, UBE3B, UBE3C and UBE3D. UBE3A (E6-AP), the first identified HECT E3 ligase and founding member of the E6-AP family, has a HECT domain of ~11 kDa (48).

The UBE3D (H10BH, UBE2CBP) gene encodes a ~43 kDa (389 amino acid) protein, and although categorized as a E6-AP HECT E3 ligase, only contains a ‘HECT-like’ domain. It is considered a HECT- like domain as it is much smaller, ~5 kDa, and shares ~60% HECT domain homology with UBE3A. The

11 UBE3D HECT-like domain contains the active cysteine residue common to all HECT domains. The UBE3D protein also contains an interaction region with UBE2C at position 235-257 on the C-terminal region of the protein. As previously stated, the E2 conjugating enzyme binding site for E1 and E3 interaction overlap causing Ub chain building to occur through a cascade of E1 and E3 binding and disassociation. The UBE2C binding site found on UBE3D allows for more efficient chain formation as UBE2C and UBE3D can maintain interaction and proximity. The current reported localization of UBE3D within the cell has been identified as cytoplasmic (Uniprot, nextprot, proteinatlas). Although very little has been reported on the characteristics and specific functional pathways of UBE3D, it has recently been implicated in Age-Related Macular Degeneration (AMD) (49).

1.2.2 Age-Related Macular Degeneration (AMD) and UBE3D

Age-related macular degeneration (AMD) is a multifaceted neurodegenerative disorder in which the photoreceptors as well as retinal pigment epithelial cells of the macula progressively degenerate over time. The macula is a region of the retina that controls central color and detailed vision and damage/malfunction in the cellular region leads to permanent central vision loss. AMD is a late-onset disease, with symptoms and progression arising slowly as the patient ages. For this reason AMD is diagnosed at ~50 years of age or older. AMD is also a highly prevalent disease as in the United States there are more than 10 million people affected and the prevalence is projected to increase to 15 million by 2020 (50). Risk factors for AMD include, obesity, smoking and familial history. To date there are very few treatment options, and those that are available focus more on delaying symptoms rather than fixative measures. For this reason early detection using mutagenic biomarkers remains a high priority.

As mentioned, AMD originates and affects the macular area of the retina. The macula is a region of the retina composed of different cell layers including a layer of sensitive photoreceptors, retinal pigment epithelial cells (RPE), the Burchs membrane followed by the choroid blood supply (from apical to basal retina, respectively). Over time as the eye ages lipids and debris released from RPE cell layer accumulate above and within the burchs membrane, these debris deposits are known as drusen. Drusen are an early sign of AMD but are mostly extracellular lipid cholesterol deposits although they are not very well described. It is hypothesized that drusen prevents proper oxygen and nutrients reaching the retina from the choroid blood vessels. In response, inflammatory cytokines (IL-6 and IL-8) are released into the blood, which recruits inflammatory cells to the macula. These inflammatory cells as well as RPE cells in response to oxidative stress then release vascular endothelial growth factors (VEGF). VEGF-A and VEGF-C cause choroid blood vessels to become angiogenic, thus creating choriocapillaries which invaginate the RPE cell

12 layer. (50). Choriocapillaries are abnormally leaky which allows fluid to seep into the layers of the macula. Fluid accumulates between the burchs membrane and the photoreceptors damage the delicate rod and cones cells. From here, if left untreated, bleeding due to this condition can cause scarring of the macula and permanent vision loss.

As mentioned, at the moment there are no effective treatment options to correct AMD, only to deal with symptoms or to slow disease progression. These include VEGF inhibitors and antioxidant vitamin and mineral supplements (AREDS). For these treatments to be as effective as possible diagnosis of early onset of AMD is crucial. Early onset has been identified through genetic AMD studies looking for loci accounting for heritability and risk factors (. The genes nearby AMD associated loci have been implicated in important biological pathways pertaining to the steps in AMD progression. The biological processes include: Cholesterol and lipid metabolism, oxidative stress pathway, complement pathway, angiogenesis and extracellular matrix pathway.

1.2.3 UBE3D implicated in East-Asian AMD

UBE3D has recently been implicated in relation to age-related macular degeneration within the East-Asian community (49). Huang et al performed full exome sequencing of non-synonymous single nucleotide variants (SNVs) on East-Asian AMD patients across different geographical cohorts. They were able to identify and validate known SNVs associated with AMD progression. The known AMD associated SNV’s identified were CFH, HTRA1, B3GALTL, C2, HMCN1, CFB, SKIV2L and validated on follow up cases and controls to confirm their results. One of their highest confidence novel missense SNV associated with AMD was found at the rs7739323 of UBE3D. The SNV conferred an alteration from a valine to a methionine at amino acid position 379. This positioning is located within the UBE3D HECT domain. The minor allele frequency (MAF), upon the initial China cohort (216 cases, 1,553 controls), of the rs773923 SNV was identified in 0.1406 of AMD cases and 0.2202 of controls (p-value 2.02 x 10-4). As well after separating AMD patients into different cohorts (Northern China, Beijing, Japan, Hong Kong) with increased cases and controls (3,772 and 6,942 total, respectively) the association between SNV rs7739323 in UBE3D and AMD was drastically strengthened (p-value: 1.46 x 10-9 odds ratio (OR)=0.74, 95% confidence interval (CI): 0.63–0.88).). SNV rs7739323 is therefore thought to be protective against AMD across the Asian cohorts compared to AMD cases.

Knockout mouse models for UBE3D were also created. The homozygous UBE3D-/- mouse was not viable. This seems to further confirm UBE3D as an essential protein as identified previously (51). The

13 heterozygous knockout mouse model for UBE3D (UBE3D+/-) was viable (49). The UBE3D+/- mice showed decreased retinal function using electroretinography and an abnormal amount of pigment granules was found in the retinal pigment epithelium/microvilli using electron microscopy. Increased pigment granules in retina is a common symptom of AMD. The findings thus demonstrate that UBE3D and the ubiquitin-proteasome system may play a role in AMD pathogenic progression.

1.3 Cilia, Dynein Motors and associated proteins

1.3.2 Cilia

Cilia are finger-like projections on the cellular surface. They are organelles that are microtubule-based projections and are found on nearly all cell types in vertebrates. The structure of a cilium can be divided into zones extending from the cytoplasm to the tip of the cilia. These subcompartments, from inner most to outer most, include the basal body, transition zone, axoneme, ciliary extension and ciliary tip. Different cell types have either a single primary cilia or large groupings of cilia (200-300 cilia) (52, 53).

Tip Complex Retrograde

Anterograde Distal Appendages

Subdistal Basal Body Appendages Daughter Centriole

Heterotrimertic IFT-Dynein IFT complex B IFT complex A Kinesin

Microtubule IFT Cargo IFT Cargo Centriolar Satellites BBS

Figure 1.1.2

Cellular cilia depiction demonstrating ciliary protein turnover via intraflagellar transport. Centriolar satellite proteins, responsible for ciliation initiation as well as ciliary maintenance are transported along microtubules via cytoplasmic dynein.

15 Cilia are complex organelles and are only assembled upon exit from the cell cycle, thus entry into the cell cycle causes cilia resorption (54, 55). The formation of cilia, ciliogenesis, initiates in response to specific proteins being targeted to the basal body. The basal body is where the pre-assembly of protein complexes needed for ciliary extension occurs (such as dynein arm assembly) (55). Cilia have many receptors along the ciliary membrane (ion channels, cilia specific receptors and signaling molecules). These receptors are used for recognizing extracellular sensory stimuli. The movement of proteins required for cilia extension, maintenance and resorption is referred to as intraflagellar transport (IFT) (56). IFT occurs along the axoneme where proteins are first loaded onto transport machinery such as kinesin or dynein motors at the ciliary cytoplasmic base.

Cilia can either be motile or immotile. The motility of cilia can be used for cellular locomotion (flagella) or for sweeping fluid across the extracellular surface (mucus movement in trachea). The motility aspect of a cilium is dependent on the structure of the axoneme. Motile cilia consist of 9 outer microtubule doublets, which surround two central single microtubules (9+2) and contain many structures used for cilia motility (example: Radial spokes and inner and outer dynein arms). The axonemal dynein motors are attached to the 9 outer microtubules. As previously mentioned, axonemal dynein heavy chains are able to exert ATP-driven ciliary movement. This movement is generated through harmonized activation and inactivation of the inner and outer dynein arms of the axonemal dynein motors along the axoneme.

Non-motile cilia similarly contain axonemes made of 9 outer microtubule doublets but lack the central microtubules (9+0) and outer and inner dynein arms. Non- Motile cilia are strictly for sensory of extracellular stimuli due to previously mentioned receptors found along the ciliary membrane. Different receptors found along the cilia plasma membrane, upon recognition of a specific stimuli, can be converted into signaling cascades. The signal cascades are transduced to the cellular body via microtubule-based retrograde transport by IFT dynein motors. Some of these signaling pathways include non-canonical Wnt signaling, platelet derived growth factor receptors, and hedgehog signaling (57, 58) (59).

Defects in cilia (known as ciliopathies) have been implicated in a wide variety of human diseases. These diseases affect respiratory, kidney, lymphoid, bladder and retinal functions. For example malfunctions in IFT have been shown to lead to retinal degeneration. Retinal photoreceptors connect their inner segment (nucleus) and outer (photo pigment membrane stacks) segment with a 9+0 cilium. Turnover and maintenance of photoreceptor proteins must be transported by IFT through the cilium. One symptom of Bardet Biedl Syndrome (BBS) is retinal degeneration (rod-cone dystrophy). All analyzed BBS proteins

16 are localized to the ciliary base or axoneme and are involved in IFT or cilia formation. As well, in Alström syndrome, retinitis pigmentosa (found in BBS) is a common feature which is attributed to the ALMS1 gene involvement in IFT.

Satellite proteins are localized around the centrosome and basal body of cilia. They are microtubule- associated membraneless electron dense granules of proteins, approximately 70 – 100 nm. The initial satellite protein identified was Pericentriolar Material 1 (PCM1) which is a large coiled-coil. Other satellite proteins have been identified based on co-immunoprecipitation/co-localization with PCM1 (86).

PCM1 considered essential in centriolar satellites as shown by satellite proteins losing their granularity upon PCM1 deletion or depletion. In retinal pigment epithelial (RPE-1) cells, PCM1 has been shown to be required for cilia formation. PCM1 is believed to prevent essential cilia protein degradation, TALPID3, by MIB1. Centriolar satellites are crucial regulators of cilia and centrosome function but exact mechanism is not fully characterized. There are three known E3 ubiquitin ligases that are satellite associated (HERC2 and MYCBP2) (86).

1.3.3 Dynein motors

Dynein motors are microtubule associated multi-protein complexes that are crucial for intracellular transport, cell division and cellular motility. Dynein motors are made up of several dynein chain proteins classified into heavy, intermediate and light. They are generally made out of 2 or more heavy chain proteins, which are responsible for the power stroke needed for locomotion. Heavy dynein chains contain a AAA+ domain which hydrolyses ATP creating the force generation along the microtubule (60).The intermediate and light chains bind the heavy chains and are responsible for cargo binding. There are three major classes of dynein motors: cytoplasmic dynein, IFT dynein and axonemal dynein. Cytoplasmic dynein and IFT dynein are responsible for intracellular transport along microtubules towards their minus end usually located near the nucleus of quiescent cells (61). Whereas axonemal dynein are the driving force in cilia motility (62).

