Hrd1 Partners In Endoplasmic Reticulum-Associated Degradation

Protein Quality Control (PQC) comprises cellular pathways that regulate the turnover of short-lived, misfolded proteins. A main component of PQC is Endoplasmic Reticulum (ER)-Associated Degradation (ERAD), which controls the degradation of proteins synthesized in the ER. Aberrations in ERAD have been linked to malignancies such as sarcomas, breast, and pancreatic carcinomas, as well as neurodegenerative disease. The machinery in this system is complex and while significant progress has been made to understand ERAD, it is not clear how the different components come together, or how they are regulated. HRD1 is a resident ubiquitin ligase that has been proposed as a metastasis suppressor. My goal is to understand how HRD1 is regulated during normal states and in disease, particularly because my own work suggests complex mechanisms that regulate this protein and, more generally, ERAD. We found that HRD1, an E3 ubiquitin ligase that functions in ERAD, along with other PQC components are differentially expressed in various cancer cell lines and in different mouse tissues, suggesting that specific types of ubiquitin chains are formed in ERAD under varying conditions. These findings led us to wonder whether HRD1 makes different types of ubiquitin-ubiquitin linkages with different E2 ubiquitin-conjugases. Through in vitro reconstituted systems, we found that HRD1 and the ubiquitin-conjugase Ube2G2 make only K48-linked polyubiquitin. This type of chain is classically tied to protein degradation by the proteasome. However HRD1, in the presence of another conjugase Ube2J1, forms unconventional K11-, K29-, and K33-linked polyubiquitin chains. These in vitro findings suggested that ERAD substrates are processed differently under specific conditions. In cells, HRD1 makes at least K48- and K63-linked polyubiquitin chains with Ube2G2 and Ube2J1, and the direct interaction of HRD1 with VCP/p97, while not required for polyubiquitin chain formation by this E3 ligase, may determine some types of ubiquitin chains formed by HRD1 with either E2 conjugase. Our findings suggest that HRD1 has specific partners in different tissues with potentially different ERAD outcomes. My work has broad implications in pathophysiology; it may emphasize specific sites of therapeutic intervention, and will provide significant clues into the functional balance of protein quality control in normal and disease states

The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis

Pathways to the proteasome in yeast

The proteasome is a highly selective and precisely regulated protease that degrades proteins essential for cellular homeostasis. This dissertation explores the molecular mechanisms by which the proteasome selects its substrates from a diverse protein pool First, the interplay between different pathways to the proteasome was investigated, revealing a hierarchy in degradation whereby substrates delivered by ubiquitin-like (UBL) domains may have priority access to the proteasome. Next, the role of spacing between the ubiquitin tag and disordered initiation region in degradation was investigated. The findings reveal another layer to substrate selection by the proteasome, and provide important considerations when determining whether a given protein may be degraded by the proteasome. Overall, this research provides new insights into how the proteasome selects its substrates in a diverse protein pool.Biochemistr

Structural Analysis of Heterodimeric and Homooligomeric Protein Complexes by 4-D Fast NMR

