published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The cellular levels of a number of proteins have been found to be regulated by the ubiquitin-proteasome pathway. In this pathway, proteins are covalently tagged (&ldquo / ubiquitinated&rdquo / ) by ubiquitin, which acts as a signal for degradation by the proteasome. A number of key cellular processes, including cell-cycle progression, transcription and DNA repair, are regulated in this way. In recent years a number of cellular proteins resembling ubiquitin in structure or function, the so-called ubiquitin-like proteins, have been identified. Ubiquitin-like proteins can be divided into two classes-the so-called &ldquo / ubiquitin-like modifiers&rdquo / , which consist of a single domain that structurally resembles ubiquitin, and &ldquo / ubiquitin-domain&rdquo / proteins, which are multi-domain proteins, which include domains that resemble ubiquitin.<br /> <br /> This thesis describes the recombinant expression, purification and full backbone assignment of the human DWNN domain, a novel ubiquitin-like domain. The DWNN domain occurs at the N-terminus of RBBP6, a protein that has been shown to interact with p53 and Rb as well as to be involved in mRNA processing and apoptosis. A bacterial expression system was used to overexpress the DWNN domain as a GST fusion protein. The domain was labelled with 15N and 13C to perform triple-resonance heteronuclear NMR experiments, from which full backbone assignments were obtained.<br /> <br /> Although full structure determination of the DWNN domain falls outside the scope of this thesis, the backbone assignments formed the basis for the subsequent structure determination, which confirmed that the DWNN domain is indeed a novel ubiquitin-like domain. The RBBP6 protein may therefore represent a novel E3 ubiquitin ligase that plays a role in regulating the cellular levels of p53 and Rb.

NEDD8 modification of proteins is extensively studied in the recent years, and the ubiquitin-like molecule has been shown to be involved in numerous signalling pathways. In addition to its well-established roles, we showed that NEDD8 responds to various stress conditions, such as inhibition of the 26S proteasome, heat shock and oxidative stress. Modification of proteins with ubiquitin and ubiquitin-like molecules is involved in the regulation of almost every biological process. Historically, each conjugation pathway has its unique set of E1, E2 and E3 enzymes that lead to activation and conjugation of their cognate molecules. We also showed the unexpected finding that the ubiquitin E1 enzyme Ube1 activates the ubiquitin-like molecule NEDD8. The above-mentioned stress conditions cause a global increase in NEDDylation. Surprisingly, this does not depend on the NEDD8 E1 activating enzyme but rather on Ube1. A common event in the tested stress conditions is the depletion of “free” ubiquitin. A decrease in “free” ubiquitin levels in the absence of additional stress is sufficient to stimulate NEDDylation through Ube1. We also performed mass spectrometric analyses to investigate NEDD8 chain formation under stress. We found that NEDD8 forms chains with itself and with ubiquitin, and these chains are recognized by proteasome receptors and shuttle factors. Our studies revealed an unprecedented interplay between NEDD8 and ubiquitin pathways, operating in diverse cellular stress conditions. In a parallel project, we characterized the role of the deNEDDylating enzyme NEDP1 in response to DNA damage. The NEDP1 ortholog in C. elegans, Ulp-3 has been previously investigated in collaboration with Anton Gartner’s group. The enzyme has been found to be required for DNA damage-induced apoptosis in the worm germ line. Our results in human cell lines showed that the role of NEDP1 is conserved, since NEDP1 knockdown resulted in impaired effector caspase activation. Moreover, we showed that the nedp1 gene is induced upon ionizing irradiation. In the absence of the enzyme, we observed increased NEDDylation that was dependent on the NEDD8 E1 enzyme.