Cytoplasmic dynein is a large protein complex (~1.5MDa) and is built around a large heavy chain (DYNC1H1) homodimer creating a double headed motor. It plays a crucial role in many diverse cellular processes throughout the cell cycle and quiescence. Its overarching mechanism is microtubule-based transport of organelles, tubules, vesicles, and molecules toward the microtubule negative end. This

17 includes mitochondria, lysosomes, lipid droplets, endosomes, cytoskeleton components, centrosome proteins and vesicles destined from the endoplasmic reticulum to the golgi apparatus. Cytoplasmic dynein has also be shown to play an important role in cell division (61). This includes proper Golgi, spindle and centrosome positioning during mitosis, spindle assembly for chromosome separation as well as acting as a checkpoint protein to ensure proper chromosome separation. This is accomplished through pulling microtubules associated with the organelles (60).

IFT dynein is assembled around a dynein heavy chain homodimer (DYNC2H1). It shares almost the same overarching role as cytoplasmic dynein in that its function is negative end microtubule-based transport but it has only been shown to function along the ciliary axoneme. The ciliary tip is where the positive ends of microtubules reside so IFT dynein is responsible for retrograde transport along the axoneme. Constant turnover of ciliary proteins is needed for signal transduction as well as maintenance. The kinesin 2 family are responsible for carrying new ciliary proteins as well as IFT dynein motors to the ciliary tip. After release of cargo proteins, IFT dynein is then responsible for trafficking proteins as well as kinesin to the ciliary base for protein turnover (63, 64).

The third class of dyneins, called axonemal dyneins, play a very different role than other dyneins and are structured differently as well. Axonemal dynein motors are more complex than other dynein motors. Outer arms consist of three heavy chains (α-, β- and γ-chain) and ~15 light-intermediate chains, and the inner arms comprise eight different dynein heavy chains. Axonemal dyneins are found within the axoneme of motile cilia and/or flagella. Their function is to cause directed movement of the cilia. As previously mentioned motile cilia axonemes consist of 9 outer microtubule doublets and a pair of central microtubules (9+2). Axonemal dyneins are categorized into outer and inner motors depending on positioning. The coordinated activation and inactivation of these opposing motors creates motility of microtubules of the axoneme (65, 66).

1.3.4 DNAAF2 and the assembly of axonemal dynein motors

The gene DNAAF2 (PF13/Kintoun) encodes a ~91kDa (873 amino acid) protein called Dynein Axonemal Assembly Factor 2 (DNAAF2). DNAAF2 is a member of the PIH1 family as it contains a PIH domain, which is necessary for DNAAF2 to interact with the HSP90 chaperone proteins. DNAAF2 has been reported to be localized in the cytosol as well as at the centrosome/cilia organization center.

DNAAF2 is responsible, in conjunction with other members of the DNAAF (PIH domain containing) family, to pre-assemble axonemal dynein motors in the cytoplasm prior to these complexes being

18 transported and assembled into the axoneme in motile cilia. It is hypothesized that DNAAF1, DNAAF2 and DNAAF3 in conjunction with HSP90/HSP70 chaperones are responsible for building these complex axonemal dynein motors. For the outer dynein arms, DNAAF2 specifically has shown to be responsible for proper folding of the three dynein heavy chain heads and assembly and stability of the dynein heavy chains (62).

DNAAF2 has been reported to interact with members of the HSP90 and HSP70 family members via its PIH domain. The HSP interaction acts as a chaperone for proper folding of dynein heavy chain for outer axonemal dynein arms by DNAAF2/DNAAF1. DNAAF2 has been shown to interact with DNAAF1, DNAAF3 and DYX1C1, which are required for proper assembly of axonemal dynein arms in the cytoplasm. DNAAF2 has also been hypothesized to play other roles in cells not containing motile cilia. DNAAF2 compared to DNAAF1 and DNAAF3 is expressed in all tissue types whereas DNAAF1 and DNAAF3 are only found in cell type containing motile cilia (lymphoid, Lung, breast, bladder)(Protein atlas). DNAAF2 has also been reported to interact with RUVBL1 and RUVBL2 (RuvB-Like-1/2). These proteins are involved in transcriptional activation, DNA repair, spermatogenesis and deubiquitylation. It is unclear how DNAAF2 and RUVBL family interact.

1.4 Identifying protein:protein interactions using BioID coupled to mass spectrometry

BioID is a technique that allows for identification of proteins in proximity with a chosen protein of interest through their biotin labeling (biotinylation) (67). A biotin ligase (BirA) found in Escherichia coli has been specifically mutated so that it promiscuously biotinylates nearby proteins via their primary amine group. The mutated biotin ligase (BirA*) loses its ability to retain bound activated biotin. Thus, BirA* prematurely releases activated biotin within a ~10nm vicinity (68). Activated biotin is highly reactive, and thus the cloud of activated biotin follows BirA* in vivo and covalently labels nearby proteins (69). Due to the covalent nature of biotinylation it allows for harsh cellular lysis conditions which are optimal for solubilization of all proteins (including membrane and inter-organelle proteins). Proteins that are biotin- labeled can be enriched through affinity purification using streptavidin-sepharose beads and then identified through LC-MS/MS.

To perform BioID for a specific protein the BirA* is fused in frame to the protein of interest and then inducibly expressed within cells in vivo). The Flp-In T-Rex system by Invitrogen allows for inducible expression of the fused BirA* and protein of interest in the presence of tetracycline. This system has

19 been successfully used across many mammalian cell lines (70). Centrosome and cilia are mostly insoluble, which has created issues in identifying a comprehensive protein-protein interaction network. The BioID technique has allowed novel insight in this system (89).

1.4.2 Analysis of protein interaction data

The BioID protocol upon LC-MS/MS analysis produces massive datasets of peptides identified in a given sample. To easily visualize the identified proteins as well as comprehend which are considered false positives and which are true interactors several statistical tools have recently been developed and implemented.

Prohits (71) is a software program that is able to perform simple comparisons between samples. It is also useful to readily visualize interactors identified through controls and those specific to your protein of interest (bait protein). Data generated on different MS instruments can also be compared and analyzed through Prohits. The initial peptide data from the MS must first be searched on Prohits comparing to known genome datasets (XTandem) as well as predictive analysis to more accurately identify the correct protein based on the peptide. The Prohits analyst module allows for these identified genes to be qualitatively viewed for easier analysis as well as be quantified based on the peptide spectral count found in your sample. Each identified peptide can also be analyzed for post-translational modifications on residues, such as the diglycine tag associated with Ub linkages, which remain on residues after tryptic digestion. Prohits also allows for filtering of data based on search thresholds, contaminant lists, and removal of non-specific background compared to control samples. This filtering allows for peptide and protein specific confidence scores to rule out false positives.

A secondary tool found within Prohits is called Significance Analysis of INTeractomes (SAINT)(72)(73). SAINT uses a Baysian algorithm to differentiate between what should be considered a high confidence interactor and background. SAINT compares sample data to that of several controls. Control samples consist of BioID data from cells only expressing the BirA* alone, cells expressing unrelated bait proteins as well as untransfected parental cells. These are used to identify false positives, such as proteins that are naturally biotinylated in the cells, interactors specific to the BirA* tag and not the bait protein as well as proteins that non-specifically stick to the sepharose beads. The SAINT algorithm first compares spectral counts for each bait-prey pair between samples. Each BioID bait sample is independently prepared in two biological replicates and each are then analyzed on the MS twice, as two technical replicates, thus creating four separate MS data analyses per bait. SAINT also compares spectral counts between all bait-prey pairs

20 compared to controls. SAINT provides a score ranging from 0 to 1. Due to large diversity between BioID experiments based on bait protein unique characteristics (localization, pathway involvement, protein structure) as well as cell type and experimental characteristics (MS settings and cell preparation) SAINT scores deemed high confidence also differ from experiment to experiment. To narrow the interactome to only high confidence interactors SAINT calculates a Bayesian FDR (BFDR) which is dataset-independent. A BFDR of 0.02 (i.e. a 2% false discovery rate) is applied in most cases (69).

Within ProHits Analyst module-Comparison page you are able to analyse peptide comparisons of each hit which allows the user to view all peptides that are present. Molecular weight modifications can be identified on specific peptides.

1.5 Research hypothesis and thesis outline

We hypothesize that a better understanding of the poorly characterized member of the HECT E3 ligase family, UBE3D, can be achieved by determining the Ub linkages created by this E3 and identifying its interacting partners. In the results section I investigate the UBE3D directed Ub linkages (section 2.1), the UBE3D interactome (section 2.2) and further investigate one UBE3D interactor, DNAAF2 (section 2.3).

2 Results 2.1 Identifying the Ub Chain Building Activity of UBE3D

To identify the Ub chain specificity of UBE3D I performed an in vitro autoubiquitylation assay and then analyzed the products by western blot and mass spectrometry.

2.1.2 Conducting an E2-UBE3D HECT E3 functional analysis

To determine the functional interactions between the E2 conjugating enzyme UBE2D3 and the UBE3D E3 ligase I used an in vitro autoubiquitylation assay. After the autoubiquitylation assay with the GST- tagged HECT domain of UBE3D (described in detail, section 2.1.4), each reaction mixture was resolved via SDS-PAGE, and ubiquitylated products were detected by western blotting using anti-Ub antibody (Thermofisher)(Figure 2.1.1).

A reaction was deemed successful when the positive control reaction lead to Ub migration in SDS- PAGE as an anti-Ub smear at a molecular weight (MW) greater than 50kDa (Figure 2.1.1). The positive control was purchased ITCH HECT E3 domain. This demonstrated that the reaction was conducted under ideal conditions as this E3 ligase was able to produce a wide array of high quantity both large and short Ub chains. The negative control demonstrated that in the absence of a HECT-domain present in the reaction there was no production of any Ub chains, demonstrating the E2 conjugating enzyme UBE2D3 is unable to produce Ub chains without the presence of a HECT domain. To classify length-type of the Ub linkages resulting from the E3 of interest, anti-Ub smears were classified into three categories (44): long Ub chains (MW>125kDa), short or monoubiquitylated Ub oligomers (MW<125kDa) or lack/failed of E3-E2 interaction demonstrated by absence of anti-Ub smear. Western blots for GST were also conducted to control for the amount of GST-E3 domain loaded into the reaction.

The UBE3D HECT domain displayed a functional interaction with the E2 conjugating enzyme UBE2D3, producing both long and short Ub chains. Loading 250ng of GST tagged UBE3D HECT domain into the reaction was shown to have proportionally higher Ub chain production as compared to when only 100ng was loaded in the reaction (Figure 2.1.1).

21 22

Figure 2.1.1: Western Blot analysis of UBE3D autoubiquitylation reaction. An autoubiquitylation reaction was performed with the E2 conjugating enzyme UBE2D3 and the UBE3D E3 ligase, and the reactions were then resolved on SDS-PAGE and blotted with an anti-Ub antibody (left panel). Lane 1: Autoubiquitylation reaction using 250ng of purified GST tagged UBE3D HECT domain; Lane 2: Autoubiquitylation reaction using 100ng of purified GST tagged UBE3D HECT domain; Lane 3: Molecular weight ladder (Thermo Fisher); Lane 4: Negative control, no HECT domain included in the reaction (No E3); Lane 5: Positive control, ITCH E3 ligase used in the reaction (ITCH). The reactions were also run on SDS-PAGE and blotted with an anti-GST antibody (right panel). Lane 1: Molecular weight ladder (Thermo Fisher); Lane 2: Autoubiquitylation reaction using 250ng of purified UBE3D HECT domain; Lane 3: Autoubiquitylation reaction using 100ng of purified UBE3D HECT domain. The western blots shown are representative of 4 separate replicates with successful reactions.

