Browsing by Subject "Ubiquitin"
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Item Calpain 9 Functions in TNF Receptor Mediated Apoptosis(2012-07-20) Kunkel, Gregory Thomas; Wang, XiaodongEvasion of apoptosis is a hallmark of cancer development. The Inhibitor of Apoptosis Proteins, IAPs, block Caspase activity and cell death. Release of the Second Mitochondria-Derived Activator of Caspases, Smac, from the mitochondria relieves IAP Caspase inhibition, activating apoptosis. Our lab has developed a small molecule Smac mimetic. Surprisingly, approximately 25% of cell lines show single agent Smac mimetic sensitivity through activation of autocrine TNF-a secretion and TNF dependent apoptosis. Using Smac mimetic sensitivity as a model system, I performed a genome-wide high-throughput siRNA screen and identified Calpain 9, CAPN9, as a novel component of TNF-alpha induced apoptosis. CAPN9 knockdown does not affect TNF-a secretion, yet is essential for downstream activation. Two splice variants are reported for CAPN9. The smaller splice, CAPN9-SP2, is required for effective TNF-a induced apoptosis. CAPN9 is essential for RIPK1 recruitment and ubiquitination at the TNFR1 upon activation with TNF-alpha. CAPN9 knockdown demonstrates previously unreported association of ubiquitinated proteins, and actin binding proteins with TNFR1 in the absence of stimulus. This interaction is CAPN9 dependent and correlates with CAPN9 regulation of TNF-a induced apoptosis.Item Characterizaton of Mcl-1 in Regulating Different Forms of Cell Death(2007-12-17) Gao, Wenhua; Wang, XiaodongProgrammed Cell Deaths (apoptosis, autophagic cell death and necrosis) play essential roles in animal development and certain diseases. Autophagy is a cellular process that provides nutrients to starved cells by digesting cellular constituents. Defects in autophagy have been implicated in cancer and neurodegeneration, but how autophagy causes cell death is not understood. In mammals, there are two apoptotic pathways: the extrinsic one through death receptors in the cell membrane and the intrinsic one through mitochondria that release death proteins. The Bcl-2 family of proteins function upstream in the intrinsic pathway. They can be subdivided into anti-apoptotic and proapoptotic proteins. Mcl-1 has two different splice variants, Mcl-1L and Mcl-1S. Mcl-1L is an anti-apoptotic Bcl-2 family protein with a short half-life, which functions in the apical step of the intrinsic apoptotic pathway and can inhibit the release of death proteins from mitochondria. After genotoxic treatment, Mcl-1L is rapidly degraded, resulting in mitochondria damage and apoptosis. Prevention of Mcl-1L degradation with proteasome inhibitors blocks apoptosis. An Mcl-1L ubiquitin ligase was identified using biochemical purification method and named Mule, which has five recognizable domains including UBA, WWE, HECT and two ARM repeats like domains. Mule also contains a region similar to the Bcl-2 homology region 3 (BH3) that allows it to specifically interact with Mcl-1L. Depletion of Mule by RNA interference stabilizes Mcl-1L, resulting in an attenuation of apoptosis induced by DNA-damaging agents. To further understand Mcl-1 function, I generated inducible Mcl-1 knockdown stable U2OS cell lines. Depletion of Mcl-1L but not Mcl-1S causes the cells to commit autophagic cell death. The autophagic phenotype can be blocked by knocking down Beclin 1, which is a known upstream component of the autophagic pathway. Mcl-1L knockdown induced cell death is accompanied by and requires the upregulation of p53 and p21. Loss-of-function experiments confirmed the involvement of Mcl-1S in inducing autophagy. Taken together, my data showed that Mcl-1 genes function in the apical step of both apoptosis and autophagic cell death pathways.Item Identification and Characterization of a New E1 That Activates Ubiquitin and FAT10(2008-05-13) Chiu, Yu-Hsin; Chen, Zhijian J.Ubiquitination is one of many post-translational modifications in eukaryotes. Three enzymes (E1, E2, and E3) are involved in conjugating ubiquitin to protein substrates. I identified a novel E1-like protein, E1-L2, which is homologous to the ubiquitin E1 and another E1 involved in the activation of the ubiquitin-like protein ISG-15 (E1-L1). E1-L2 activates both ubiquitin and FAT10, a ubiquitin-like protein. Interestingly, E1-L2 can transfer ubiquitin to only a subset of E2 enzymes, Ubc5 and Ubc13, but not Ubc3 and E2-25K, suggesting that E1-L2 may function in certain ubiquitination reactions. E1-L2, but not E1 