Fanconi anemia (FA) is a genome instability syndrome that is clinically manifested by bone marrow failure, congenital defects, and elevated cancer susceptibility. The FA pathway is known to regulate the repair of DNA interstrand crosslinks in part through DNA homologous recombination (HR) repair. Up to today 16 FA proteins have been discovered that may participate in the common pathway. Cells that have mutations in the FA genes are hypersensitive to DNA damaging agents and display chromosome instability. A key regulatory event in the FA pathway is monoubiquitination of FANCD2-FANCI heterodimer that is mediated by a multi-component E3 ubiquitin ligase complex called FA core complex. Current model suggests that once the FANCD2-FANCI heterodimer is monoubiquitinated it relocates to chromatin where it interacts with other key repair proteins to facilitate DNA repair. More than 90% of the FA cases are presumed to be associated with defects in the monoubiquitination reaction, suggesting the significance of the modification in the pathogenesis of the disease. Despite the significance, the molecular interplay between the FA core complex and the FANCD2-FANCI heterodimer remains enigmatic. We are interested in the assembly mechanism of the various FA subcomplexes into the core complex, and we are actively investigating how the FANCD2-FANCI heterodimer is recruited to these putative subcomplexes. As the FA pathway is a crucial determinant for cellular resistance to DNA damaging agents, there have been hypotheses that disruption of this pathway may be beneficial in enhancing chemosensitivity of certain cancer cells. In collaboration with Dr. Cai’s chemistry lab, we will develop a screen platform to identify a small molecules to interrupt the monoubiquitination reaction. Completion of these studies will enhance the much-needed knowledge of the key enzymatic reaction in the pathway, and perhaps the information can be used for development of novel chemotherapeutic strategies.

Fanconi anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer predisposition. Cell lines derived from FA patient exhibit chromosomal instability and sensitivity to DNA interstand crosslinkers (ICLs) like mitomycin (MMC). The key event in Fanconi anemia pathway is the regulated ubiquitination and deubiquitination of FANCD2 and FANCI. Upon DNA damage, FANCD2 and FANCI are monoubiquitinated by FA core complex. They then move into the chromatin and serve as the landing site for downstream players, like FANCP/SLX4 and FAN1. USP1, the deubiquitinating enzyme (DUB), removes ubiquitin from FANCD-Ub/FANCI-Ub, and this step is required for the integrity of FA pathway. This dissertation addresses how USP1 is regulated in the cell. In Chapter 2, we discovered UAF1/WDR48 as a critical binding partner for USP1, by activating its enzymatic activity in vitro and in vivo. We then generated DT40 knockout cell lines of USP1 and UAF1. We showed that USP1/UAF1 complex is functionally required for homologous recombination (HR). Interestingly, PCNA-Ub is also a substrate for USP1. We discovered that hELG1, through its binding to USP1/UAF1 complex, regulates the deubiquitination of PCNA-Ub and translesion DNA synthesis (TLS). Then in Chapter 3, we discovered a tandem repeat of SUMO-like domains (SLD1 and SLD2) in the C terminus of UAF1. SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 of UAF1 or mutation of the SIM of FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination. The SLD2 sequence of UAF1 also binds to a SIM on hELG1, and targets the USP1/UAF1 complex to its PCNA-Ub substrate. We proposed the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD-SIM interactions coordinates HR and TLS. Originating from USP1/UAF1 complex, we worked out a general mechanism of DUB regulation by WD40 proteins, which involved in two more DUBs, USP12 and USP46 (discussed in Chapter 4 and Appendix A). Lastly in Chapter 5, through bioinformatic analysis we identified a series of novel proteins containing ubiquitin-binding zinc fingers (UBZ). We then focused on SNM1A and FAAP20/C1orf86, and characterized their function in DNA crosslink repair.