Figure 2.1.2: Prohits peptide comparison and modification analysis. Represented are three separate MS runs of successful autoubiquitylation reactions (UBE3D – 100ng, UBE3D – 250ng and ITCH respectively) looking specifically at modifications to the each Ub , trypsin cleaveged, gene amino acid peptide (A). The black numbers within the olive boxes represent spectral counts (B) for specific peptide sequences (C) found within Ub protein. (D) Represents previously described modifications to specific peptide sequences (Modifications include: DiGlycine (K); Acetylation (Protein N-term); deamination (N,Q) and variable oxidation (M). This enables the reader to visualize where within the protein, the modifications exist (E). The UBE3D HECT domain (predicted 5.08kDa) tagged with Glutathione S-Transferase (GST, Predicted 26kDa) together form a GST-UBE3D HECT domain structure with a summative predicted molecular size of ~32kDa. To confirm proper size and amount of GST-fusion protein, a western blot using an anti-GST antibody was performed (Figure 2.1.1, right panel).

24 2.1.3 Characterization of Ub chain linkages in UBE2D3 E2 – UBE3D HECT E3 reactions

To determine if the HECT domain can specify Ub chain linkage types, autoubiquitylation reactions deemed successful (via SDS-PAGE anti-Ub smears migrating >50-125125kDa) were then subjected to tryptic proteolysis. The tryptic peptides were analyzed using nanoflow liquid chromatography electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS). The Ub linkage specificity was recognized using database searching by including a mass shift equivalent to the Ub diglycine remaining on the lysine following tryptic digest (+114.0492) (74). The Ub linkage specificity was also identified using a spectral library of Ub/Ub-lysine modifications derived from all 7 of the Ub chain linkage types (75).

Analysis of the UBE3D-HECT domain autoubiquitylation assay demonstrated that K63 diglycine modifications were identified in the samples (Figure 2.1.2E). Thus, this demonstrates that K63 chains were the only Ub linkage identified via this methodology for UBE3D.

2.1.4 Summary

These results show that the UBE3D-HECT domain has the ability of creating Ub linkage, through K63, without the full protein present. Ub K63 linkages, as previously described, have been shown to contribute to the regulation of endocytosis, aggresome formation and DNA damage response (Figure 2.1.2) (15); (76).

2.1.5 Experimental Details

Plasmids

UBE3A, UBE3B, UBE3C, UBE3D, UBE3D-AMD* HECT domain open reading frames were amplified by PCR from pcDNA5 and inserted using the Addgene restriction enzyme buffer system into a pGEX6P1 vector containing a GST-tag, with Not1 and Sma1 located upstream (N-terminal) of the cDNA insert.

UBE2D1, UBE2D2, UBE2D3 open reading frames were amplified by PCR from pcDNA5 and inserted using the Infusion system into a pET28 vector containing a 6xHis-tag using Ndel and Xhol restriction site located upstream of the cDNA insert.

25 Protein purification

Proteins were expressed in E. coli BL21 (DE3) Gold (Stratagene, LaJolla, CA) grown in 5ml Terrific Broth (TB) medium with 50 µg/ml ampicillin at 37ºC for 16-18 h. They were then added and grown in 500ml-1L TB to an OD600 of 0.8-1 and then expression of the tagged cDNA induced with 0.1 mM IPTG (isopropyl-1-thio-D-galactopyranoside) and further grown for 16-18 h at 12ºC. Following induction bacteria were pelleted and washed using PBS and re-pelleted and stored at -80°C.

Pellets were resuspended in ice cold PBS with 2% Triton X-100 (25ml per 250ml pellet). To remove proteins aggregations, samples were sonicated (4-6x 30 second bursts, amplitude 35 with 1 minute rest on ice between sonications). Samples were transferred to a 40ml tube and centrifuged at 4000rpm for 30 minutes at 4°C. Supernatant was transferred to a tube containing 150ul prewashed (in PBS) MagneGST beads, 1ul benzonase nuclease (Novagen) and 40ul protease inhibitor cocktail (Sigma-Aldrich) and incubated at 4oC for 3 h end-over end. Beads were then pelleted and washed 3 times in cold PBS. 600ul GST elution buffer was added to the beads and incubated end-over end for 30minutes at 4oC. Supernatant was collected, dialyzed against 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 2 mM dithiothreitol (DTT) and stored at -80°C.

GST elution buffer: 150mM NaCl, 50mM Tris-HCl pH8.0, 930mg dry glutathione, 40mL water with a final pH of 8.0. Buffer was filtered through a 45um syringe filter.

Autoubiquitylation Assays

E3 HECT domain and E2 protein quantification assays were carried out in a volume of 10µl protein purification sample containing 2µl 2x loading dye (ThermoScientific). Reaction mixtures were boiled for 3 minutes at 95ºC, then separated by 10-15% gradient SDS-PAGE gels (Invitrogen, Grand Island, NY) along with a BSA concentration gradient of 250ng, 500ng, 750ng, 1 µg, 1.5 µg, and visualized by Coomassie Brilliant Blue (ThermoFisher).

Autoubiquitylation reactions were performed in a volume of 60 µL in a buffer of 50 mM Tris pH 7.5, 5 mM MgCl2, 2 mM ATP and 2 mM DTT, containing human recombinant E1 (50ng; Boston Biochem, Cambridge, MA), E2 (6xHis tagged, 100ng), ubiquitin (5µg), and E3 (GST-tagged HECT domain proteins, 0.5µg). After incubation at 30°C for 90 min, 20 µl reactions were stopped by addition of 10 µl

26 loading dye and boiling at 95ºC for 3 minutes and 40 µl samples were lyophilized by speedvac. 20µl samples (including positive and negative controls) were then resolved on 10% SDS-PAGE gels and visualized by western blotting, using a mouse monoclonal antibody directed against Ub (ThermoFisher), an HRP-conjugated goat-anti-mouse secondary antibody, and ECL (Bio-Rad, Hercules, CA).

Samples for MS analysis: 40 µl lyophilized sample was resuspended in 50mM ammonium bicarbonate and 2 µl Trypsin at 37ºC for 16-18 h. Samples were then subjected to another 1 µl Trypsin at 37ºC for 2 hours and then lyophilized by speedvac in preparation for MS analysis.

Controls included:

Negative controls: Absence of E3 HECT Domain: Performed in a volume of 20 µL in a buffer of 50 mM

Tris pH 7.5, 5 mM MgCl2, 2 mM ATP and 2 mM DTT, containing human recombinant E1 (50ng; Boston Biochem, Cambridge, MA), E2 (6xHis tagged, 100ng), ubiquitin (5µg). After incubation at 30°C for 90 min, 20 µl reactions were stopped by addition of 10 µl loading dye and boiling at 95ºC for 3 minutes.

Absence of Ub: Performed in a volume of 20 µL in a buffer of 50 mM Tris pH 7.5, 5 mM MgCl2, 2 mM ATP and 2 mM DTT, containing human recombinant E1 (50ng; Boston Biochem, Cambridge, MA), E2 (6xHis tagged, 100ng), E3 (GST-tagged HECT domain proteins, 0.5µg). After incubation at 30°C for 90 min, 20 µl reactions were stopped by addition of 10 µl loading dye and boiling at 95ºC for 3 minutes.

Positive control: Presence of ITCH E3 HECT Domain: Performed in a volume of 20 µL in a buffer of 50 mM Tris pH 7.5, 5 mM MgCl2, 2 mM ATP and 2 mM DTT, containing human recombinant E1 (50ng; Boston Biochem, Cambridge, MA), E2 (6xHis tagged, 100ng), ubiquitin (5µg) and ITCH E3 GST-tagged HECT domain proteins (0.5µg). After incubation at 30°C for 90 min, 20 µl reactions were stopped by addition of 10 µl loading dye and boiling at 95ºC for 3 minutes.

Mass Spectrometry

For mass spectrometric analysis, autoubiquitilation reactions were scaled up 2-fold. Samples were lyophilized by speedvac for 16-18 h. They were then resuspended in 50mM ammonium bicarbonate with 11 ul ul trypsin at 37°C for 12-16h. They were then subjected to 1 ul of trypsin at 37°C for 2 h. Samples were then lyophilized in a vacuum-centrifuge in preparation for MS analysis.

The analytical LC column (Acclaim PepMapTM RSLC nanoViper (75mm x 50cm, 3 mm) and pre column (Acclaim PepMapTM 100 nanoViper (75mm x 2cm, 3 mm) were made in lab. They were made from fused

27 silica capillary tubing (InnovaQuartz) and filled with 100Å C18 silica particles (Magic, Michrom Bioresesources). Lyophilized samples were resuspended in 0.1% HCOOH and then centrifuged at 13,000 rpm for 30 minutes. Samples were separated by nanoflow liquid chromatography with a 90-minute reverse phase buffer gradient (10-30% acetonitrile, 0.1% HCOOH mobile phase). Then the Proxeon EASY-nLC pump, running at 250nL/min, sprayed into a Q Exactive HF (ThermoFisher) orbitrap mass spectrometer. The Orbitrap performed the initial parent ion scan (within 390-1800 m/z range; 60,000 FWHM resolution @ 200 m/z). For MS/MS HCD fragmentation, up to the twenty highest intensity peaks were selected (minimum ion count of 1000 for activation). Q Exactive ion trap detected the fragmented ions with an exclusion list of 5 seconds.

Protein Identification:

.RAW files from LC-MS/MS peptide analysis converted into .mzXML format using Proteowizard. They were searched using X!Tandem and Comet which compares peptide sequences from the samples to the Human RefSeq Version 45. X!Tandem (77); (78), a part of the Trans-Protein Pipeline (TPP), used a parent MS tolerance of 15ppm on MS and MS/MS fragment tolerance of 0.4Da for MS/MS (up to two missed trypsin cleavages). Each bait was analyzed using two biological replicates, and each biological replicate was analysed in two technical replicates (therefore 4 samples for each bait and induction condition) (79). A Protein Prophet threshold cut off of 0.85 was implemented and proteins which passed were analyzed by SAINT version 3.3 (72), (73) with a unique peptide of 2+. SAINT was configured so total peptides> 2, nburn 2,000, niter 5,000, lowMode 0, minFold 1, normalize 0.

2.2 Identifying potential UBE3D Protein Interactors

As previously stated, little is known about the functional characteristics and specific biological pathways that UBE3D is involved in. Using the BioID technique our goal was to better understand the gaps in knowledge about UBE3D.

2.2.2 Using BioID to identify UBE3D protein interactors

As described in detail below (section 2.2.5), Flag-BirA* was fused to the N-terminal end of UBE3D. This Flag-BirA*-UBE3D construct was introduced, stably selected and then expressed in response to tetracycline using the Flp-In system in HEK 293 Flp-In T-REx cells. After exogenous biotin was added

28 to the media in the presence of tetracycline, cells were harvested for BioID based affinity-purification mass spectrometry analysis using streptavidin to pull-down biotinylated proteins in proximity to the bait Flag-BirA*-UBE3D.

2475 proteins were identified in at least 1 of the 4 BioID replicates by LC-MS/MS analysis. To narrow this massive dataset certain statistical cut offs were used. These were filtered using SAINT (72) by comparing the data to 16 negative controls, in which BioID was performed on cells expressing the FLAG-BirA* tag alone. Interactions with AverageP>0.8 and MaximumP>0.9 and average spectral counts >5 were considered part of the bait proximity interactome. A SAINT score cut off of 0.85 (~2% false discovery rate) was implemented, which decreased the list of putative interactors to 246 proteins. 55 out of 246 unique proteins found in this interactome have been previously reported to be associated with the microtubule and ciliogenesis organization center (Go enrichment) (Figure 2.2.1).

As an internal positive control for BioID protocol efficacy it is expected that the Bait protein (UBE3D, in this case) be identified in the spectral count data. The number of peptides detected will depend on how many tryptic sites are present in the protein as well as the size of the protein. This was confirmed as the UBE3D spectral counts across all 4 MS/MS runs averaged 472 as compared to only containing 1 UBE3D peptide in the spectral count from the BirA* alone control (Table 2.2.1).