<p>A molecular depiction of the assembly, interaction and regulation of protein complexes is essential to the understanding of biological functions of protein complexes. Structural analysis of protein complexes by Nuclear Magnetic Resonance (NMR) has relied heavily on the detection and assignment of intermolecular Nuclear Overhauser Effects (NOEs) that define the interactions of protons at the molecular interface. Intermolecular NOEs have traditionally been detected from 3-D half-filtered NOE experiments by suppressing intramolecular NOEs prior to NOE transfer. However, due to insufficient suppression of undesirable signals and a lack of dispersion in the H dimension, data analysis is complicated by the interference of residual intramolecular NOEs and assignment ambiguity, both of which can lead to distorted or even erroneously packed protein complex structures. Leveraging the recent development of fast NMR technology based on sparse sampling in our lab, we developed a strategy for reliable identification and assignment of intermolecular NOEs using high resolution 4-D NOE difference spectroscopy. Spectral subtraction of individually labeled components from a uniformly labeled protein complex yields an "omit" spectrum containing only intermolecular NOEs with little signal degeneracy. </p><p>The benefit of such a strategy is first demonstrated in structural analysis of a homooligomeric protein complexes, the foldon trimer. We show that intermolecular NOEs collected from the 4-D omit NOE spectrum can be directly utilized for automated structural analysis of the foldon trimer by CYANA, whereas intermolecular NOEs derived from 3-D half-filtered NOE experiments failed to generate a converged structure under the same condition. </p><p>Such a strategy was further demonstrated on a heterodimeric protein complex in translesion sysnthesis (TLS), a DNA damage tolerance pathway. The TLS machinery consists of several translesion DNA polymerases that are recruited to the stalled replication fork in response to monoubiquitinated proliferating cell nuclear antigen (PCNA) in order to bypass DNA lesions encountered during genomic replication. The recruitment and assembly of translesion machinery is heavily dependent on ubiquitin-binding domains, including ubiquitin-binding motifs (UBMs) and ubiquitin-binding zinc fingers (UBZs) that are found in translesion DNA polymerases. Two conserved ubiquitin-binding motifs (UBM1 and UBM2) are found in the Y-family polymerase (Pol) &iota, both of which contribute to ubiquitin-mediated accumulation of Pol &iota during TLS. Although the Pol&iota UBM2-ubiquitin complex has been previous reported by our lab and others, the Pol &iota UBM1-ubiquitin complex has remained a challenge due to significant signal overlap in conventional 3-D NOE spectroscopy. In order to determine the molecular basis for ubiquitin recognition of Pol &iota, we solved the structures of human Pol &iota UBM1 and its complex with ubiquitin by 4-D fast NMR, revealing a signature helix-turn-helix motif that recognizes ubiquitin through an unconventional surface centered at L8 of ubiquitin. Importantly, the use of 4-D omit NOE spectroscopy unambiguously revealed an augmented ubiquitin binding interface that encompasses the C-terminal tail of UBM1.</p><p>4-D omit NOE spectroscopy was also used to study the Fanconi anemia associated protein 20 (FAAP20)-ubiquitin complex within the Fanconi Anemia (FA) complexes required for efficient repair of DNA interstrand crosslinks (ICLs), a process that is mediated by the ubiquitin-binding zinc finger (UBZ) domain of FAAP20. Unexpectedly, we show that the FAAP20-ubiquitin interaction extends beyond the compact UBZ module and is accompanied by transforming the disordered C-terminal tail of FAAP20 into a rigid &beta-loop, with the invariant C-terminal tryptophan (W180 of human FAAP20) emanating toward I44 of ubiquitin for enhanced binding. Accordingly, alanine substitution of the absolutely conserved C-terminal tryptophan residue of FAAP20 abolishes ubiquitin binding and impairs FA core complex-mediated ICL repair <italic>in vivo<italic>.</p><p>Reliable detection and unambiguous assignment of intermolecular NOEs is essential to NMR-based structure determination of protein complexes. The development of 4-D omit NOE spectroscopy in this thesis overcomes many limitations of conventional 3-D half-filtered experiments to allow for reliable detection and unambiguous assignment of intermolecular NOEs of heterodimeric complexes and homooligomeric complexes. These advantages render such a strategy particularly attractive for structural studies of protein complexes by biomolecular NMR.</p>Dissertatio

CHARACTERIZATION OF THE ROLE OF K63-LINKED UBIQUITINATION IN PROTEIN AND MITOCHONDRIAL HOMEOSTASIS: IMPLICATIONS FOR PARKINSON'S DISEASE

Ph.DDOCTOR OF PHILOSOPH

Ubiquitin Ligase Trim32 and Chloride-sensitive WNK1 as Regulators of Potassium Channels in the Brain

The voltage-gated potassium channel Kv1.2 impacts membrane potential and therefore excitability of neurons. Expression of Kv1.2 at the plasma membrane (PM) is critical for channel function, and altering Kv1.2 at the PM is one way to affect membrane excitability. Such is the case in the cerebellum, a portion of the brain with dense Kv1.2 expression, where modulation of Kv1.2 at the PM can impact electrical activity of neurons and ultimately cerebellum-dependent learning. Modulation of Kv1.2 at the PM can occur through endocytic trafficking of the channel; however mechanisms behind this process in the brain remain to be defined. The goal of this dissertation was to identify and characterize modalities endogenous to the brain that influence the presence of Kv1.2 at the neuronal plasma membrane. Mass spectrometry (MS) was used to first identify interacting proteins and post-translational modifications (PTM) of Kv1.2 from cerebellar tissue, and the roles of these interactions and modifications on Kv1.2 function were evaluated in two studies: The first study investigated Trim32, a protein enzyme that catalyzes ubiquitylation, a PTM involved in protein degradation, but also in non-degradative events such as endocytic trafficking. Trim32 was demonstrated to associate and localize with Kv1.2 in cerebellar neurons by MS, immunoblotting (IB), and immunofluorescence (IF), and also demonstrated the ability to ubiquitylate Kv1.2 in vitro through purified recombinant proteins. Utilizing cultured cells through a combination of mutagenesis, biochemistry, and quantitative MS, a working model of Kv1.2 modulation was developed in which Trim32 influences Kv1.2 surface expression by two mechanisms that both involve cross-talk of ubiquitylation and phosphorylation sites of Kv1.2. The second study investigated WNK1, a chloride-sensitive kinase which regulates cellular homeostasis. Using MS, IB, and IF, WNK1 was demonstrated to associate and localize with Kv1.2 in the cerebellum, and a combination of mutagenesis and pharmacology in both wild-type and WNK1-knockout cultured cells produced a working model whereby WNK1 modulates surface Kv1.2. Activation of the downstream target SPAK kinase, also identified by MS to associate with Kv1.2 in the brain, by WNK1 was additionally found to influence the manner of WNK1 modulation of Kv1.2. In addition to providing new models of Kv1.2 modulation in the brain, these studies propose novel biological roles for Trim32 and WNK1 that may ultimately impact neuronal excitability

