Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains viii, 72 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 68-72).

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, March 2000. / Includes bibliographical references. Also available on the Internet.

Universiẗat, Diss., 2002--München. / Enth. 1 Sonderabdr. aus : Nature ; Vol. 419. 2002.

Thesis (M.S.)--University of Missouri-Columbia, 2004. / Typescript. Includes bibliographical references (leaves 69-81). Also available on the Internet.

Thesis (M.S.)--University of Missouri-Columbia, 2004. / Typescript. Includes bibliographical references (leaves 69-81). Also available on the Internet.

<b>Background:</b> Aryl Hydrocarbon Receptor Interacting Protein-Like 1 (AIPL1) interacts with NUB1 and restricts the entry of NUB1 protein into the nucleus. The interferon-induced NEDD8 ultimate buster (NUB1) protein causes degradation of neddylated and FAT10ylated proteins through the ubiquitin proteasome system. We observed AIPL1 were frequently down-regulated in various cancers compared to normal tissues. The mechanistic roles of AIPL1 and NUB1 protein in cancer cell cycle regulation remain unexplored. <b>Results:</b> Meta-analysis of cancer databases revealed that expression transcripts of chaperones, including AIPL1, were down-regulated in lung, pancreatic cancer and breast cancer relative to the adjacent normal tissues. Opposite levels of both AIPL1 and NUB1 transcripts were observed in the breast cancer. So it triggers the in vitro experiments using breast cancer cells. METABRIC breast cancer clinical cohort highlighted that patients with low NUB1 transcripts had poor survival in the ER-negative subgroup (but not in ER-positive) of breast cancer patients: hazard ratio (HR)=0.66, 95% confidence interval (CI)=0.5-0.87, p=0.003 and triple negative subgroup of breast cancer patients: HR=0.67, 95% CI=0.47-0.96, p=0.028. NUB1 silencing significantly inhibits in vitro cell growth in MDA-MB-231 and MCF7 under hypoxia. AIPL1 protein forms multimers in cancer cells. NUB1 protein moved into the nucleus in hypoxia (0.1% O2 48hrs) with final confluency at 80-90%. p21 (marker of senescence) &amp; p27 (marker of cell cycle arrest) accumulated in NUB1-silent MDA-MB-231 and RCC4 cells. It suggested that low NUB1 nuclear localisation in hypoxia cause cancer cell cycle arrest. In MDA-MB-231 cell, upon hypoxia, neddylation inhibitor (MLN4924) treated and siNUB1 transfected cells showed decreased CUL1 and further accumulated p21 &amp; p27. The evidence suggested lower neddylated CUL1 and reduced NUB1 cooperatively stabilise p21 and p27 as the substrate of CUL1-ubiquitin ligase. The neddylation inhibitor MLN4924 treated and NUB1 knockdown group exhibited more cells in sub-G1 stage as compared to the control group. In connection to higher p21/p27, it is associated with prolonged arrested cellular aging with depletion. After silencing of NUB1, the increases in cell death of cancer cells upon hypoxia happen through the neddylation-dependent CUL1-p27-p21 and CUL2-VHL axis. We then demonstrated that HIF1&alpha; protein could be both neddylated and FAT10ylated upon reoxygenation. In a tissue microarray study of breast cancer, lower cytoplasmic expression (n=57) had worse overall survival than higher cytoplasmic expression (n=57): HR=1.779, 95% CI=1.006-3.346, p=0.048. <b>Conclusions:</b> AIPL1 and NUB1 proteins exert a role in cell cycle regulation in breast cancer. Low cytoplasmic NUB1 levels are observed in the G₁-S transition of cancer cells. NUB1 depletion causes G₀/G₁ phase arrest due to CUL1 and CUL2 ubiquitin E3 ligase-dependent pathways.