Figure 2.2.1: Protein proximity interaction network for UBE3D HECT E3

Self-organized BioID interaction map for bait protein UBE3D (based on 4 MS runs which is made up of 246 unique polypeptides. Bait Protein represented by yellow node, proximity interactors represented by blue nodes. Interaction nodes separated based on biological functionality through PANTHER/Gene ontology Database: Ciliation; Cytoskeletal; Satellite; Microtubule organization center and Other. CYTOSCAPE (To visualize the protein interaction network for UBE3E bait protein, Cytoscape 3.6.0 was used (http://www.cytoscape.org).

TABLE 2.2.1

Table demonstrating comparative analysis of self-selected interactor spectral counts for BioID proximity ligation for bait UBE3D. A: Gene ID associated with spectral counts in corresponding row B: Control column demonstrating highest spectral count for associated gene from 10+ non-specific control runs; C: Corresponds to BioID spectral counts for the biological replicates and subsequent technical replicates of those biological replicates for Bait Protein UBE3D; D: Corresponding SAINT score associated with each gene ID.

31 The top interactor, indicated through a SAINT score of 1, as well as the highest spectral count-fold change compared to the controls, was DNAAF2. The confidence in this potential UBE3D:DNAAF2 interaction is further increased as several of the known DNAAF2 interactors were also subsequently identified in the UBE3D BioID interactome: Hsp90AA1, Hsp90AB1, and HSPA4 (Protein atlas, Nextprot and STRING Database)

Many of the top UBE3D interactors, defined by both SAINT score as well as spectral count fold change compared to the control spectral count for each protein seemed to be involved in either or both the cytoskeletal/microtubule organization center and ciliogenesis.

These included: - CYLD: A K63 deubiquitinase required for initiation and maintenance of cilia (80) - ALMS1 (alstrom syndrome protein 1): Required for pericentriolar material (PCM1) dependent intracellular transport; and for ciliogenesis initiation and cilia maintenance (81) - OFD1: Centriolar satellite protein and a negative regulator of ciliogenesis (82) - CEP192: rRequired for proper assembly of pericentriolar material (83) - STIP1 (stress induced phospho-protein): Coordinates the function of HSP70 and HSP90 protein folding, both required as chaperone proteins involved in ciliogenesis (84)

2.2.3 Using IF to identify UBE3D subcellular localization

To identify localization of UBE3D in the Flag-BirA*UBE3D tagged HEK293 stable cell lines used for the BioID protocol, they were grown in 6-well culture plates on glass coverslips. They were then either treated with DMSO to act as a negative control or with tetracycline (1ug/mL) for 24 hours to induce Flag- BirA*UBE3D expression for IF analysis. The FLAG-BirA*UBE3D HEK293 cell line was probed with anti-FLAG as well as antibody to a satellite protein marker (anti-PCM1) for IF analysis. These cells were also collected and lysed for western blot analysis to confirm bait expression.

Proper expression in response to 24 hour tetracycline induction of Flag-BirA* tagged UBE3D was demonstrated in HEK293 T-REx cells (Figure 2.2.2A; Lane 4). The negative control, which consisted of the same cells not treated with tetracycline, demonstrated no FLAG-BirA* expression (Figure 2.2.2A; Lane 2). IF analysis of FLAG-BirA*-UBE3D expression was imaged using anti-FLAG and was shown

32 to co-localize with PCM1 (anti-PCM1). This localization data coincides with the UBE3D interactome containing several satellite proteins as top putative interactors (e.g. OFD1, CEP192).

Figure 2.2.2: IF of FLAG-BirA*-UBE3D (Anti-Flag) in the HEK293 inducible cell line. A: IF analysis of HEK293 T-Rex cells with tetracycline induced Flag-BirA*-UBE3D expression with the corresponding antibodies under cycling, non-ciliation conditions. B: Anti-Flag Western blot: Lane 1: BioRad Precision Plus Protein Ladder; Lane 2: Cycling HEK293 T-Rex cells; Lane 3: 24hour Tetracycline-induced Flag- BirA* Tagged DNAAF2 HEK293 T-Rex cells; Lane 4: 24hour Tetracycline-induced Flag-BirA* Tagged UBE3D HEK293 T-Rex cells.

The negative control, which consisted of the same cells not treated with tetracycline, demonstrated no FLAG-BirA* expression (Figure 2.2.2A; Lane 2). IF analysis of FLAG-BirA*-UBE3D expression was imaged using anti-FLAG and was shown to co-localize with PCM1 (anti-PCM1). This localization data coincides with the UBE3D interactome containing several satellite proteins as top putative interactors (e.g. OFD1, CEP192).

To determine localization of endogenous UBE3D in RPE-1 cells as well as test the specificity of the anti- UBE3D antibody, IF and western blotting was performed on hTERT RPE-1 cells. This cell line was

33 chosen for IF analysis as they actively produce primary cilia. In addition, I performed the IF work in the Laurence Pelletier Lab (The Lunenfeld-Tanenbaum Research Institute), which routinely uses this cell line for IF characterization of cilia/centriolar proteins. hTERT RPE-1 cells were grown under non-ciliated conditions (cycling cells) on glass coverslips in 6-well culture plates for IF analysis and also collected for western blot analysis. The cells were prepared according to the immunofluorescense methods section (Triton extracted methanol fixation) and stained using anti-UBE3D, anti-PCM1, anti-gamma-tubulin and DAPI. Anti-PCM1 was chosen as FlagBirA* tagged UBE3D seemed to co-localize with satellite proteins, using PCM1 as a marker in HEK293 cells (Figure 2.2.3). Anti-gamma-tubulin is an antibody used for detection of centrioles. IF analysis using both PCM1 and gamma-tubulin antibodies was done to identify if endogenous UBE3D co-localizes with satellite proteins or centrioles.

UBE3D was demonstrated to have endogenous expression in cycling HeLa, HEK293 T-REx and hTERT RPE-1 cell lines using anti-UBE3D antibody (2.2.4A). Immunofluorescence analysis of UBE3D (anti- UBE3D) in hTERT RPE1 cycling cells (2.2.3) showed co-localization with satellite proteins (anti-PCM1), which was similar to IF results in HEK293 T-REx cells (2.2.1). To fully confirm UBE3D co-localized with satellite proteins (anti-PCM1) and not centrioles, IF images were captured in hTERT RPE-1 cells undergoing different steps of mitosis (prophase, metaphase, anaphase, telophase as well as interphase). This analysis was necessary as satellite proteins localize to centrioles as well as cellular periphery during interphase (85, 86) whereas during mitosis satellite proteins dissipate and centriolar proteins remain co- localized (86). IF during mitosis in hTERT RPE-1 cells demonstrated that UBE3D dissipates similarly to PCM1 (Figure 2.2.3.3 A white boxes) and is distinct from centrioles (anti-gamma tubulin) during the different steps of mitosis. Thus demonstrating that UBE3D must specifically co-localize with satellite proteins rather than centrioles.

34 A

Figure 2.2.3: IF analysis of UBE3D (anti-UBE3D) in cycling hTERT RPE1 cells A: Anti-UBE3D (company) Western blot confirming cell line expression: Lane 1: Cycling HeLa cells; Lane 2: Cycling HEK293 T-Rex cells; Lane 3: Cycling hTERT RPE1 cells; Below: Gamma-Tubulin control.B:

35 immunofluorescence analysis of UBE3D (anti-UBE3D) in hTERT RPE1 cycling cells triton extracted with methanol fixation with corresponding antibodies (anti-PCM1, anti-gamma-tubulin, DAPI).

Figure 2.2.4: IF analysis of UBE3D (anti-UBE3D) in mitotic hTERT RPE1 cells

A: Immunofluorescence analysis of UBE3D (anti-UBE3D) in hTERT RPE1 cells undergoing corresponding mitotic steps (descending: prophase; metaphase; anaphase; telophase) prepared by triton extracted with methanol fixation and corresponding antibodies (anti-PCM1, anti-Gamma Tubulin, DAPI).

37 2.2.4 Using siRNA to evaluate UBE3D function in ciliation

To evaluate whether UBE3D regulates ciliation, UBE3D knockdown was performed and analysed by western blotting (Figure 2.2.5A). UBE3D expression was evident in control cells (HeLa, HEK, RPE-1) and in RPE-1 cells treated with non-targeting siRNA specific for luciferase (GL2 duplex, negative control) and siRNA to Cep164, a centriole appendage protein required for cilia formation (positive control) (87). Figure 2.2.5A also demonstrates that silencer select siRNA #2 for UBE3D successfully knocked down the endogenous protein in hTERT RPE-1 cells, whereas silencer select UBE3D siRNA #1 and #3 were deemed unsuccessful.

The quantification of ciliation was performed under ciliation starvation conditions (n=3) as well as non- ciliation cycling conditions (n=2). The method for quantification of percent ciliation was conducted through manual visual counting of 300 cells (via nuclei, DAPI) and identifying how many cilia are present through anti-ARL13B staining (Cilia marker,).

Under ciliation starvation conditions three siRNA were tested for percent ciliation: Non-Targeting siRNA (GL2 Duplex), Cep164 siRNA and UBE3D siRNA. Under cycling conditions two siRNA were tested for percent ciliation: Non-Targeting siRNA (GL2 Duplex) and UBE3D siRNA (Figure 2.2.5 C).

About 45% of hTERT RPE-1 cells ciliate in response to serum starvation (previously described) (88). Figure 2.2.5B (left) and 2.2.5C (left) use of a non-targeting siRNA exemplifies this natural ciliation of hTERT RPE1 cells as they are shown to ciliate at 43% (300 cells, n=3). The positive control, Cep164 siRNA, demonstrated siRNA transfection efficacy as there were negligible cilia present upon knockdown (Figure 2.2.5C (left). An increased ciliation phenotype was identified upon successful UBE3D siRNA (Silencer Select #2) knockdown (Figure 2.2.5B (right) and 2.2.5C (left). hTERT RPE1 cells under ciliation starvation conditions demonstrated ~65% ciliation phenotype upon UBE3D siRNA knockdown in cells (300 cell counted, n=3). This demonstrates that UBE3D knockdown under ciliation starvation conditions leads to an increase in percent ciliation of ~22% (P-value 0.0044).

Only ~5-8% hTERT RPE-1 cells ciliate during cycling conditions (previously described) (88). In Figure 2.2.5C (right) the non-targeting siRNA (GL2 Duplex) exemplifies this natural ciliation of hTERT RPE1 cells as they are shown to ciliate at 5% (300 cells, n=2). An increased ciliation phenotype was identified upon successful UBE3D siRNA (Silencer Select #2) knockdown. hTERT RPE1 cells under cycling non- ciliation conditions demonstrated ~17% ciliation phenotype upon UBE3D siRNA knockdown in cells (300

38 cell counted, n=2). This demonstrates that UBE3D knockdown under non-ciliation cycling conditions leads to an increase in percent ciliation of ~12% (P-value 0.0009).