Lysine methylation is an important post-translational modification found on histones that is added and removed by histone lysine methyltransferases and demethylases, respectively. Lysine methylation occurs in a specific and well-regulated manner, and plays key roles in regulating important biological processes such as transcription, DNA damage and cell cycle. Regulation of the protein abundance of these methylation enzymes particularly by the ubiquitin-proteasome system has emerged as a key mechanism by which the histone methylation status of the cell can be regulated, allowing cells to respond rapidly to specific developmental and environmental cues. In my thesis, I focus on two histone lysine demethylases, KDM4A and PHF8, both of which appear to be regulated by E3 ligases; this regulation impacts their function in the cell. Chapter 2 shows that KDM4A is targeted for proteasomal degradation by the SCFFBXO22, and mis-regulation of KDM4A results in changes in global histone 3 lysine 9 and 36 (H3K9 and H3K36) methylation levels and impacts the transcription of a KDM4A target gene, ASCL2. Chapter 3 shows how PHF8 is targeted for proteasomal degradation by the APCCDC20 via a novel, previously unreported LxPKxLF motif on PHF8. I also found that similar to other APCCDC20 substrates like Cyclin B, PHF8 is an important G2-M regulator, loss of which results in cell cycle defects such as prolonged G2 and defective M phases. To further interrogate PHF8 biology, Chapter 4 describes the generation of a PHF8 conditional knockout mouse. PHF8 biology is interesting and relevant to human disease, as mutations are found in X-linked intellectual disability and autism. Complete loss of PHF8 by full body knockout in the mouse appears to be embryonically lethal, underscoring its key role in early development. This mouse model would allow us to extensively study the biochemistry and biology of PHF8 in the context of development and especially in brain function, where it is anticipated to play key roles. Overall, my dissertation work provides mechanistic and biological insights into how histone demethylases are dynamically regulated by the ubiquitin-proteasome system, providing an extra dimension to our understanding of how chromatin marks can be regulated.

Ubiquitination is a post-translational modification that can take several forms. Some proteins are modified with a single ubiquitin molecule, while others are modified with polyubiquitin chains. Each type of ubiquitination is thought to have distinct biological functions. The best-characterized types of ubiquitin modification are K48-linked polyubiquitination, which serves as a signal for proteasomal degradation and K63-linked polyubiquitination, which has non-proteolytic functions such in DNA repair, signaling, and endocytosis. HECT ubiquitin ligases (HECT E3s) form a class of E3s, defined by a C terminal catalytic domain. Several lines of evidence suggested that the HECT E3s assemble a polyubiquitin chain in a sequential manner with one molecule of ubiquitin at a time being conjugated to the distal ubiquitin of the chain. In the process of chain elongation, not all HECT E3s target a common internal lysine of ubiquitin, leading to diversification of chain type specificity in HECT E3s. For example, yeast Rsp5 forms K63 chains, while human E6AP forms K48 chains. Two important mechanistic questions were addressed in my work: 1) what are the determinants of chain type specificity of HECT E3s, and 2) what allows the distal ubiquitin of a chain to be continuously oriented near the active site of the HECT domain in the course of a sequential polyubiquitination reaction? I have determined that the chain type specificity of Rsp5 is a function solely of the HECT domain. Further, through the generation of chimeric HECT E3s, I demonstrated that chain type specificity determinants are located within the last 60 amino acids of the C lobe of the HECT domain. To address the second question, we solved the structure of Rsp5 HECT domain in complex with non-covalently bound ubiquitin in collaboration with Jue Chen’s laboratory (Purdue University). From the structure, we found that the N lobe of the HECT domain binds ubiquitin in a manner distinct from other known ubiquitin binding domains, and I have shown that Rsp5 proteins defective for ubiquitin binding are defective for chain elongation. We hypothesize that the ubiquitin binding site functions in the recruitment of the distal ubiquitin of polyubiquitin chain for efficient polyubiquitination. / text

