Ubiquitination is a form of post-translational modification of proteins, in which the 76-residue ubiquitin polypeptide is conjugated to the lysine residues of either the substrate protein or the preceding ubiquitin molecule. Ubiquitination requires the sequential action of three types of ubiquitin enzymes: the ubiquitin activating enzyme (E1), the conjugating enzyme (E2) and the ligase (E3). In human genome, there are two different E1s, ~40 E2s and more than 600 E3s. Ubiquitination regulates a variety of cellular processes, including cell cycle progression, induction of inflammatory response, or intracellular protein trafficking. The anaphase-promoting complex/cyclosome (APC/C) and the SCF are two important E3 ligases regulating cell cycle progression. Understanding how they recognize their substrates and how their enzymatic activity is regulated will provide valuable clues for inhibiting unchecked cell proliferation such as in cancer. The objective of this dissertation is twofold: 1) Chapter 1: understanding how APC/C forms ubiquitin chains on its substrates. We found that APC/C preferentially uses lysine11 of ubiquitin to assemble ubiquitin chains on its substrates. By recognizing the same motif present in both the substrates and ubiquitin, APC/C switches from initial modification of the substrate to processive chain elongation, leading to the rapid turnover of its substrates. 2) Chapter 2 to 5: identifying Cul3Klhl12, a structural relative of SCF, as an essential ubiquitin ligase in embryonic stem cells (ESCs). In an siRNA screen, we found that Cul3Klhl12 affects cell adhesion and proliferation of ES cells by regulating COPII-mediated protein trafficking. Cul3Klhl12 ubiquitinates Sec31A, a coat protein of COPII vesicles, and causes the enlargement of the vesicles. This enables the COPII-dependent transport and proper deposition of collagen, a major component of extracellular matrix (ECM).

The covalent attachment of ubiquitin to a protein controls a range of cellular processes in eukaryotes by altering the protein's stability, localization, or activity. Ubiquitylation requires the activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). This enzymatic cascade modifies substrates with either a single ubiquitin or polyubiquitin chains linked through lysine residues of ubiquitin. Because ubiquitin contains seven lysines, different chain types are possible, and chain topology determines the fate of the modified protein. For example, Lys48-linked chains target proteins to the proteasome, while non-proteolytic Lys63 chains are involved in complex reorganization and signaling pathways. Although the physiological roles and formation of these chain types has been extensively studied, the characterization of other chain types is lacking. In this dissertation, I present work describing the biological function and assembly of Lys11-linked ubiquitin chains.

Lys11-linked chains are formed on substrates of the human anaphase-promoting complex (APC/C), an E3 ligase essential for proliferation, promoting their degradation by the proteasome. Because this chain type was proposed to be essential for regulating mitosis, we undertook an extensive investigation into how Lys11 chains are build by the APC/C, with a focus on the role of E2 enzymes. In the Chapters below, we report that APC/C-dependent formation of Lys11- linked chains requires the activity of two E2 enzymes: a chain-initiating E2 Ube2C and a Lys11- specific, chain-elongating E2 Ube2S. These E2s likely bind the APC/C at the same time, representing a unique way for the APC/C to achieve processivity.

In most instances, linkage-specific ubiquitin chain assembly is promoted by E2s, yet this process has only been well characterized for Lys63-linked chains. Therefore, we next investigated the molecular mechanism underlying the Lys11-specificity of Ube2S. We report that Ube2S engages in essential non-covalent interactions with ubiquitin that are required for processive and specific ubiquitylation. Lys11-specificity is generated by substrate-assisted catalysis, in which an acidic residue of the substrate ubiquitin is essential for generating a catalytically competent active site.

To solidify the role of Lys11-linked chains and the APC/C in mitotic control, we developed a Lys11-specific antibody. Using this antibody, we show that the level of Lys11 chains in HeLa cells increases dramatically as cells pass through mitosis, while the level of Lys48 chains remains constant. Importantly, knockdown of the Ube2C/Ube2S E2 module inhibits formation of Lys11 chains, suggesting that the APC/C and its E2s are the major enzymatic machinery responsible for forming K11 chains in mitosis.