Alpha

Non Targeting SiRNA UBE3D SiRNA Knockdown

Fixing conditions: Triton conditions: Fixing extracted C B Triton conditions: Fixing extracted RPe1 Cells RPe1 - RPe1 Cells RPe1 methanol fixed - methanol fixed

DAPI/ARL13B Ciliated conditions DAPI/ARL13B Ciliated conditions

RPe1 UBE3D knockdown Ciliation: RPe1 UBE3D knockdown Ciliation: C Ciliation conditions Non-Ciliation conditions 70 20 60 * * 50 15 40 % ciliation 10 30 %ciliation N = 3 N = 2 20 5 300 cells 300 cells 10

0 0 *P-value: 0.0009 NT SiRNA UBE3D SiRNA2 Cep164 SiRNA * P-value: 0.0044 NT UBE3 SiRNA2

Figure 2.2.5 Analysis of UBE3D knockdown. A. Western blot analysis of UBE3D Silencer select siRNA efficacy in hTERT RPE-1 cells against anti-UBE3D: Lane 1: Anti-UBE3D Western blot confirming cell line expression and evaluating siRNA UBE3D efficacy in cycling hTERT RPE-1 cells: Lane 1: Non- targeting (NT) siRNA (GL2 Duplex); Lane 2: siRNA UBE3D 1 (Silencer Select); Lane 3: siRNA UBE3D 2 (Silencer Select); Lane 4 siRNA UBE3D 3 (Silencer Select); Lane 5: siRNA Cep 164; Below: anti-

40 Alpha-Tubulin control. B, C. IF quantification of hTERT RPE1 ciliation under ciliation and non-ciliation conditions, B: IF of hTERT RPE1 cell under ciliation conditions in response to non-targeting siRNA (GL2 duplex; left) and upon successful siRNA knockdown of UBE3D (right), stained with the cilia marker anti- ARL13B. Blue arrows identifying ARL13B stained cilia are indicated. C (left): Graph comparing percent- ciliation per 300 counted hTERT RPE-1 cells (n=3) under ciliation starvation conditions against non- targeting siRNA (GL2 duplex), successful UBE3D siRNA knockdown (silencer select #2) and Cep164 siRNA knockdown (positive control). C (right): Graph comparing percent-ciliation per 300 counted hTERT RPE-1 cells (n=2) under cycling non-ciliation conditions against non-targeting siRNA (GL2 duplex) and successful UBE3D siRNA knockdown (silencer select #2).

2.2.5 Summary

The UBE3D protein interactome was determined using mass-spectrometry coupled BioID analysis. The UBE3D interactome garnered novel insight into the localization of UBE3D, function and involvement in key pathways such as cellular ciliation. The intracellular localization of UBE3D was analysed in HEK293 T-REx and hTERT RPE1 cell lines showing that UBE3D co-localizes with satellite proteins. UBE3D siRNA knockdown was executed in hTERT RPE1 cells and an increased ciliation phenotype was identified. These results suggests further work must be performed to fully understand the connection of UBE3D with cilia.

2.2.6 Experimental Details

Expression Constructs

Using standing cloning procedures, UBE3D, DNAAF2, DNAAF2 C-term and DNAAF2 PIH Domain were cloned into the FLAG-BirA* pcDNA5/FRT/TO vectors for mammalian expression. Sequences were amplified by PCR from human teste cDNA. PCR products were digested using restriction sites Not1 and Asc1 and cloned into the appropriate plasmid using the Gateway system. The tags are all fused to the N- terminus of the open reading frame. For immunofluorescence purposes, full-length UBE3D and DNAAF2 as well as DNAAF2-PIH Domain and DNAAF2 C-terminal were cloned into GFP- and pcDNA5/FRT/TO vectors for mammalian expression.

Stable Cell lines

HEK293 Flp-In T-Rex cells (Invitrogen) were grown in media containing: Dulbecco’s Modified Eagle’s Medium (DMEM) with the addition of 10% fetal bovine serum (FBS) and penicillin-streptomycin (P/S). FLAG-BirA* fusion protein-expessing stable cell lines were selected using hygromycin (200ug/ml).

HeLa cells were grown in media containing: DMEM with the addition of 10% FBS and P/S. GFP-UBE3D cells lines for IF were selected for using hygromycin (200ug/ml).

42 hHERT RPE-1 cells were grown in media containing: DMEM/F12 with the addition of 10% FBS, 1xGlutaMax (35050-079, Thermo Fisher Scientific). All cells and cell-lines were cultured in an incubator at 37°C with 5% CO2.

BioID stable cells lines:

Transfection: Plasmids were co-transfected with Flp recombinase expression vector pOG44 into HEK293 Flp-In TREx cells. Transfections were executed using Lipofectamine 2000 (Invitrogen) system. Post- transfection, cells that incorporated the plasmid were selected using hygromycin (200ug/ml).

Characterization of cell lines:

BioID

To confirm proper fusion protein expression cells were subjected to three different induction conditions for 24 h. Cell lines were either incubated with: Tetracycline (1ug/ml, BioShop) and biotin (Sigma-Aldrich) (50uM) or no tetracycline or biotin added. Samples were then lysed using Laemmli SDS-sample buffer for western blot analysis. Samples were separated by SDS-PAGE gel (10%-15%) followed by transfer to a PVDF membrane (immoblion-P, Millipore). 1:1000 Mouse M2 anti-FLAG (Sigma-Aldrich) was used to identify FLAG-BirA* fusion proteins.

GFP:

To confirm proper fusion protein expression cells were subjected to three different induction conditions for 24 h. Cell lines were either incubated with: Tetracycline (1ug/ml, BioShop) or no tetracycline added. Samples were then lysed using Laemmli SDS-sample buffer for western blot analysis. Samples were separated by SDS-PAGE gel (10%-15%) followed by transfer to a PVDF membrane (immoblion-P, Millipore). 1:3000 Mouse GFP (Sigma-Aldrich) was used to identify GFP fusion proteins.

43 BioID Ciliation Conditions

To induce ciliation in HEK293 cells a serum starvation protocol was used. HEK293 cells ciliate in response to media lacking FBS (89). For ciliated conditions for BioID samples, HEK293 cells were grown to 80% confluency in growth medium and then were subjected to culture medium lacking FBS for 48 h. Post-48 h serum starvation cells were induced for an additional 24 h containing 1ug/ml tetracycline and 50uM biotin.

BioID protocol

Each BioID pellet was derived from 5 x 150mm2 cell culture plates of a specific HEK293 FLAG-BirA* fusion protein cells lines. Plates were grown to ~80% confluency and then subjected to 24 h of growth medium containing tetracycline (1ug/ml) or ciliation conditions (as previously described). Cell plates (post 24 h tetracycline induction) were collected and pelleted then washed with PBS and re-pelleted after 24 h tetracycline induction. Pellets were stored at -80°C up to 1 month.

Cell pellets were resuspended in 10ml RIPA buffer followed by 1 h 4°C end-over-end rotator. To disrupt any remaining cellular clumps, samples were subjected to brief sonication (2x bursts of 15% amplitude for 15 seconds). Samples were then centrifuged at 15,000 rpm for 30min at 4°C.

RIPA lysis buffer: 50mM Tris-HCL pH 7.5, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 0.1% SDS, 1:500 protease inhibitor cocktail (Sigma-Aldrich), 1:1000 benzonase nuclease (Novagen).

Supernatant was then transferred to a fresh 15ml tube and incubated for 3 h for 4°C end-over end rotation with 30ul pre-washed Streptavidin sepharose beads (GE). Beads were pelleted by centrifuge for 2 minutes at 2000rpm. Beads were transferred to a keratin free (KF) eppendorf tubes using fresh 50mM ammonium bicarbonate ~8 pH (ABC). Beads were washed 3 more times using 50mM ABC (pellet between washes, 30 seconds 3000rpm). Beads were transferred to a fresh tube and washed twice more and centrifuged for 5 minutes at 3000 rpm. Beads were then resuspended in 200ul 50mM ABC (50% fresh ABC + 50% old ABC ~pH 8.3) containing 1ug MS-grade TPCK trypsin (Promega, Madison, WI) and incubated 16-18 h at 37°C end-over end. Following incubation an additional 0.5ug MS-grade TPCK trypsin was added and incubated at 37°C waterbath for 2 h. Beads were pelleted by centrifuge (2min at 3000rpm). Supernatant was transferred to a fresh KF eppendorf tube and beads were washed twice with 150ul 50mM ABC and

44 supernatants were pooled with initial eluate. Whole sample was subjected to speedvac to be lyophilized (~8-16h). Lyophilized samples were then resuspended in 0.1% formic acid (buffer A). Each sample is run through MS twice for two technical replicates (1/5th sample shot per technical replicate). Each bait and condition was repeated in two biological replicates resulting in 4 MS runs per bait and condition.

Mass Spectrometry

BioID coupled to mass spectrometry and protein-protein interactions

BioID is a technique that allows for identification of proteins in proximity with a chosen protein of interest through their biotin labeling (biotinylation) (67). A biotin ligase (BirA) found in Escherichia coli has been specifically mutated so that it promiscuously biotinylates nearby proteins via their primary amine group. The mutated biotin ligase (BirA*) loses its ability to retain bound activated biotin. Thus, BirA* prematurely releases activated biotin within a ~10nm vicinity (67). Activated biotin is highly reactive, and thus the cloud of activated biotin follows BirA* in vivo and covalently labels nearby proteins (69). Due to the covalent nature of biotinylation it allows for harsh cellular lysis conditions which are optimal for solubilization of all proteins (including membrane and inter-organelle proteins). Proteins that are biotin labeled can be purified and then identified through LC-MS/MS.

To perform BioID for a specific protein the BirA* is fused using restriction enzymes to the protein of interest and be inducible in vivo. The Flp-In T-Rex system by Invitrogen allows for inducible expression of the fused BirA* and protein of interest in the presence of tetracycline. This system has been performed on many mammalian cell lines (70).

Mass Spectrometry

The analytical LC column (Acclaim PepMapTM RSLC nanoViper (75mm x 50cm, 3 mm) and pre column (Acclaim PepMapTM 100 nanoViper (75mm x 2cm, 3 mm) were made in lab. They were made from fused silica capillary tubing (InnovaQuartz) and filled with 100Å C18 silica particles (Magic, Michrom Bioresesources). Lyophilized samples were resuspended in 0.1% HCOOH and then centrifuged at 13,000 rpm for 30 minutes. Samples were separated by nanoflow liquid chromatography with a 90-minute reverse phase buffer gradient (10-30% acetonitrile, 0.1% HCOOH mobile phase). Then the Proxeon EASY-nLC

45 pump, running at 250nL/min, sprayed into a Q Exactive HF (ThermoFisher) orbitrap mass spectrometer. The Orbitrap performed the initial parent ion scan (within 390-1800 m/z range; 60,000 FWHM resolution @ 200 m/z). For MS/MS HCD fragmentation, up to the twenty highest intensity peaks were selected (minimum ion count of 1000 for activation). Q Exactive ion trap detected the fragmented ions with an exclusion list of 5 seconds.

Protein Identification

.RAW files from LC-MS/MS peptide analysis converted into .mzXML format using Proteowizard. They were searched using X!Tandem and Comet which compares samples to the Human RefSeq Version 45. X!Tandem (Beavis, 2006; Craig and Beavis, 2004) a part of the Trans-Protein Pipeline (TPP), used a parent MS tolerance of 15ppm on MS and MS/MS fragment tolerance of 0.4Da for MS/MS (up to two missed trypsin cleavages). Each bait was analyzed using two biological replicates and each biological replicate was repeated in two technical replicates (therefore 4 samples for each bait and induction condition) (79). A Protein Prophet threshold cut off of 0.85 was implemented and proteins which passed were analyzed by SAINT version 3.3 (72), (73) with a unique peptide of 2+. SAINT was configured so total peptides> 2, nburn 2,000, niter 5,000, lowMode 0, minFold 1, normalize 0.

Analysis of protein interaction data

The BioID protocol upon LC-MS/MS analysis produces massive datasets of the peptides that are identified in your sample. To easily visualize the identified genes as well as comprehend which are considered false positives and which are true interactors several statistical tools have recently been implemented.

Prohits (90) is a software program that is able to perform simple comparisons between samples as well as for better visualization between interactors identified through controls and those specific to your protein of interest (bait protein). Data from the MS can be stored and viewed through Prohits based on which machine the sample was shot. Prohits also contains many features to analyze and present the data. The initial peptide data from the MS must first be searched on Prohits comparing to known genome datasets (XTandem) as well as predictive analysis to more accurately identify the correct protein from the peptide. The Prohits analyst module allows for these identified genes to qualitatively viewed for easier analysis as well as be quantified based on peptide spectral count found in your sample. Each identified peptide can be analyzed for post-translational modification residues, such as the diglycine tag left through tryptic

46 digestion of Ub linkages. Prohits also allows for filtering of data based on search thresholds, contaminant lists, removal of non-specific background compared to control samples. This filtering allows for peptide and protein specific confidence scores to rule out false positives.