Protein ubiquitination regulates various cellular events, such as protein degradation, immune signalling and DNA repair. This functional versatility arises from the ability of ubiquitin (Ub) to form distinct polymers linked through one of its eight primary amines (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63 and the N terminus of Met1). Discrimination of these differently linked polyUb chains by linkage-specific ubiquitin-binding domains (UBDs) is crucial to ensure an appropriate cellular response to a particular stimulus. While Lys48- and Lys63-linked chains have been studied extensively and their functions are well-described, the remaining, so-called atypical Ub chains have largely resisted characterisation. The broad aim of this thesis was to better our understanding of atypical Ub modifications with a focus on Ub recognition by linkage-specific binders. In the first part, the UBD family of Npl4-like zinc fingers (NZFs) will be discussed, as this family has previously been shown to be capable of linkage-specific recognition. A candidate-based approach was used to screen UBDs for linkage-preference for atypical Ub chains. This revealed that TRABID NZF1 is specific for Lys29/Lys33 chains and is the first UBD to specifically recognise these linkages. Moreover, [a protein] was found to be specific for Lys6/Lys63 chains and is the first UBD with specificity for Lys6 chains. Biochemical and structural characterisation of these UBDs in complex with their preferred chain types uncovered the molecular mechanisms that confer linkage specificity. Using insights gained from studying NZF UBDs, further investigations into the versatility of the NZF scaffold were performed. Unexpectedly, this revealed that [a protein] is able to recognise substrate ubiquitination of [a protein] in a substrate- and site-specific manner and this interaction was structurally and biochemically characterised. In the second part, the UBDs of all selective autophagy receptors were screened for interesting interactions with Ub. None of the UBDs tested exhibited linkage-specific interaction with Ub, except for OPTN, which prefers Met1-linked chains. Moreover, because autophagy receptors can also act in mitophagy, these UBDs were tested for preferential interaction with Ub phosphorylated on Ser65. However, none of the UBDs tested prefer phosphorylated Ub. Two structures of these UBDs in complex with Ub are presented, which together with previously published structures, allowed us to rationalise the observed binding behaviours. In the final part, the generation of linkage-specific tools for Lys6- and Lys33-/Lys11-linked chain is described. In collaboration with Avacta, linkage-specific affimer binders against Lys6- and Lys33/Lys11-linked polyUb chains have been developed. Crystal structures of the affimers in complex with their cognate diUb linkages revealed the basis of linkage specificity and further allowed structure-guided improvements. These improved affimers prefer their cognate diUb >106-fold over other Ub linkages. Furthermore, affimers were validated in a number of applications: using the Lys6-affimer as a pull-down reagent allowed enrichment of Lys6 chains over 100-fold compared to other linkages. This led us to identify the HECT E3 ligase HUWE1 as a major source of cellular Lys6 chains. Furthermore, the Lys6-affimer was used in confocal imaging where it localised to depolarized mitochondria in a Parkin-dependent manner, consistent with Parkin assembling Ub chains, including Lys6 linkages, upon mitochondrial damage. Together, these affimers are the first linkage-specific tools for these atypical Ub chains and enable targeted investigations into the biological functions of specific atypical Ub linkages. Taken together, the research presented in this thesis identified novel, linkage-specific components of the Ub system. Moreover, the developed tools will lay the basis for further, mechanistic investigations into the functions of atypical Ub linkages in important signalling pathways which will hopefully shed light on the biological roles of these understudied chain types.

In addition to apoptosis, metacapases function to regulate various other processes that promote and sustain life. For example, the Saccharomyces cerevisiae metacaspase Yca1 promotes cellular fitness by regulating insoluble protein levels. However, the mechanism(s) that regulate this proteostasis function for Yca1 have remained elusive. Here, using proteomics coupled to protein interaction studies, we describe a role for Yca1 in restraining deposition to the insoluble proteome and further identify a post-translational regulatory mechanism for modulating Yca1 function. Our initial analyses uncovered a role for Yca1 in aggregate assembly where Yca1, in coordination with the Cdc48 chaperone, regulates the composition of the insoluble proteome. Interestingly, loss of Yca1 was correlated with reduced sequestration of proteins related to ribosomal and translational processes in the insoluble protein fraction during heat stress. Subsequent proteomic analyses identified a regulatory mechanism for Yca1 mediated by the ubiquitin system, a feature that was instrumental for limiting insoluble protein content. Specifically, we noted K355 ubiquitination and S346 phosphorylation as key modifications that directed Yca1 function to maintain proteostasis. Loss of function mutations at these sites led to increased retention of insoluble protein and increased vacuolar structures. Surprisingly, loss of Yca1 also affected ubiquitin homeostasis in vivo as observed by reduced levels of low molecular weight free ubiquitin. Upon further analysis, we observed that the ubiquitin precursor protein Rsp31 was cleaved by Yca1 suggesting a possible role for Yca1 in de novo ubiquitin synthesis. Together, these analyses suggest that post-translational modifications of Yca1 are critical regulatory features for this protease, and that this enzyme regulates cell proteostasis in combination with other chaperone and protein degradation machinery.

AMPA-type glutamate receptors (AMPARs) play a critical role in mediating the majority of fast excitatory synaptic transmission in the brain, where alterations in receptor expression, distribution and trafficking have been shown to underlie synaptic plasticity and higher brain function. However, the molecular mechanisms regulating AMPAR surface expression and turnover are still not fully understood. We report that mammalian AMPARs are subject to post-translational modification by ubiquitin, and identify Nedd4 as the E3 ligase responsible for mediating this process. AMPAR ubiquitination enhanced receptor degradation and reduced AMPAR cell-surface expression; conversely, inhibition of proteasomal activity caused AMPAR accumulation. Using site-directed mutagenesis we replaced each of four lysine residues available as putative ubiquitination sites on the AMPAR subunit GluA1 C-terminal with an arginine and identified critical residues for ubiquitination and receptor degradation. Consistent with the role of protein ubiquitination, lysine mutation reduced the efficiency of AMPAR endocytosis. We further investigated the molecular mechanisms involved in the internalization of ubiquitinated AMPARs. We find that the endocytic adaptor protein Eps15 plays a critical role in this process. siRNA-mediated suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interaction with AMPARs requires Nedd4-mediated GluA1 ubiquitination along with the ubiquitin interacting motif (UIM) of Eps15. Consistent with ubiquitination-mediated receptor internalization, knockdown of Eps15 suppresses GluA1 internalization of wild-type GluA1, but not a mutant GluA1 lacking ubiquitination sites, indicating a crucial role for Eps15 in the trafficking of ubiquitinated AMPARs. These findings reveal novel regulatory mechanisms in the control of glutamate receptor amount and distribution dynamics, which are key factors implicated in higher brain functions and neurological disorders.