Posttranslational modification of proteins with ubiquitin, ubiquitylation, is essential in all eukaryotes. Ubiquitylation is accomplished by an enzymatic cascade: E1 activates ubiquitin and transfers it to E2, which works with E3 to transfer ubiquitin to substrate residues, usually lysines. Once attachment of the first ubiquitins to substrate, chain initiation, has occurred, E2 and E3 modify one of the seven lysine residues in ubiquitin itself, resulting in formation of polymeric ubiquitin chains, chain elongation. Ubiquitylation is involved in nearly every cellular process, and is central to cell cycle control. Ubiquitin-dependent protein degradation is especially important during mitosis, as cells must turn over many proteins within a short time-window. The anaphase-promoting complex (APC/C) is an essential E3 that targets cell cycle regulators for degradation during mitosis. The APC/C must degrade many substrates within a short period of time, and likely acts under saturation. Furthermore, APC/C substrates are required at different times during mitosis. Therefore, the APC/C cannot degrade all substrate at once, but rather must order substrate degradation so that substrate proteins can accomplish their functions. The balance between efficiency and regulated degradation is the APC/C's major challenge as it orchestrates mitotic exit.

In this dissertation I address how human APC/C targets its many substrates for degradation. First, I report our finding that APC/C functions by decorating its substrates with Lys11-linked ubiquitin chains. Lys11-linked chains target substrates to the 26S proteasome and are essential for cell division in human cells, Drosophila cells, and Xenopus tropicalis embryos. The human APC/C uses a dedicated E2, Ube2C/UbcH10, to perform chain initiation while a second E2, Ube2S, elongates these first modifications using exclusively Lys11 on ubiquitin. Ube2C and Ube2S likely function simultaneously, thus allowing APC/C to overcome its massive substrate workload. Efficient chain initiation by Ube2C is made possible by conserved initiation motifs in substrates. Initiation motifs do not regulate substrate binding to APC/C, but instead control the efficiency with which Ube2C and APC/C attach the first ubiquitin moieties to substrates. I will show that chain initiation by Ube2C is the rate-limiting, regulated step during Lys11-linked chain formation by the APC/C. Once Ube2C has initiated chains on APC/C substrates, Ube2S takes over, rapidly elongating Lys11-linked ubiquitin chains that target substrates to the 26S proteasome. Finally, I address how use of two E2 enzymes performing different functions allows APC/C to overcome its major challenge: rapid yet regulated turnover of essential cell cycle regulators. The separation of ubiquitin chain formation by the APC/C into initiation and elongation steps, with initiation being rate-limiting and tightly regulated, draws parallels between ubiquitylation and other processive reactions such as transcription and translation.

Metazoan development hinges on pivotal signaling events that intricately regulate its diverse cellular processes. Posttranslational modifications (PTMs), such as ubiquitylation, play an important role by precisely modulating protein stability and turnover, ensuring protein homeostasis is maintained in a precise spatiotemporal manner throughout development. Ubiquitylation involves a meticulously orchestrated enzymatic cascade, beginning with an E1 ubiquitin activating enzyme, followed by a ubiquitin transfer to an E2 conjugating enzyme, and ending with an E3 ligase, which attaches ubiquitin to its target substrates. This cascade forms an elaborate and signaling code, forming monoubiquitin or polyubiquitin chains that govern diverse biological decisions based on cellular context and regulatory need of the modified substrate. Despite considerable advancements in discovering ubiquitin-dependent mechanisms in both health and disease, numerous questions remain unanswered. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. In Chapter 2, I describe the identification of a gene expression quality control pathway, where E3 ligase UBR5 degrades orphan transcriptional regulators which encompass a dynamic network of protein centered on oncoprotein c-Myc. Through cellular, biochemical, and structural experiments we show that UBR5 recognizes, binds, and degrades motifs that only unveil themselves upon complex dissociation. Rapid turnover of orphan subunits by UBR5 establishes dynamic interactions between transcriptional complexes to allow execution of gene expression. In Chapter 3, we begin exploring the relationship and pathway of UBR5 and master regulator of pluripotency, OCT4. I describe our preliminary understanding of how UBR5 recognizes OCT4 by generating a degron mutant and dimerization mutant of OCT4. Additional data which I will describe in this section shows that UBR5, OCT4, and c-MYC are part of a single gene expression circuit controlled by ubiquitylation. Further investigation is needed to fully uncover this quality control pathway important for preserving pluripotency. Finally, Chapter 4 unveils the discovery of a proteasome-dependent pathway involved in clearance of Huntingtin (HTT) oligomers. Using an imaging-based screen, we uncovered the key ubiquitin regulators of an oligomer clearance pathway. E3 ligase RNF126, its E2 UBE2K (also known as Huntingtin-interacting protein 2), and the VCP disaggregase are required for efficient proteasomal clearance of early HTT aggregates. These studies offer valuable insight into quality control networks crucial for preserving pluripotency, ensuring efficient execution of transcriptional programs, and facilitating clearance of Huntingtin oligomers. The discovery of these pathways paves the way for the development of target protein degradation strategies, leading to the creation of novel therapies aimed at improving patient outcomes.