A secondary tool found within Prohits is called Significance Analysis of INTeractomes (SAINT)(71, 72); (73, 90). SAINT uses a statistical algorithm used to differentiate between what should be considered a high confidence interactor and background. SAINT compares sample data to that of several controls (n =+10). Control samples consist of BioID data from, cells only expressing the BirA* alone, cells expressing unrelated bait proteins as well as untransfected parental cells. These are used to identify proteins that a naturally biotinylated in the cells as well as interactors specific to the BirA* tag and not the bait protein. SAINTs algorithm first compares spectral counts for each bait-prey pair between repeated samples consisting of 2 biological replicates per bait and condition of which each are shot on the MS twice. SAINT also compares spectral counts between all bait-prey pair compared to those found in the controls. Through SAINT provides a score ranging from 0 to1 in which a SAINT score >0.79 are considered high confidence interactors for that bait protein (~2% false discovery rate). (69).

Immunofluorescence

Immunofluorescense (IF) staining was conducted on cells cultured on glass coverslips which were fixed by submerging the glass coverslip into pure methanol at 20°C followed by incubating at - 20°C for at least 15 minutes. This is followed by submerging glass coverslips in PBS with 0.2% gelatin from cold water fish skin (PBS-FSG) to block the cells. Primary antibodies were diluted in PBS-FSG prior to cell application. Glass coverslips were inverted onto 30ul droplets of primary antibody-PBS- FSG solution on fresh parafilm (ParaFilm M) and incubated at room temperature for 60 minutes. Coverslips are then incubated for 15-30minutes in a PBS-FSG bath, to remove non-specific primary antibodies. Secondary antibodies are also prepared in PBS-FSG in a 1/1000 dilution (along with DAPI (Sigma) at 1ug/ml) for 60 minutes. Coverslips are then incubated for 45 minutes in a PBS-FSG bath, to remove non-specific secondary antibodies. A second round of washing in fresh PBS-FSG occurs for 30 minutes. Coverslips are then inverted onto glass microscope mounts using mounting media (Invitrogen) and then are sealed with clear nail polish to stop dehydration of coverslips. All sample preparation as well as microscopy settings (such as light intensity, number of z-sections and exposure time) were remain constant throughout experiments to have continuity throughout imaging. 3D-SIM imaging was executed using 3D-SIM (OMX Blaze v4, GE Biosciences PA) with 405, 445, 488, 514, 568 and 642 nm

47 diode lasers on a 60X/1.4A plan apochromat oil objective (Olympus), 2x binning on a Deltavision Elite DV (GE Healthcare-Applied Precision). The camera used was a 2048x2048 sCMOS (GE Life Sciences, PA). The program SoftWoRx (6.0) was used to create all .tiff files.

RNA interference siRNA knockdown experiments in hTERT RPE-1 cells was done using Lipofectamine RNAiMAX tranfection protocol (Invitrogen). The negative control for siRNA was done using the non-targeting Luciferase GL2 Duplex (Darmacon). UBE3D siRNA knockdown was performed in hTERT RPE-1 cells. Cells were seeded at 1x105 in a 6-well plate and were transfected with 50nM of 3 unique UBE3D siRNA (silencer select). Medium was changed 24 h after transfection with fresh growth medium or serum-starved medium for ciliation analysis. ~72 hours post-transfection cells were collected and boiled at 95°C using Laemmli SDS-sample buffer for western blot analysis. Samples were separated by SDS-PAGE gel (10%- 15%) followed by transfer to a PVDF membrane (immoblion-P, Millipore). 1:500 rabbit anti-UBE3D was used to identify proper knockdown compared to non-targeting siRNA.

2.3 Characterization of the relationship of DNAAF2 with UBE3D

As not much is known about the functional characteristics and specific biological pathways that DNAAF2 is involved in, my goal was to use BioID to fill this gap.

2.3.2 Using BioID to identify DNAAF2 protein interactors

Using DNAAF2 as the bait protein for BioID, 2399 proteins were identified in at least 1 of the 4 BioID analyses performed. Following SAINT analysis the list of putative interactors was reduced to 221 proteins. Of these, 97 were identified as being involved in pathways associated with DNAAF2 cellular function and predicted localization, these included: Microtubule organization center (19 genes); Actin related (27 genes); Myosin related (14 genes); Vesicle transport (14 gene’s); ARP2/3 complex (8 genes); R2TP/Prefoldin complex (11 genes); Ub pathway (4) (gene ontology,) (Figure 2.3.1).

As expected, the spectral counts for DNAAF2 was high, averaging 1542 across all 4 MS/MS runs as compared to the top control only containing 4 DNAAF2 spectral counts (Table 2.3.1). As well, 7 out of

48 11 reported interactors of DNAAF2 were recovered with high confidence in the BioID data, these included: SPAG1 (91); RUVBL1 & RUVBL2 (62); HSPA4L (62) (Table 2.3.1).

Figure 2.3.1: Protein proximity interaction network for DNAAF2

Self-distributed BioID interaction map for bait protein DNAAF2 (based on 4 MS runs which is made up of 221 unique polypeptides. Bait Protein represented by yellow node, proximity interactors represented by blue nodes. Interaction nodes separated based on biological functionality/complexes, individually researched as well as PANTHER/Geneontology Database: Microtubule organization center; Actin related; Myosin related; Vesical transport; ARP2/3 complex; R2TP/Prefoldin complex; Ubiquitin pathway.

Biological Replicate 1 Biological Replicate 2 DNAAF2: Full Length C A B D Gene I.D. Control DNAAF2 DNAAF2 DNAAF2 DNAAF2 SAINT Score Average Technical Technical Technical Technical Replicate 1 Replicate 2 Replicate 1 Replicate 2 DNAAF2 4 1505 1802 1492 1368 1 UBE3D 1 82 79 83 86 1 SPAG1* 1 17 19 20 19 1 RUVBL2* 90 132 141 127 118 1 PIH1D1 14 20 18 16 17 1 ARPC2 1 16 16 15 16 1 MYH10 5 896 919 1267 1246 1 HSPA4L* 3 6 11 13 10 1 OFD1 4 3 5 15 13 0.86

TABLE 2.3.1

Table demonstrating comparative analysis of interactors of interest spectral counts for proximity ligation for bait DNAAF2.

Several of the highest confident/most interesting interactors from BioID experiments for DNAAF2. A: Protein associated with spectral counts in corresponding row B: Control column demonstrating highest spectral count for associated gene from 10+ nonspecific control runs; C: Corresponds to BioID spectral counts for the biological replicates and subsequent technical replicates of those biological replicates for Bait Protein DNAAF2; D: Corresponding to SAINT score associated with each gene ID.

One of the top putative interactors, indicated through a SAINT score of 1 as well as the average spectral count-fold change compared to the controls was UBE3D. As previously demonstrated, the top putative interactor of UBE3D when using the BioID protocol was DNAAF2 (Table 2.2.1). Thus, by recovering UBE3D as a top putative interactor in the reverse BioID interactome for DNAAF2 further demonstrated a potential relationship between the two proteins.

Many of the top interactors, defined by both SAINT score as well as spectral count fold change compared to the top control spectral count for each protein seemed to be involved in either or both microtuble organization center, Myosin/Actin as well as the large ARP2/3 and R2TP/Prefoldin Complexes.

The top putative interactor for DNAAF2 identified by average spectral count fold change as compared to the top control was MYH10, a myosin component. As well of the top 18 putative interactors, 11 of them

51 are myosin/actin components (MYH10, ACTN1 MYO1D, MYO18A, MYO5A, MYH9, MYL6B, MYH14, MYO5B, ACTN4, ACTG1).

R2TP/Prefoldin complex is made up of ~12 proteins and the DNAAF2 BioID assay was able to recover 10 of these proteins as putative interactors. For the R2TP complex this includes RUVBL1, RUVBL2, PIH1D1, RPAP3, ECD, HSP90, and for the Prefoldin complex this includes URI1, PDRG1, UXT, WDR92, WDR1 (92).

2.3.3 DNAAF2 Localization in hTERT RPE1 cells

To identify localization of DNAAF2 in RPE-1 cells IF was performed using anti-DNAAF2 antibody (Darmacon) using methods previously described for UBE3D (IF section in 2.2.5; Figure 2.3.2) with anti- DNAAF2, anti-PCM1, anti-Gamma Tubulin and DAPI. Anti-PCM1 was chosen as UBE3D, which had DNAAF2 as its top putative interactor, seemed to co-localize with satellite proteins (PCM1 used as marker) in hTERT RPE1 cells (Figure 2.2.3/2.2.4). Antibody to the centriole marker protein, gamma tubulin, was also used for IF as DNAAF2 BioID results identified a large number of high confident centriole associated putative interactors (microtubule organization center) (Figure 2.3.1). The goal was to identify if DNAAF2 co-localizes with centrioles or satellite proteins.

Figure 2.3.2:

IF analysis of DNAAF2 (anti-DNAAF2) in hTERT RPE1 cycling cells which were triton extracted with methanol fixation and probed with corresponding antibodies (anti-PCM1, anti-Gamma Tubulin) and DAPI stain. (White boxes: co-localization of gamma tubulin and anti-DNAAF2)

Figure 2.3.3: IF analysis of DNAAF2 (anti-DNAAF2) in mitotic hTERT RPE1 cells

A: IF analysis of DNAAF2 (anti-DNAAF2) in hTERT RPE1 cells undergoing corresponding mitotic phase of the cell cycle. Cells were treated with triton extracted with methanol fixation and corresponding antibodies (anti-PCM1, anti-Gamma Tubulin) and DAPI stain.

DNAAF2 is known to have endogenous expression in cycling HEK293 T-REx and hTERT RPE-1 cell lines using anti-DNAAF2, IF analysis of DNAAF2 (anti-DNAAF2) in hTERT RPE1 cycling cells demonstrated co-localization with centriole proteins (anti-Gamma Tubulin) (Figure 2.3.2B). To further confirm DNAAF2 co-localized with centriole proteins (anti-gamma tubulin) and not satellite proteins, immunofluorescence was performed in hTERT RPE-1 cells in the mitosis phase of the cell cycle (Figure 2.3.3). This analysis was necessary as satellite proteins and centrioles localize to similar locations during interphase whereas during mitosis satellite proteins dissipate and centriolar proteins remain co-localized. IF during mitosis in hTERT RPE-1 cells demonstrated that DNAAF2 remains co-localized to centrioles (Figure 2.3.3 white boxes) as well as being distinct from satellite proteins (anti-PCM1) during the mitosis phase of the cell cycle.

2.3.4 DNAAF2 N-terminal (PIH domain) BioID Results

The N-terminal domain of DNAAF2 is classified as part of the PIH domain family (protein interacting with Heat shock proteins). As previously described, it is located at position 43-207 of the 837 amino acid DNAAF2 protein. The PIH domain although identified in 8 different proteins has yet to fully understood in terms of biological function. By performing BioID on the N-terminal PIH domain of DNAAF2 our goal was to better understand the gaps in general research. As well we hope to be able to map out specific putative interactors, identified in the DNAAF2 full length BioID data, to specific regions of DNAAF2 (PIH domain & C-terminal portion).

Using DNAAF2 PIH domain as the bait protein for BioID, 1221 proteins were identified as having greater than 5 average spectral counts across all four BioID replicates (Figure 2.3.4). These were filtered using SAINT (72) where only interactions with AverageP>0.8 and MaximumP>0.9 were considered part of the bait proteins proximity interactome. A SAINT score cut off of 0.85 (~2% false discovery rate) was implemented and decreased the list of putative interactors to 193 Gene ID’s.