or E1-L1, forms a thioester with FAT10 in vitro. The formation of the thioester bond requires the active site cysteine residue of E1-L2 and the C-terminal diglycine motif of FAT10. In addition, endogenous FAT10 forms a thioester with E1-L2 in cells stimulated with tumor necrosis factor-alpha and interferon-gamma, which induce FAT10 expression. Silencing of E1-L2 expression by RNAi impaired the formation of FAT10 conjugates in cells. Furthermore, E1-L2 deficient embryos died before embryonic day 13.5, suggesting that E1-L2 is essential for early embryonic development. Since the FAT10-deficient mice develop normally, it is likely that specific ubiquitination reactions catalyzed by E1-L2 play an important role in animal development.Item Mechanistic Studies of the Activation of Ubiquitin-Conjugating Enzymes by Ring-Type Ubiquitin Ligases(2006-05-15) Özkan, Engin; Deisenhofer, JohannUbiquitination, modification with ubiquitin, is a post-translational regulation of proteins in eukaryotes. Ubiquitin-activating enzymes (E1) activate ubiquitin and form thioester linkages with ubiquitin, which are then transferred onto ubiquitin-conjugating enzymes (E2). Ubiquitin-conjugating enzymes, with the help of ubiquitin ligases (E3) transfer ubiquitin onto ubiquitination targets to form isopeptide linkages between the targets and ubiquitin. The function of ubiquitin ligases in the ubiquitination process has not been clearly understood. Recently, it has been shown that ubiquitin ligases are modular proteins or protein complexes that bring together the ubiquitination targets and the E2 enzyme. In this study, we show that E3 enzymes without their target-binding domains are effective activators of the ubiquitination reaction. We demonstrate that E3 binding to E2 enzymes increases the rate of the breakdown of the E2-ubiquitin thioester bond. Using the Statistical Coupling Analysis, a pairwise residue covariation analysis, we observe that E2 enzymes have a set of residues that vary as a group throughout evolution. Extensive mutagenesis of the E2 enzyme UbcH5b, including its covarying residues, yield mutants that can accept ubiquitin, break the thioester linkage, bind E3 enzymes, but cannot be stimulated to release their ubiquitin thioesters in the presence of an E3 enzyme. We also show that thioester release measurements agree with polyubiquitination of physiological targets. We have structurally characterized our model E2 enzyme UbcH5b, it mutants, and its interaction with E3 enzymes. Taken together, our data strongly demonstrate that E3 enzymes are, apart from their scaffolding function, activators of E2 enzymes to ubiquitinate targets. We discuss possible physical mechanisms of this activation, and hypothesize a long-distance communication pathway between the E3-binding surface and the thioester-forming active site of E2 enzymes. We also report attempts to crystallize several E2 and E3 enzymes and their complexes.Item Modulation of Transcription Factor Activity by Mono-Ubiquitin(2008-09-12) Archer, Chase Tanner; Kodadek, Thomas J.The Ubiquitin-Proteasome Pathway plays both proteolytic and non-proteolytic roles in the regulation of transcription. We recently reported that the ATPases of the 26S proteasome can destabilize activator-DNA complexes in a non-proteolytic manner that requires direct interactions between the Rpt4 and 6 subunits with the activation domain of the activator. Remarkably, mono-ubiquitylation of the activator blocks this repressive activity. In this study, we probe the mechanism of this protective effect. Using novel label transfer and chemical cross-linking techniques, we show that ubiquitin contacts the ATPase complex directly, apparently via Rpn1 and/or Rpt1, and that this interaction results in the dissociation of the activation domain-ATPase complex via an allosteric process. We also provide in vivo evidence demonstrating the importance of monoubiquitylation in inhibition of activator-DNA destabilization. A model is proposed in which activator mono-ubiquitylation serves to limit the lifetime of the activator-ATPase complex interaction and thus the ability of the ATPases to unfold the activator and dissociate the protein-DNA complex.Item Signal Specific Ubiquitination and Degradation of IkBa(2003-10-08) Hakala, Kevin William; Kodadek, Thomas J.The transcription factor Nuclear Factor kB (NF-kB) is retained in the cytoplasm by the action of its inhibitor IkB. Upon phosphorylation by the IKK complex, IkB is rapidly ubiquitinated and targeted for 26S proteasome mediated degradation, thus