Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Intranuclear rodlets (INRs) are poorly understood intranuclear bodies originally identified within neuronal nuclei on the basis of their unique morphology. Their mechanism of formation, biochemical composition and physiological significance are largely unknown. To gain insight into the molecular regulators of INR formation, mice with a conditional adult β cell-specific knockout of the master regulator of β-cell metabolism, Lkb1 protein kinase (LABKO mice) were studied. The proportion of beta cells containing INRs was significantly reduced in LABKO mice. Further examination ruled out mTOR and Mark2 as downstream effectors of Lkb1 knockout INR phenotype. Instead it identified the mTOR pathway as an independent regulator of INR formation. To investigate INR changes in a pathophysiological context, β cell INRs were examined in two models of human metabolic syndrome: (1) mice maintained on a high-fat diet and (2) leptin-deficient ob/ob mice. Significant INR reduction was observed in both models. Taken together, our results support the view that INR formation in pancreatic β cells is a dynamic and regulated process. The substantial depletion of INRs in LABKO and obese diabetic mice suggests their relationship to β cell function and potential involvement in diabetes pathogenesis. The significance of these findings was further underscored by the demonstration of INRs in human endocrine pancreas, suggesting their potential relevance to the development of metabolic syndrome in humans. The existence of biochemically distinct subtypes of INRs has been suggested by previous reports of differential immunological staining of INRs in neurochemically distinct neuronal populations. Here, a novel variant of INR has been identified that is immunoreactive for the 40kDa huntingtin associated protein and ubiquitin; and evidence was provided for the existence of additional INR subtypes sharing ubiquitin immunoreactivity as a common feature. Selective association of these INRs with melanin concentrating hormone and tyrosine hydroxylase immunoreactive neurons of the hypothalamus and the locus coeruleus was described. It was also demonstrated for the first time that biochemically distinct INR subtypes can co-exist within a single nucleus where they engage in non-random spatial interactions. These findings highlight the biochemical diversity and cell type specific expression of these enigmatic intranuclear structures. On the basis of these findings and previous literature a hypothesis is proposed as to the overall functional significance of INRs in the cell nucleus.

Development requires the acquisition of different cell fates. A major conserved pathway required for cell fate determination is the Notch signalling pathway. Neuralized is a key regulator of the Notch pathway and is essential for embryonic development in Drosophila melanogaster. I have been studying the role of Neuralized during Drosophila development, focusing on the regulation of this protein. Neuralized is an E3 ubiquitin ligase that targets Notch ligands for ubiquitination and endocytosis in the signal sending cell. This endocytic event is required for signal transduction, and cells lacking Neuralized fail to signal through Notch. I have identified a conserved interaction between Neuralized and phosphoinositides that is essential for the ability of Neuralized to promote ligand endocytosis and Notch signalling. Interactions between Neuralized and phosphoinositides are not required for ligand ubiquitination, identifying a role for Neuralized in downstream aspects of ligand trafficking. I have also determined that Neuralized is dynamically regulated through a combination of tissue specific expression, subcellular trafficking, protein interactions and posttranslational modification. Neuralized contains two related protein domains of unknown function called Neuralized homology repeats (NHR). To gain insight into the function of the NHR domain, I characterized another NHR containing protein, CG3894. CG3894 is required for development and preliminary data indicate that NHR domains dimerize, suggesting a possible interaction between Neuralized and CG3894. The study of Neuralized in Drosophila has contributed to our understanding of this essential protein both at a developmental and cellular level, and has provided a means through which to ask questions about regulation of Notch signalling in a relatively simple context. Given the importance of Notch signalling to development, and contributions that aberrations in signalling make to cancer and diseases of the nervous system, expanding our understanding of the regulation of Notch signalling is essential to understanding how this important pathway functions.

Skeletal muscle (SM) atrophy complicates many illnesses, diminishing quality of life and increasing disease morbidity, health resource utilization and health care costs. In animal models of muscle atrophy, loss of SM mass results predominantly from ubiquitin-mediated proteolysis and ubiquitin ligases are the key enzymes that catalyze protein ubiquitination. We have previously shown that ubiquitin ligase Nedd4-1 is up-regulated in a rodent model of denervation-induced SM atrophy and the constitutive expression of Nedd4-1 is sufficient to induce myotube atrophy in vitro, suggesting an important role for Nedd4-1 in the regulation of muscle mass. In this study we generate a Nedd4-1 SM specific-knockout mouse and demonstrate that the loss of Nedd4-1 partially protects SM from denervation-induced atrophy confirming a regulatory role for Nedd4-1 in the maintenance of muscle mass in vivo. Nedd4-1 did not signal downstream through its known substrates Notch-1, MTMR4 or FGFR1, suggesting a novel substrate mediates Nedd4-1’s induction of SM atrophy.