Figure 2.3.4: Protein proximity interaction network for DNAAF2 N-terminal PIH domain

Self-distributed BioID interaction map for bait protein DNAAF2 N-terminal PIH domain (based on 4 MS runs which is made up of 193 unique polypeptides). Bait Protein represented by small blue central node, proximity interactors represented by blue nodes. Interaction nodes separated based on biological functionality/complexes, individually researched as well as PANTHER/Geneontology Database: Microtubule organization center; Vesical transport; R2TP/Prefoldin complex; Ciliary dyskinesia.

Of the 193 filtered genes, 40 were identified as proteins being involved in pathways associated with DNAAF2 general function and predicted localization, these included: Microtubule organization center (21 genes); Vesicle transport (11 genes); R2TP/Prefoldin complex (6 genes); Ciliary dyskinesia (2 genes) (gene ontology)

CYTOSCAPE (To visualize the protein interaction network for FB-DNAAF2 bait protein, Cytoscape 3.6.0 was used.

As internal control for BioID protocol efficacy it is expected that the Bait protein (DNAAF2 N-terminal PIH domain) be identified in the data with high spectral counts. This is confirmed as DNAAF2 spectral counts across all 4 MS/MS runs averaged 538 as compared to the top control only containing 4 DNAAF2

56 spectral count (Table 2.3.2). The spectral counts for DNAAF2 have decreased compared to the full-length protein BioID spectral counts (1542, Table 2.3.1) as the N-terminal PIH domain only accounts for 25% of the full length protein.

5 out of 11 of DNAAF2’s reported interactors were recovered with high confidence in the BioID data, these include: SPAG1; RUVBL1 & RUVBL2; HSPA4; PRPF38A (Table 2.3.2).

Many of the top interactors, defined by both SAINT score as well as spectral count fold change compared to the top control spectral count for each protein were identified in the full length DNAAF2 BioID data. These include: SPAG1; RUVBL1; RUVBL2; HSPA4; OFD1; MYH10. These genes were not identified in the DNAAF2 c-terminal BioID data (see below).

Of the 11 genes associated with R2TP/Prefoldin complex identified in the DNAAF2 full length BioID data (Figure 2.3.1, Table 2.3.1) 6 of these were identified in the DNAAF2 N-terminal PIH domain BioID data (RUVBL1, RUVBL2, RPAP3, URI1, HSPA4, WDR92) and none of the 11 were identified in the DNAAF2 C-terminal BioID data (see below).

TABLE 2.3.2

Table demonstrating comparative analysis of interesting interactor spectral counts for bait DNAAF2 N- terminal PIH domain following BioID.

58 Several of the highest confident/most interesting interactors from BioID experiments for DNAAF2 N- terminal PIH domain. A: Gene ID associated with spectral counts in corresponding row B: Control column demonstrating highest spectral count for associated gene from 10+ non specific control runs; C: Corresponds to BioID spectral counts for the biological replicates and subsequent technical replicates of those biological replicates for Bait Protein DNAAF2 N-ternimal PIH domain; D: Corresponding to SAINT score associated with each gene ID.

The DNAAF2 N-terminal PIH domain also had many putative interactors associated with the microtubule organization center (21 genes) (Figure 2.3.4, Table 2.3.2). Some major ones included Cep192 and OFD1 (Figure 2.3.4, Table 2.3.2).

Interestingly, the DNAAF2 N-terminal PIH domain BioID data was completely absent of any spectra counts for UBE3D (Figure 2.3.4, Table 2.3.2), which was deemed one of the top putative interactors for the full length DNAAF2 interactome.

2.3.5 DNAAF2 C-Terminal BioID Results

The C-terminal of DNAAF2 (208-837aa) has not had any known domains recognized or has been researched at all in the literature. BioID was performed on the C-terminal of DNAAF2 to better map out which putative interactors identified in the full length DNAAF2 interactome were specific to the N- terminal PIH domain (section 2.3.3) and which were specific to the C-terminal.

Using DNAAF2 C-terminal domain as the bait protein for BioID, 826 proteins were identified having greater than 5 average spectral counts across all four BioID replicates (Figure 2.3.5, Table 2.3.3). To narrow this massive dataset certain statistical cut offs were used. These were filtered using SAINT (72) where only interactions with AverageP>0.8 and MaximumP>0.9 were considered part of the bait proteins proximity interactome. A SAINT score cut off of 0.85 (~2% false discovery rate) was implemented and decreased the list of putative interactors to 44 Gene IDs.

Of the 44 filtered genes, 21 of them were identified as protein being involved in pathways associated with the general function and predicted localization of DNAAF2. These included: Actin related (4 genes); Vesical transport (9 genes); Ciliogenesis (4 genes); Ubiquitin system (3 genes) (gene ontology,).

CYTOSCAPE (To visualize the protein interaction network for UBE3E bait protein, Cytoscape 3.6.0 was used (http://www.cytoscape.org).

59 One of the top putative interactors, indicated through a SAINT score of 1 as well as the average spectral count-fold change compared to the controls was UBE3D. As previously demonstrated, the top putative interactor of UBE3D using BioID was DNAAF2 (Table 2.2.1). Thus, by recovering UBE3D as a top putative interactor in the reverse pull down for DNAAF2 further confirms there is a relationship between the two proteins.

Figure 2.3.6 demonstrates the distinct differences in putative interacters between BioID Interactomes for DNAAF2 full length, DNAAF2 N-Terminal (PIH domain) and DNAAF2 C-terminal.

Figure 2.3.5: Protein proximity interaction network for DNAAF2 C-terminal domain

Self-distributed BioID interaction map for bait protein DNAAF2 C-terminal (based on 4 MS runs) is made up of 44 unique polypeptides. Bait Protein represented by yellow central node, proximity interactors represented by blue nodes. Interaction nodes separated based on biological functionality/complexes, individually researched as well as PANTHER/Gene Ontology Database: Actin related; Vesical transport; Ciliogenesis; Ubiquitin system.

TABLE 2.3.3

Table demonstrating comparative analysis of interesting interactor spectral counts for proximity ligation for bait DNAAF2 C-terminal.

Several of the highest confident/most interesting interactors from BioID experiments for DNAAF2 C- terminal. A: Gene ID associated with spectral counts in corresponding row B: Control column demonstrating highest spectral count for associated gene from 10+ non-specific control runs; C: Corresponds to BioID spectral counts for the biological replicates and subsequent technical replicates of those biological replicates for Bait Protein DNAAF2 C-Terminal; D: Corresponding to SAINT score associated with each gene ID.

62 2.3.6 Summary

A protein interaction profile for DNAAF2 was created using the BioID technique. The DNAAF2 interactome garnered novel insight into key pathways and protein complexes (such as the R2TP/Prefoldin complex) DNAAF2 may have some involvement with. The intracellular localization of DNAAF2 was analysed in HEK293 T-Rex and hTERT RPE1 cell lines resulting in identifying DNAAF2 co-localizes with centrioles (Gamma-Tubulin). To further understand the protein interaction profile of DNAAF2, BioID experiments were performed on the N-terminal PIH domain of DNAAF2 and the C-terminal portion of DNAAF2. Using these data sets DNAAF2 interactors could be mapped to specific domains of the protein.

63 2.3.7 Experimental Details

Refer to 2.2.5

Figure 2.3.6: DNAAF2 Domain Specific Interactome Comparison Heatmap

Above is a heatmap representing a BioID interactome comparison of baits : DNAAF2 C-terminal (205- 873aa), DNAAF2 Full Length and DNAAF2 N-terminal. Each line represents a gene found in the corresponding rows interactome with a SAINT score >0.85. The darkness of the lines are based on a Log2 fold change of spectral counts per gene compared to the highest spectral count control for that gene.

3 Discussion 3.1 Novel insights on the regulation of ciliation by UBE3D

In this thesis my goal was to better understand the ill-defined UBE3D HECT E3 ligase. To this end I first investigated whether UBE3D could direct specific Ub linkages. Using an in vitro autoubiquitylation assay, which was confirmed by western blot and then analyzed by mass spectrometry, I have shown that UBE3D HECT domain has the ability to create K63-linked Ub chains with just the HECT domain. To better understand the regulatory complex in which UBE3D participates a proximity- dependent biotin identification (BioID) assay was implemented. Through UBE3D BioID it was uncovered that UBE3D has connections with cilia. The UBE3D protein interactome included potential ciliogenesis regulatory proteins and protein involved in dynein motor production which is necessary for ciliogenesis. By identifying the localization of UBE3D within the cell we could further uncover how and the manner in which UBE3D interacts with these cellular processes and specific putative interactors. We uncovered that UBE3D co-localizes with satellite proteins located in the pericentriolar area in HEK293 T-Rex, hTERT RPE1 and HeLa cells. Satellite proteins are crucial for initiation and maintenance of cilia. This is further supported the UBE3D BioID interactome data and the association of UBE3D in cilia, microtubules and with DNAAF2.

To further investigate the role in which UBE3D plays in ciliogenesis we performed a UBE3D siRNA knockdown in hTERT RPE1 cells. These cells were chosen as they are proven to ciliate. Upon successful UBE3D siRNA knockdown a phenotype of increased ciliation was identified both under WT ciliation (cycling) and ciliation (arrested) cellular conditions. This identified hypotheses that UBE3D may negatively regulate ciliation specific proteins and/or a set of ciliation related protein interactors that are crucial to ciliogenesis progression. The BioID interactome data for UBE3D identified DNAAF2 as top candidate of UBE3D as it significantly scored as the top interactor of UBE3D. DNAAF2 has been shown to be involved in dynein motor assembly which is crucial for cilia regulation (Figure 2.2.1). We thus, performed BioID on DNAAF2 and was able to recover UBE3D as one of its top interactors (Figure 2.3.1) further directing us to the connection between UBE3D, DNAAF2 and ciliogenesis.

The first of many interesting results was identifying the ability of UBE3D to produce K63 ubiquitin chains. K63 ubiquitin chain conjugation onto protein substrates can have several different potential downstream effects. These include: proteasomal degradation of the substrate as well as regulation of DNA repair, kinase activity, chromosome segregation and protein synthesis. The IF experiments conducted on UBE3D

64 65 (in HEK293, HeLa and hTERT RPE1 cell) showed that UBE3D co-localized with the satellite protein PCM1 (Figure 2.2.2 and 2.2.3). Due to their co-localization occurring around the pericentriolar area and ciliary basal body further evidence that UBE3D has a role in ciliogenesis was established. Also, other K63 regulated processes were not represented as high confidence potential interactors, such as known nuclear, ribosomal or DNA repair proteins. Thus, it is unlikely that UBE3D would target substrates via K63 Ub chain conjugation to regulate DNA repair, chromosome segregation or protein synthesis.

It is my working hypothesis that UBE3D is a negative regulator of ciliogenesis for several reasons. The BioID interactome of UBE3D contained high confidence putative interactors involved in cilia maintenance (CYLD, ALMS1 SPATA2, DNAAF2), ciliogenesis promoters (DNAAF2, CEP192) and negative regulators of ciliogenesis (OFD1, DYNLRB1). Secondly, upon siRNA knockdown of UBE3D in hTERT RPE1 cells, an increase of 22% ciliation was seen under high ciliation (arrested; pvalue 0.0044) and an increase of 12% ciliation under low ciliation (cycling; pvalue 0.0009) conditions. These observations led to the hypothesis that UBE3D through conjugation of K63 Ub chains directs substrate(s) for proteasomal degradation and these substrates are crucial to the initiation and maintenance of ciliogenesis.