Deregulation of platelet-derived growth factor receptor (PDGFR) signaling is a driving event in glioblastoma, promotes tumor progression epithelial to mesenchymal transition (EMT) in multiple cancers, modulates the tumor stroma to facilitate tumorigenesis and reduces tumor uptake of chemotherapeutics. Previous studies identified the 105 kDa isoform of ankyrin3 (Ank105) as a binding partner of the PDGFR signaling machinery and demonstrated that expression of Ank105 promoted PDGFR degradation (Ignatiuk et al., 2006)(Ignatiuk et al., 2006)(Ignatiuk et al., 2006). Receptor tyrosine kinases are targeted for degradation via endocytosis and ubiquitin-dependent trafficking to the lysosome. It was hypothesized that Ank105 promoted the constitutive degradation of the PDGFR and attenuation of PDGFR signaling by facilitating endocytosis of the PDGFR and targeting the PDGFR for lysosomal degradation via an ubiquitin-dependent mechanism. The studies in this thesis characterized the effects of Ank105 expression on PDGFR signaling and protein expression levels, determined the endocytic pathways involved in Ank105-mediated PDGFR degradation and studied the role of ubiquitin binding in Ank105 function. The most robust effect of Ank105 expression on the PDGFR was constitutive degradation as PDGFR protein expression levels in Ank105-expressing cells were significantly reduced compared to NIH 3T3 cells in the absence of PDGF ligand. Low constitutive PDGFR levels resulted in attenuated pro-proliferative AKT and mitogen-activated protein kinase (MAPK) signaling in response to ligand stimulation. To determine the endocytic requirements for Ank105-mediated constitutive PDGFR degradation, a constitutive PDGFR degradation assay was developed and the effects of several small molecule endocytosis inhibitors were evaluated. Additionally, the small molecule endocytosis inhibitors were validated by determining the effects of these inhibitors on low density lipoprotein (LDL) uptake and ligand-induced PDGFR degradation in Ank105-expressing cells. Both LDL uptake and ligand induced PDGFR degradation are known to proceed by a clathrin and dynamin dependent mechanism of endocytosis. In Ank105-expressing cells, both LDL uptake and ligand incuded PDGFR degradation were dependent upon clathrin and dynamin function. Interestingly, constitutive PDGFR degradation in Ank105-expressing cells was not dependent upon CME, but required dynamin activity. Expression of Ank105 may promote clathrin-independent, dynamin-dependent, constitutive endocytosis of the PDGFR. Additionally, acute inhibition of either lysosomal or proteasomal degradation strongly impaired constitutive PDGFR degradation, whereas ligand-induced PDGFR degradation was less sensitive to protein degradation inhibitors, while LDL uptake was unaffected. It was unclear if PDGFR was degraded in the proteasome or if the proteasome was involved in sorting of PDGFR to the lysosome for degradation. Ubiquitination of receptors is required to target them for degradation. Ank105 was assayed for the ability to interact with ubiquitin and ubiquitinated proteins. Interestingly, Ank105 bound ubiquitin in vitro via the spectrin binding domain and co-immunoprecipitated with several ubiquitinated proteins, suggesting a role for Ank105 in the sorting of ubiquitinated proteins for degradation. Furthermore, Ank105 co-immunoprecipitated with a number of high and low molecular weight proteins in the absence of PDGF stimulation. Identification of Ank105 binding partners would provide further insight in the mechanism of Ank105-mediated constitutive PDGFR degradation. In summary, Ank105 promoted the attenuation of PDGFR signaling via alteration of constitutive PDGFR endocytosis and targeting of constitutive PDGFR for degradation, potentially through interaction with ubiquitin and ubiquitinated proteins. Reduction of constitutive PDGFR levels in cancers with PDGFR driver mutations, acquired PDGF responsiveness and stromal expression of PDGFR, could significantly reduce tumor proliferation, tumorigenesis and increase effectiveness of chemotherapeutics.