A systematic analysis of human transmembrane E3-RING proteins

The reversible covalent conjugation of the small highly conserved ubiquitin protein modifier to selective substrates plays central roles in countless proteolytic and non-proteolytic cellular functions. Substrate protein ubiquitination is co-ordinated by the sequential activity of three distinct classes of proteins: (i) E1-activating enzymes, (ii) E2-conjugating enzymes, and (iii) E3-protein ligases. Really Interesting New Gene (RING) proteins represent the largest family of E3-proteins comprising over half of predicted human E3-ligases. As such, E3-RING proteins play pivotal roles in controlling both specificity and functionality within the ubiquitin system. E3-RING proteins function as catalytically inactive molecular scaffolds that position Ub~E2 and substrate proteins in close proximity for ubiquitination to occur. Within the active ligase complex, E3-RING proteins and E2 conjugating enzymes are believed to select protein substrate(s) and the form of conjugated ubiquitin upon them, respectively. Whilst E3-RING/E2 partners have been investigated in recent HTP screen approaches, a key area of data paucity exists for integral membrane E3-RING (TM-E3-RING) proteins. As such, high throughput yeast-two-hybrid assays were performed for the entire complement of TM-E3-RING proteins and E2-conjugating enzymes. A broad subset of TM-E3-RING/E2 positive and negative Y2H interactions was re-tested in secondary luciferase protein complementation assays (PCAs), which increased confidence in Y2H-derived interactions and extended network coverage. Data from these studies was collated with previously published binary TM-E3-RING/E2 interaction data to provide a high-confidence TM-E3-RING/E2 network consisting of 312 unique binary interactions. In vitro auto-ubiquitination assays were employed to assign functional activity to TM-E3-RING/E2 protein pairs, revealing high verification rates for both positive and negative Y2H or PCA binary interaction data. Furthermore, novel trends in the generation of different forms of ubiquitin modifications were identified between selective TM-E3-RING/E2 pairs. Finally, Y2H screens were also performed to identify TM-E3-RING dimerization events, which represent an emerging theme in ubiquitin system regulation. In total 71 TM-E3-RING/TM-E3-RING interactions were reported demonstrating high incidence of these binding events. Novel data was combined with known interactions to generate a TM-E3-RING network containing >500 binary interactions, encompassing both components of the core ubiquitin cascade and non-ubiquitome proteins. This TM-E3-RINGcentric network provides a valuable tool for the investigation of specificity and regulation of TM-E3-RING proteins and specific ubiquitin cascades

Role of the Polyadenylation Factor CstF-50 in regulating the BRCA1/BARD1 E3 Ubiquitin (Ub) Ligase Activity

The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. The studies presented in this dissertation focus on the determination and characterization of the role of mRNA processing factor CstF-50 and escort protein p97 in the regulation of the BRCA1/BARD1 E3 ubiquitin (Ub) ligase activity during the DNA damage response (DDR). As part of the studies presented in Chapter II, I determined that the polyadenylation factor CstF plays a direct role in DDR, specifically in transcription-coupled repair (TCR), and that it localizes with RNA polymerase II (RNAP II) and BARD1 to sites of repaired DNA. My results also indicated that CstF plays a role in the UV-induced ubiquitination and degradation of RNAP II. In Chapter III, I determined that the carboxy-terminal domain of p53 associates with factors that are required for the ultraviolet (UV)-induced inhibition of the mRNA 3\u27 cleavage step of the polyadenylation reaction, such as the tumor suppressor BARD1 and the polyadenylation factor CstF-50. These results were part of a study that identified a novel 3\u27 RNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking RNA processing to the p53 network. In addition, in Chapter IV I showed that CstF-50 can interact not only with BRCA1/BARD1 E3 Ub ligase but also with ubiquitin (Ub), the escort-factor p97 and some of BRCA1/BARD1 substrates, such as RNAP II, H2A and H2B. I also demonstrate that CstF-50-associated p97 activates the BRCA1/BARD1-dependent RNAP II poly-ubiquitination, H2A and H2B monoubiquitination as well as BRCA1/BARD1 autoubiquitination. Together my results provide evidence that CstF-50-associated p97 regulates BRCA1/BARD1 Ub ligase activity during DDR, helping in the assembly and/or stabilization of the ubiquitination complex. Extending these studies, in Chapter V, I showed that UV-treatment induces changes in the localization of BRCA1, BARD1, CstF-50, p97 and some of BRCA1/BARD1 substrates in different nuclear fractions, and that these changes depend on BRCA1/BARD1 and CstF-50 expression. Further, my results demonstrate that the content of monoubiquitinated H2B in the chromatin of genes with different levels of expression changes during DDR and this is mediated by BRCA1/BARD1 and CstF-50. The data presented in this chapter show new insights into the role of mRNA 3\u27 processing factor CstF-50 in regulating the Ub pathway, resulting in epigenetic control during DDR. Finally, in Chapter VI, I identified the RNA binding protein HuR as a new substrate for BRCA1/BARD1/CstF-50/p97 Ub ligase activity in different cellular conditions. All together, the studies presented in this dissertation revealed unexpected insights into the role of the RNA processing factor CstF-50, tumor suppressors BRCA1/BARD1 and p53, the Ub pathway and chromatin structure during DDR