Bio-Functional Analysis of Ubiquitin Ligase UBE3D by 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 © Copyright by Adam William Penn 2020 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 iv 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 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. 3 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).