<p>Cells thrive on sustaining order and balance to maintain proper homeostatic functions. However, the primary machinery involved in protein quality control including chaperones, ubiquitin proteasome system, and autophagy all decline in function and expression with age. Failures in protein quality control lead to enhanced protein misfolding and aggregation. Efficient elimination of misfolded proteins by the proteasome system is critical for cellular proteostasis. However, inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, which is a hallmark of numerous neurodegenerative diseases. Due to the post-mitotic nature of neurons, they are more susceptible to the collapse in proteostasis correlated with age. </p><p> </p><p>Here, we propose a cell based model of aggresome clearance using a reversible proteasome inhibitor, MG132, to identify the precise molecular machinery involved in proper processing of inclusions. It is known that once misfolded proteins are aggregated, the proteasome system can no longer degrade them. Furthermore, the continuous accumulation of aggregates often leads to aggresome formation, which results in amalgamated inclusion bodies that are simply too large for autophagosomes to engulf and degrade. Although, studies have shown that aggresomes can eventually be cleared by autophagy, the molecular mechanisms underlying this process remain unclear. </p><p>Our research reveals that regardless of impaired proteolysis, proteasomes can still stimulate autophagy-dependent aggresome clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the deubiquitinating enzyme Poh1. Unanchored ubiquitin chains activate ubiquitin-binding histone deacetylase 6, which mediates actin-dependent disassembly of aggresomes. This crucial de-aggregation of aggresomes allows autophagosomes to efficiently engulf and eliminate the protein aggregates. Interestingly, the canonical function of Poh1 involves the cleavage of ubiquitin chains en bloc from proteasomal substrates prior to their degradation by the 20S core, which requires intact 26S proteasomes. In contrast, here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained as an efficient K63 deubiquitinating enzyme complex. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Ultimately, we hope to apply these molecular markers of inclusion body processing to identify the underlying lesion in aggregate prone neurodegenerative disease.</p> / Dissertation

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Nervenzellen sind hochspezialisierte Zellen, die an Synapsen miteinander verbunden sind, was die Übertragung von neuronalen Informationen erlaubt. Die Entwicklung von Synapsen und die Informationsverarbeitung und Gedächtnisbildung bei reifen Synapsen erfordert eine dynamische Umorganisation von neuronalen Netzwerken. Das beinhaltet die Bildung und Entfernung von Synapsen, Umsatz von synaptischen Proteinen und die Veränderung und Anpassung von synaptischer Erregungsübertragung. U. a. Ubiquitinierung, als regulatorische, posttranslationale Modifikation von Proteinen, könnte eine entscheidende Rolle für solche komplexe, synaptische Umorganisationen spielen. Nedd4-1, eine HECT-Typ E3 Ubiquitin Ligase, reguliert und fördert die Entwicklung von Nervenzellfortsätzen durch die Ubiquitinierung von Rap2. Um die Bedeutung von Nedd4-abhänginger Ubiquitinierung im entwickelten Gehirn zu untersuchen, wurden Mausmodelle generiert und analysiert, in denen Nedd4-1 und dessen nächstes Homolog Nedd4-2, speziell in Nervenzellen ausgeschaltet wurde. Ich habe herausgefunden, dass Nedd4-1 und Nedd4-2 wichtige regulatorische Proteine für die neuronale Morphogenese und die synaptische Plastizität, insbesondere die Aufrechterhaltung von LTP, darstellen. Desweiteren habe ich festgestellt, dass Synaptopodin (SYNPO), ein Prolin-reiches, Aktin-assoziiertes Protein, von Nedd4-1 und Nedd4-2 in vitro ubiquitiniert wird. Dieses Ergebnis deutet daraufhin, dass SYNPO in dem Mechanismus eine Rolle spielt, durch den Nedd4-1 und Nedd4-2 LTP aufrechterhalten. Diese Studie wirft ein neues Licht auf die funktionelle Rolle von Nedd4-abhänginger Ubiquitinierung bei höheren Funktionen des Gehirns von Säugetieren sowie der neuronalen Entwicklung.

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