Protein modification by ubiquitination regulates protein abundance, function and localisation. Specificity of ubiquitination is largely determined by ubiquitinprotein ligases (E3s). The Nedd4–family proteins are a group of E3s containing a conserved domain structure of a C2 domain, multiple WW domains and a carboxyl terminal HECT domain, which is responsible for E3 activity. The prototypical member of this family, Nedd4, is known to down-regulate the epithelial Na+ channel (ENaC) by ubiquitination. This process requires interactions between ENaC and specific WW domains of Nedd4. Mutation or deletion of WW domain binding sites in ENaC leads to Liddle's syndrome, an autosomal dominant form of hypertension. At the beginning of this study there was evidence to suggest that Nedd4–2, a Nedd4–family protein closely related to Nedd4, could also regulate ENaC. The focus of this study was to characterise the ability of Nedd4–2 to regulate ENaC and other potential substrates. Two major splice variants of Nedd4–2 were identified, which were both found to down–regulate ENaC in Xenopus oocytes. In vitro binding studies and whole cell functional analysis showed that interactions between ENaC and Nedd4– 2 occur via two of the four Nedd4–2 WW domains. The E3 activity of Nedd4–2 was further examined, revealing that it can use the same ubiquitin–conjugating enzymes as Nedd4 and exhibits strongest activity in the presence of UbcH5b. An in vitro ubiquitination assay and whole cell functional analysis provided evidence that Nedd4–2 down-regulates ENaC via ubiquitination. The possibility that Nedd4 and Nedd4–2 could down-regulate a number of voltage–gated Na+ channels (Navs) by a similar mechanism to regulation of ENaC was investigated. Not only were Nedd4 and Nedd4–2 found to interact with seven Navs, but these channels and ENaC have conserved WW domain binding specificity. Ubiquitination studies indicated that these channels can be ubiquitinated by Nedd4 and Nedd4–2. Co–expression of Nedd4 or Nedd4–2 with neuronal Navs in Xenopus oocytes reduced channel activity to varying degrees. These data indicate that Nedd4 and Nedd4–2 are likely to be key regulators of neuronal Nav channels in vivo. / Thesis (Ph.D.)--Department of Medicine, 2004.

Three of the best-characterized prions of Saccharomyces cerevisiae are [PSI+], [URE3], and [PIN+]. This study focuses on the prions [PSI+] and [PIN+]. [PSI+] is the prion isoforms of the protein Sup35 that functions as a eRF3 translational termination factor. The presence of [PSI+] is detected by the partial loss of function of Sup35. The prion [PIN+] is the isoform of the protein Rnq1, and this proteins function is unknown. The presence of the prion [PIN+] is necessary for the de novo formation of the prion [PSI+] (Derkatch et al., 1997). The chaperone, Hsp104, belongs to an evolutionary conserved Hsp100 family of proteins that participate in a various number of cellular processes (Schirmer et al., 1996). Hsp104, in particular, is responsible for the cells adaptation to heat shock, it controls spore viability and the long-term viability of starving vegetative cells. (Sanchez and Linquist, 1990; Sanchez et al.,1992) It is an ATPase that has been shown to promote solubilization of aggregated protein (Parsel et al., 1991). A unique relationship exists between Hsp104 levels within the cell and the maintenance of the prion [PSI+]. The over production of Hsp104 eliminates [PSI+] (Chernoff 1995). This seems logical considering Hsp104 is a disaggregase, and it is reasonable to assume that the over production provides sufficient resources to break the aggregates into portions that are accessible to either other chaperones which would facilitate the proper folding or perhaps the system responsible for the elimination of unusable proteins, such as the ubiquitin-proteasome system. This study examines the role of the ubiquitin-proteasome system in curing of [PSI+] by Hsp104. The role of alternate pathways, in which the prion isoform is refolded into it correct, functional conformation by the action of the chaperones Ssb1 and Ssb2 is examined. These results suggest that the combination of both the degradation pathway and the refolding of proteins are involved in curing of [PSI+] by Hsp104 over production.

TSG101 exhibits multiple functions, including vesicular trafficking, cell growth, differentiation, and transcriptional regulation. However, the cellular signaling that regulates TSG101 functions remains unexplored. Our previous result indicates that TSG101 can be phosphorylated by PKC and GSK3£]. In this thesis, we further investigate the detail phosphorylated amino acid residues by using in vitro kinase assay in conjunction with MALDI-TOF and peptide array analysis. The results indicate that S13, S48, S103 and S367, T383 residues could be phosphorylated by PKC, S182 and S212 by GSK3£]. Coiled-coil domain of TSG101, which contains 2 consensus CK¢º phosphorylation sites,could be phosphorylated by CK¢º. These results indicate the functions of TSG101 might be regulated by these kinases. Proteasome mediated protein degradation is important to maintain proper cellular functions including cell growth, differentiation and signaling associated with cell cycle control. Impaired function of this system has been implicated in human diseases associated with neurodegeneration and cancer. The inhibitor of proteasome has been successfully used in treatment of these related diseases. In this thesis, we successfully established Ub-X-GFP reporter cell lines, which could be used in the future study on the functional role of TSG101, an E2 variant, in the proteasome mediated degradation pathway. Furthermore, these cell lines will serve a useful cellular platform for screening new proteasome inhibitors.