(Figure 2.2.1). - ALMS1 is a crucial protein involved in microtubule organization particularly in the formation and maintenance of cilia. ALMS1 is located in the cytosol and the ciliary basal body. The ciliary basal body is in very close proximity to the pericentriolar/satellite proteins. As well UBE3D had ALMS1 as a putative interactor with a SAINT score of 1 as well as a 10 fold spectral count change compared to the top control (Figure 2.2.1). - CYLD is a deubiquitinase that cleaves K63 linkages which through UBE3D degradation could create a harmonious relationship for proper cilia formation maintenance and decomposition. In this facet if a cell requires ciliation CYLD would cleave K63 Ub chains from proteins required for ciliation that have been ubiquitylated by UBE3D for K63 Ub chain proteasomal degradation. As well, if a cell requires cilia decomposition, UBE3D could cause proteasomal degradation of CYLD via K63 ubiquitylation. CYLD is localized to the microtubule organization center which accepts satellite proteins for cilia formation and maintenance. Its function is to recruit the basal body to the base of the cilia to initiate ciliogenesis. Thus, siRNA knockout of UBE3D leading to increased ciliation is likely due to CYLD to uncontrollably initiate ciliation through basal body recruitment. - SPATA2 (spermatogenesis associated protein) is responsible for recruitment of CYLD.

U b U s U U b b b k63 k63 C-lobe s U U b b E2 k63 k63 U b k63 Cilia N-lobe protein Cilia Sub protein Binding

Proteasome

B. C. U b s U U b b s k63 U U s b b U k63 s b C-lobe k63 U b E2 k63 Cilia protein Cilia N-lobe CYLD protein Sub Binding

UBE3D +/-

U b s U b k63 U b k63

Cilia Cilia protein protein CYLD

67 Figure 3.1.1

Figure demonstrating working hypothesis for UBE3D mechanism of action for ciliation. A. UBE3D lysine 63 (K63) ubiquitylation linkage formation onto cilia-related substrate leading to substrate specific 26s proteasome mediated protein degradation. B. Working hypothesis demonstrating UBE3D knockout mouse stops function of UBE3D to lead to downstream proteasomal degradation of ciliation specific substrates leading to increased ciliation as demonstrated through UBE3D knockout ciliation phenotype. C & D. Working hypothesis demonstrating the ability of the deubiquitinizing enzyme CYLD to remove K63 ubiquitin linkages from ciliation-related substrates which have been destined for proteasomal degradation due to UBE3D ubiquitylation.

3.2 UBE3D in Association with Age-Related Macular Degeneration

A small nucleotide variant found in the UBE3D gene has been implicated in early onset of age-related macular degeneration (50). The UBE3D small nucleotide variant implicated in age related macular degeneration occurs within the HECT domain (49). HECT domains are very sensitive to mutations and often cause inactivity if amino acid alteration occur (28). The UBE3D SNV described in age-related macular degeneration alters a valine to a methionine (49). I hypothesize that loss of function of UBE3D protein due to the described AMD-SNV causes early onset of AMD. When the UBE3D gene is fully knocked out from a genome mouse models are not viable. A heterozygous knockout mouse for UBE3D demonstrated symptoms of AMD (decreased retinal function) (49). My research has demonstrated UBE3D has some involvement in cilia maintenance and or functionality. Photoreceptors contain a specialized central cilium which connects the inner portion used for blood supply and maintenance and outer portion which controls sensitivity. By disrupting the functionality of UBE3D via SNV, I hypothesize that the central cilium has decreased maintenance of proteins which are normally regulated by UBE3D. This causes distress in the photoreceptors leading to inflammation which is the first sign of AMD (50).

68 3.3 New insights of DNAAF2 association of Axonemal Dynein assembly

Another goal of this thesis was to better define DNAAF2 and the landscape of the axonemal dynein arm assembly as well as its role in non-motile cilia cell types. After identifying DNAAF2 as the highest confidence interactor in the UBE3D BioID interactome (Figure 2.2.3) (this was based on SAINT score and spectral count fold changed over the highest control spectral count for DNAAF2) we chose to further investigate the functional interaction landscape of DNAAF2. I performed BioID to create an interactome for DNAAF2 and identified UBE3D as a top interactor. I was also able to identify many interesting protein functional clusters and complexes. Many of the putative interaction clusters revolved around intracellular motility: Actin/Myosin; microtubule organization center; vesical transport. As well as the R2TP/Prefoldin complex, in which 10 of the 12 protein components were deemed high confidence putative interactors. To better understand the role of DNAAF2 in these functional cellular areas as well as with R2TP/Prefoldin complex, IF was performed to determine the cellular localization of DNAAF2. It was identified that DNAAF2 co-localizes with centrioles (anti-gamma tubulin) in HEK293 T-Rex and hTERT RPE1 cells (Figure 2.3.3). To further confirm this finding IF directed cellular localization analysis was performed for DNAAF2 during cell cycle. This was important as satellite proteins, UBE3D now believed to be one, dissipate during cell cycle whereas centrioles don’t. DNAAF2 was demonstrated to remain co-localized with centrioles during cell cycle (Figure 2.3.4). This gave insight into why DNAAF2 seemed to have an interactome containing many putative interactors involved in actin/myosin and microtubule organization center regulation. To better understand the relationship between putative interactors and DNAAF2 we chose to perform BioID on smaller portions of the full length DNAAF2. BioID analysis was performed on both the PIH domain of DNAAF2 (which represented approximately 25% of the most N-terminal region) and the C-terminal portion. Through this method we were able to map out specific interactors to unique sections of DNAAF2 while also uncovering new insight into the role of PIH domains. These results led us to new insights into how DNAAF2 functions in axonemal dynein arm assembly as well as map interaction profiles for both the PIH domain and C-terminus of DNAAF2.

BioID analysis for DNAAF2 identified a novel putative interaction landscape which led to many interesting findings. The first of many interesting results was identifying UBE3D as one of the top interactors of DNAAF2. This was interesting as during UBE3D BioID analysis, DNAAF2 was identified to be its top interactor. This increased confidence in UBE3D and DNAAF2 being potential interactors. As internal control of the BioID efficacy most (7 of 11) of the described interactors of DNAAF2 from the literature were identified with high confidence in the interactome (62). The DNAAF2 interactome also

69 was found to have a large cluster of high confidence microtubule and centrosome proteins. These results coincided with the IF results demonstrating DNAAF2 co-localized with centrioles (anti-gamma tubulin). One of the most described DNAAF2 interactors in the literature, SPAG1, was also found with high confidence in the interactome (94). Using FRET analysis in vivo, SPAG1 (sperm associated antigen 1) has been described in the literature to demonstrate a proximity interaction between HEATR2, SPAG1 and DNAAF2 in an early phase complex, that initiates preassembly of the late phase axonemal dynein assembly complex (DNAAF1, DNAAF2, DNAAF3, DYX1C1, PIH1D3, LRRC6, ZYMND1) (94). HEATR2 and SPAG1 have been shown to interact, and SPAG1 and DNAAF2 have been shown to interact, but no interaction between HEATR2 and DNAAF2 has been shown. As well DNAAF2 has been shown to disassociate with SPAG1 prior to late phase axonemal dynein complex formation. This interaction scenario coincides with the DNAAF2 interactome data as SPAG1 is a high confidence interactor of DNAAF2 and HEATR2 was not identified as an interactor. The gap in the literature is the transition from early phase complex (HEATR2-SPAG1-DNAAF2) to late phase complex leading to axonemal dynein assembly. The R2TP/Prefoldin complex, another interaction group identified in the DNAAF2 interactome may be the bridge between this early and late phase transition. The R2TP/Prefoldin complex is a co-chaperone complex involved in the assembly of several important large-multiprotein complexes (RNA polymerases, snoRNPs and PIKK-containing complexes) (94). Of the 12 associated proteins that make up the R2TP/Prefoldin complex, the interactome of DNAAF2 contained 10 with high confidence. Two of the well described DNAAF2 interactors (RUVBL1, RUVBL2) are members of this complex (62). As well the R2TP/Prefoldin complex co-chaperones with HSP90 proteins (HSP90AA1, HSP90AB1, HSPA4) for complex assembly. HSP90AA1, HSP90AB1 and HSPA4 are also well defined DNAAF2 interactors (62).

I propose that axonemal dynein assembly occurs in the region at the base of cilia. DNAAFs have been proposed to form R2TP-like complexes (93). Due to several DNAAFs containing TPR and PIH domains DNAAFs have been proposed to form R2TP-like complexes that work upstream of canonical R2TP complex chaperoning and pre-assembling portions of dynein subunits. Studies have shown the R2TP complex is required for dynein assembly. Mutant analysis of DNAAF2 and DNAAF3 (62) demonstrated outer and inner arms missing in dynein motors leading to ciliary dyskinesia.

It has been proposed the R2TP-like complex functions in the assembly of heterodimeric dynein intermediate chains 1 and 2 (DNAI1/2) via their PIH-domains (DNAAF2, DNAAF6, PIH1D2) which bind RUVBL1, RUVBL2, SPAG1 which contains a RPAP-domain and DNAAF4. DNAAF4 could then bind

70 HSP90 via its TRP-domain. (94). These folded intermediate chains are then passed on to canonical R2TP complexes which then combine the other dynein subunits, which are then transported into the cilium via intraflagellar transport.

Furthermore, by performing BioID analysis on the N-terminal PIH domain and C-terminal of DNAAF2 we were able to map both SPAG1 and 6 of the R2TP/Prefoldin complex proteins as well as HSPA4 specifically to the PIH domain of DNAAF2. Interestingly we were also able to map UBE3D interaction to strictly the C-terminus of DNAAF2. These novel connections could give new insight into the complex process of axonemal dynein assembly.

PIH domains are not well described and understudied. By looking creating a PIH domain interactome using BioID I was able to elucidate many putative interactors and better understand the functionality of the domain itself. When comparing the interactomes of the C-terminal and PIH domain of DNAAF2 there is a clear distinction that the PIH domain is the more active portion of the protein. This was demonstrated as the PIH domain had ~100 high confidence interactors whereas the C-terminal domain had ~25. Several complexes, R2TP, as well as known DNAAF2 interactors, SPAG1, were identified to specifically interact with the PIH domain

Figure 3.2.1

Working model depiction for the hypothesized R2TP-like complex (DNAAF4, SPAG1, PIH domain containing proteins and RUVBL1 & RUVBL2) compared to the R2TP complex (RPAP3, PIH1D1 and RUVBL1 & RUVBL2)

72 Future directions:

Several novel insights into UBE3D, DNAAF2 and ciliogenesis were uncovered throughout the research performed and presented in this thesis. To confirm these insights further work must be completed. The BioID interactome analysis for both proteins was performed in HEK293 cells, which do not produce motile cilia. DNAAF2 has been implicated in the literature as being a crucial protein involved in axonemal dynein motors found in motile cilia. It would be interesting to perform BioID protein interactome analysis of UBE3D and DNAAF2 in cells that produce motile cilia, such as brain, lung or reproductive organs. These types of investigations may further uncover their role and mechanism in motile ciliogenesis.

To further confirm the increased ciliation phenotype upon UBE3D siRNA knockdown an siRNA rescue assay must be performed. This would entail creating an inducible UBE3D expression vector that is resistant to the siRNA and introducing it into cells. If the increased ciliation phenotype is negated upon expression of the siRNA resistant UBE3D this will confirm that UBE3D knockdown is the reason for the increased ciliation phenotype. I would also perform siRNA knockdown of DNAAF2 to identify if it too has an effect on ciliation. Although I evaluated 7 siRNAs to UBE3D, only one siRNA effectively knocked down UBE3D expression, which was used in these studies. Going forward I would use multiple independent siRNAs targeting the expression of each protein to overcome potential off-target effects.

I would validate a connection between UBE3D and DNAAF2 using independent assays, such as a proximity ligation assay or co-immunoprecipitation assay.

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