Cellular signaling relies on many proteins that form dimers and the correct pairing of each dimer is critical for their activity. Despite the fact that there are numerous recurrent and modular dimerization domains, including BTB domains and leucine zippers, whether cells have mechanisms to detect and eliminate dimers of aberrant composition is not known. Here, by searching for regulators of the BTB family, we discovered a quality control pathway that ensures functional dimerization [dimerization quality control (DQC)]. Key to this network is the E3 ligase SCF-FBXL17, which selectively binds and ubiquitylates BTB dimers of aberrant composition to trigger their clearance by proteasomal degradation. Underscoring the physiological importance of DQC, SCF-FBXL17 is required for the differentiation, function, and survival of neural crest and neuronal cells in Xenopus laevis. Thus, metazoan organisms actively monitor BTB dimerization.

After identifying DQC, one key question that remained was how a single enzyme, SCF-FBXL17, could recognize hundreds of aberrant heterodimers but leave active homodimers intact. In order to understand how DQC can distinguish complexes based on their composition, we used cryo-electron microscopy and X-ray crystallography to determine structures of aberrant BTBs alone and bound to SCF-FBXL17. We find that SCF-FBXL17 encircles a monomeric BTB domain through a multipartite interface that can accommodate significant sequence variation. Distinct from most quality control systems, SCF-FBXL17 does not detect protein misfolding, but probes dimer stability through a domain-swapped and rapidly evolving β-strand within BTB domains. Our findings suggest that SCF-FBXL17 uses a mechanism of subunit exchange to establish quality control of complex composition, and we predict that other quality control proteins may similarly monitor other recurrent oligomerization domains.

The small protein ubiquitin is a post-translational modification that is essential for regulating many cellular processes in eukaryotes. In a series of enzymatic steps, ubiquitin is activated by the ubiquitin-activating enzyme E1 and then transferred to the active site of an E2 ubiquitin-conjugating enzyme. An E3 ubiquitin ligase interacts with the ubiquitin-charged E2 and the substrate protein to facilitate transfer of ubiquitin to a substrate residue, typically lysine. Ubiquitin modifications can affect substrate protein interactions, localization, or stability. With such versatile outcomes, ubiquitylation regulates countless cellular processes, including immune signaling, transcriptional regulation, DNA damage response, and the cell cycle. The Anaphase-Promoting Complex (APC/C) is a RING domain-containing E3 ligase that regulates mitotic progression by building ubiquitin chains on its cell cycle regulator substrates, targeting them for proteasomal degradation. APC/C works with two E2 enzymes to first initiate substrate ubiquitylation using Ube2C and then to build ubiquitin chains with Ube2S. How APC/C coordinates the activity of these E2 enzymes to processively build chains on substrates is not well understood. Additionally, it not known how these E2s contribute to mitotic progression: while APC/C is essential for mitotic exit, depletion of its cognate E2 enzymes does not have dramatic effects. In this dissertation, I present work determining how APC/C works with its E2s to build ubiquitin chains and how this is important for regulating cell cycle progression.