Abstract SMT3 (Suppressor of MIF2 3 protein) was identified as a mutation suppressor in yeast centromere protein MIF2. It is also known as an ubiquitin-like protein due to the smilarities of their primary structures that is very conserved during the eukaryotic evolution. Although only one SMT3 was found in low eukaryotes such as in yeast, three members of SMT3 (SMT3A, SMT3B and SMT3C) have been identified in high eukaryotes. It has been known that SMT3C plays an important role in post-translational modification. However, the functions of SMT3A and SMT3B are not well studied yet and the relationship among the SMT3 families remains unclear. In the present study, Daxx, a Fas binding protein, was demonstrated to bind to SMT3B using yeast two-hybrid assay. It was found that the N-terminal domain of Daxx (Daxx 1) and the C-terminal domain of Daxx (Daxx 4), respecifitively, bound to all members of human SMT3 families (including SMT3A, SMT3B and SMT3C). Neverthless, mechanisms of interactions between the SMT3 families and Daxx domains remined unclear. Studies on truncated human SMT3 families have shown that two glycines on the C-terminal end of human SMT3 families were required in the interaction between SMT3 and Daxx domains, for example, SMT3A and SMT3B required C-terminal two glycines on the Daxx 4 domain where as SMT3C required C-terminal two glycines on the Daxx 1 domain. Morever, truncated SMT3C and Daxx 1 domain point mutations have also indicated that the the linkage of glycine97 of SMT3C and the lysine60 of Daxx 1, in which the SMT3C/ SUMO-1 consensus sequence £ZKXE was found. Further, SMT3C was the only member of the SMT3 families capable of self-reacting. Results also suggested that similar mechanism of interaction between SMT3A/B and Daxx 1, which is not in accordance with the model proposed in this study regarding the interaction mechanism between SMT3C and Daxx 1. Although two glycines on the C-terminal end of SMT3A/B were necessary for the interactions with Daxx 4 domains, the SMT3C/SUMO-1-consensus sequence £ZKXE was not detected in the Daxx 4 domain. It is therefore, suggested that the mechanism of the interaction between SMT3A/B and Daxx 4 is similar to that of SMT3C and Daxx 1, that may required different binding sequences that is specific for SMT3A/B.

Abstract Tumor susceptibility gene TSG101 plays an important role in cellular functions including intracellular protein sorting, vesicular trafficking, and transcription regulation. Our previous results from yeast two-hybrid screening show that TSG101 interacts with a novel transcriptional repressor protein, KLIP1. In this study, we demonstrated in vivo interaction between TSG101 and KLIP1 in nucleus of 293 cells using co-immunoprecipitation assay and confocal imaging. In addition, we found KLIP1 could be modified in a modality of either poly- and mono-ubiquitination when exogenously expressed in 293 cells in conjunction with either wild type His-tagged ubiquitin or a mutant His-tagged ubiquitin (K0-Ub) which has no capability of forming polyubiquitin chain. Furthermore, we found that TSG101 could increase 60 kDa-KLIP1, but decrease 71 kDa-KLIP1 levels of monoubiquitinated KLIP1 protein species in a dose dependent manner. These results indicate that TSG101 might regulate KLIP1 protein function through affecting its monoubiquitin modification status. Further investigation using wildtype pHA-KLIP1 and mutant pHA-KLIP1-M6 containing mutation in its 6 lysine residues for possible ubiquitin modification revealed that wildtype HA-KLIP1, but not HA-KLIP1-M6, could inhibit transcription activity of thymidine kinase (TK) promoter. In conclusion, our results support that TSG101 interacts and acts as a transcriptional co-repressor of KLIP1 by keeping it in 60 kDa-monoubiquitinated status in the nucleus, where KLIP1 functions as a transcription repressor for TK promoter. Further experiment using mutant HA-KLIP1 expression plasmid containing single mutation in the 6 lysine sites should reveal the exact location of ubiquitin-modified lysine site for monoubiquitinated species of KLIP1 protein.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Department of Molecular Genetics and Cell Biology, June 2001. / Includes bibliographical references. Also available on the Internet.