Development requires the acquisition of different cell fates. A major conserved pathway required for cell fate determination is the Notch signalling pathway. Neuralized is a key regulator of the Notch pathway and is essential for embryonic development in Drosophila melanogaster. I have been studying the role of Neuralized during Drosophila development, focusing on the regulation of this protein. Neuralized is an E3 ubiquitin ligase that targets Notch ligands for ubiquitination and endocytosis in the signal sending cell. This endocytic event is required for signal transduction, and cells lacking Neuralized fail to signal through Notch. I have identified a conserved interaction between Neuralized and phosphoinositides that is essential for the ability of Neuralized to promote ligand endocytosis and Notch signalling. Interactions between Neuralized and phosphoinositides are not required for ligand ubiquitination, identifying a role for Neuralized in downstream aspects of ligand trafficking. I have also determined that Neuralized is dynamically regulated through a combination of tissue specific expression, subcellular trafficking, protein interactions and posttranslational modification. Neuralized contains two related protein domains of unknown function called Neuralized homology repeats (NHR). To gain insight into the function of the NHR domain, I characterized another NHR containing protein, CG3894. CG3894 is required for development and preliminary data indicate that NHR domains dimerize, suggesting a possible interaction between Neuralized and CG3894. The study of Neuralized in Drosophila has contributed to our understanding of this essential protein both at a developmental and cellular level, and has provided a means through which to ask questions about regulation of Notch signalling in a relatively simple context. Given the importance of Notch signalling to development, and contributions that aberrations in signalling make to cancer and diseases of the nervous system, expanding our understanding of the regulation of Notch signalling is essential to understanding how this important pathway functions.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Tumor susceptibility gene 101¡]TSG101¡^encodes an inactive ubiquitin conjugating E2 enzyme implicated in regulation of protein sorting, vesicular trafficking, transcription activation of nuclear receptor, cell growth and differentiation. Previous studies showed that TSG101 can be mono- or poly- ubiquitinated, which is relevant to its functional status. There are seven Lysine (K) sites, K6, K11, K27, K29, K33, K48 and K63, on ubiquitin (Ub). Polyubiquitination using different Ub K sites confers differential function for protein degradation, DNA damage repair, endocytosis and protein sorting. AIP4 E3 ubiquitin ligase modifies its substrates involved in erythroid and lymphoid lineage differentiation and the associated immune responses. Mutation in AIP4 gene resolves in multisystemic autoimmune disease. TSG101 was recently shown to be a molecular checkpoint for T cell receptor downregulation. Here we investigate the ubiqutination status of TSG101. The ubiquitin-conjugated protein in lysate of cells co-transfected with pHA-TSG101 and His-tagged wild type Ub or each K site mutant ubiquitin expression plasmids was purified on nickel beads and then subjected to western blotting using antibodies against HA-TSG101 or His-tag. The results showed that K series mutant had differential effect on the steady-state of HA-TSG101. Proteasome inhibitor could alleviate its degradation especially in the K63 ubiquitin expression group, implying K63 ubiquitination E3 ligase is critical in maintaining HA-TSG101 level. Our coimmunoprecipitation result demonstrated the interaction between AIP4 and HA-TSG101, implying that TSG101 might be a substrate for AIP4. The ectopic overexpression of AIP4 increased the amount of HA-TSG101 in an E3 ligase activity depended manner. Taken together, these results indicated that AIP4 activity mediating Ub K63 modification might be critical for regulating cellular TSG101 protein level. Further experiment should clarify this issue.

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ABSTRACT TRANSCRIPTION PATTERN COMPARISON OF TWO UBIQUITIN SPECIFIC PROTEASES (USP6 AND USP32) Akhavan Tabasi, Shiva M.Sc., Department of Biology Supervisor: Assist. Prof. Dr. A. Elif Erson August 2007, 93 pages Breast cancer is the most common type of cancer among women worldwide. The incidence of breast cancer is 1 in 8 among women. Usually loss of tumor suppressor genes and overexpression of proto-oncogenes are known to be involved during mammary tumorigenesis. USP32 (Ubiquitin Specific Protease 32) gene is located on chromosomal band 17q23, a region of amplification in breast cancer. Gene amplification is known to be a common mechanism in breast cancer cells, through which proto-oncogenes are overexpressed and contribute to tumor progression. Presence of multiple oncogene candidates on 17q23 requires individual characterization of these genes. USPs (Ubiquitin Specific Protease), have various roles in protein degradation pathways (e.g / by editing the ubiquitin chains, recycling of ubiquitin, v deubiquitinating the target proteins and inhibiting their degradation by the proteasome). Deregulated expression of USPs is likely to interfere with the degradation of many key regulatory proteins in the cell. Therefore, USP32 becomes an interesting oncogene candidate that may have roles in protein degradation pathways based on the fact that it is located on an amplicon region and that it is overexpressed in breast tumors. On the other hand, USP6 (Ubiquitin Specific Protease 6), a known oncogene on 17p13, is also a deubiquitinating enzyme, with conserved histidine and cysteine domains, which are also shared by USP32. Interestingly there is a 97% sequence similarity between bases 3,197 to 7,831 of USP6 and 2,390 to 7,024 of USP32 gene. In this study, we aimed to investigate the expression patterns of USP32 and USP6 (including alternative transcripts) in breast tissue to avoid any possibility of overlapping functions of two enzymes due to their high sequence similarity. In addition, we sub-cloned USP32 gene into TOPO-TA vector, so that further functional studies (e.g / localization and overexpression) can be performed. Further characterizations of Ubiquitin Specific Protease 32, may help us understand its importance in the protein degradation pathway during breast tumorigenesis.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references.