APC/C ubiquitylates numerous cell cycle regulators at particular times in mitosis to control spindle assembly, inactivation of the Spindle Assembly Checkpoint, and mitotic exit. While APC/C interacts with both substrates and with E2 enzymes at defined interfaces, it is unclear how chain formation occurs. In order to add a new ubiquitin molecule to the growing chain, ubiquitin-charged Ube2S must be in close proximity to the distal end of the chain. In Chapter 2, we report that APC/C stimulates chain formation by increasing the affinity between Ube2S and an acceptor ubiquitin at the end of the substrate-conjugated chain. Our finding support a model in which APC/C tethers the end of the growing chain close to the E2 active site to promote efficient chain formation without adjusting interactions with substrate or E2.

To further understand how APC/C-specific E2 enzymes regulate mitosis, we investigated the role of Ube2S in governing APC/C activity at the significant transition from early mitosis with active Spindle Assembly Checkpoint signaling to late mitosis and mitotic exit. In Chapter 3, we found that Ube2S affects dynamics of the spindle checkpoint and is found in mitotic checkpoint complexes with the phosphatase PP2AB56, which has a well-described role in stabilizing kinetochore-microtubule attachments and turning off the checkpoint. We report that PP2AB56 promotes ubiquitin chain formation by APC/C, suggesting that there is collaboration between phosphatase and ubiquitylation activities to couple spindle checkpoint inaction with APC/C activation.

Metazoan development is dependent on the robust spatiotemporal execution of stem cell cell-fate determination programs. Although changes in transcriptional and translational landscapes have been well characterized throughout many differentiation paradigms, their regulatory mechanisms remain poorly understood. Ubiquitin has recently been found to be a key modulator of developmental programs. Ubiquitylation of target proteins occurs through a cascade of enzymatic reactions beginning with a ubiquitin activating enzyme (E1) which transfer the ubiquitin moiety to a ubiquitin conjugating enzyme (E2). The reaction is finalized by the transfer of ubiquitin to its target protein by a ubiquitin ligase (E3). Post-translational modification of proteins can lead to several different outcomes, depending on the context of the modification, known as the ubiquitin code. The precise spatiotemporal execution of ubiquitylation is critical for organismal development and homeostasis. Due to the modular and reversible nature of ubiquitylation, it is an ideal moiety in the control of a plethora of cellular processes.

Cell-cell fusion is a frequent and essential event during development, whose dysregulation causes diseases ranging from infertility to muscle weakness. Critical to this process, cells repeatedly need to remodel their plasma membrane through orchestrated formation and disassembly of cortical actin filaments. In Chapter 2, I describe the identification of a ubiquitin-dependent toggle switch that establishes reversible actin bundling during mammalian cell fusion. My work identified KCTD10 as a modulator of the EPS8-IRSp53 complex, which stabilizes cortical actin bundles at sites of cell contact to push fusing cells towards each other. This work highlights how cytoskeletal rearrangements during development are precisely controlled, raising the possibility of modulating the efficiency of cell fusion for therapeutic benefit.

Organismal development must rely on the timely and robust execution of quality control responses. However, how these responses modulate metazoan development is poorly understood. Showcasing the versatility of ubiquitin signaling, Chapters 3 and 4 provide insight into the role of ubiquitin in controlling stress and quality control responses. Chapter 3 describes the reductive stress response, in which FEM1B senses and reacts to persistent depletion of reactive oxygen species. Loss of ROS is detrimental for development, as it inhibits myogenesis. Concomitant to this stress response is the identification of multimerization quality control, regulated by BTBD9. MQC surveys multimeric BTB complex composition, ensuring that multimeric complexes contain the correct stoichiometries and compositions. MQC is critical for development, as loss of MQC als prevents myogenesis. These two chapters showcase the integration of ubiquitin signaling, stress/quality control pathways, and development. These writings provide a more holistic understanding into the robust regulatory underpinnings of organismal formation