liberating NF-kB for transport to its nuclear destination. The current project was initiated to reconstitute this pathway in vitro by using the purified ubiquitination and degradation machinery to degrade IkBa, and activate NF-kB. While signal dependant IkBa ubiquitination was achieved early in the project, this substrate was not degraded by a number of different 26S protein preparations. Instead, an integral or associated isopeptidase activity was observed with each 26S preparation. The development of new 26S protein purification methods has enabled the isolation of highly purified 26S proteins that exhibits low degradative activity towards the ubiquitinated IkBa substrate without excess isopeptidase activity. In an effort to increase substrate degradation, the IkBa ubiquitination reaction was carefully scrutinized. The current literature reports that Ubch5 is the relevant E2 that works in conjunction with the IkBa SCFᔲCP E3 complex, however, Cdc34/Ubc3 can also ubiquitinate IkBa, and may also be a relevant E2. While both E2s carry out in vitro signal dependant ubiquitination of IkBa, the ubiquitin conjugates made by Ubc3 are specific for Lysine-48 linked isopeptide bonds, whereas Ubch5 is able to utilize a variety of ubiquitin surface Lysine residues in isopeptide bond formation. Because K-48 linked ubiquitin conjugates are believed to target substrates for 26S mediated degradation, it was not surprising to find that my 26S proteasome preparations exhibited higher levels of IkBa degradation when ubiquitin conjugation reactions were carried out with Ubc3 instead of Ubch5. Using small interfering RNA to knock down the protein levels of each E2 in vivo, we have found that Ubc3 has no effect on IkBa degradation, whereas the Ubc5/7 double knockdown exhibits partial inhibition of IkBa degradation which is comparable to knocking down the levels of the IkBa E3 specificity factor ᔲCP. The completion of this project has established an in vitro ubiquitination and degradation system that will be instrumental for future studies aimed at determining how the 26S proteasome unfolds and degrades its protein substrates.Item Ubiquitin Mediated Regulation of NF-kappa B Signaling(2008-05-13) Pineda, Gabriel; Chen, Zhijian J.NF-κB signaling is involved in many vital cellular functions such as immunity, cell proliferation, inflammation, and apoptosis. The activation of NF-κB signaling requires the process of ubiquitination. K63-and K48-linked ubiquitin chains have been shown to have distinct roles and biological function in NF-κB signaling. K63-linked ubiquitin chains are required for the activation of TAK1, which leads to the activation of IKK. Activation of IKK leads to K48-linked ubiquitination, and the subsequent proteasomal degradation of IκBalpha . Two important areas of research focusing on ubiquitin regulation of NF-κB signaling are addressed in this dissertation. The areas addressed include understanding how ubiquitinated substrates are targeted for proteasomal degradation and how CYLD negatively regulates NF-κB signaling. In these studies, I investigated the molecular mechanisms involved in the regulation of IκBalpha degradation. Using a siRNA approach, NPL4 was shown to be required for IκBalpha degradation. In vitro proteasomal degradation assays demonstrated that the NPL4 complex is required for IκBalpha degradation. Evidence from both in vitro and in vivo studies suggest NPL4 is required for IκBalpha degradation, but not for IKK activation. These results suggest NPL4 is working at a step after ubiquitination of IκBalpha , but before proteasomal degradation. I propose that ubiquitinated IκBalpha is targeted to the proteasome by an interaction between the NPL4 complex that is mediated through the zinc finger domain of NPL4. The cylindromatosis tumor suppressor gene (CYLD) encodes a 110 kDa deubiquitination enzyme that negatively regulates NF-κB signaling. Loss-of-function mutations in CYLD lead to the disease Familial Cylindromatosis, which is characterized by the formation of benign skin tumors that originate from the head and neck of individuals afflicted with the disease. Here I present in vitro evidence that CYLD inhibits both TAK1 and IKK activation by TRAF6 in a cell free system. I also demonstrate, using a highly purified in vitro system, that CYLD specifically cleaves K63 linked ubiquitin chains and harbors endoproteolytic activity. Furthermore, the third CAPGLY domain of CYLD was shown to be a novel ubiquitin binding domain. My results provide biochemical evidence that CYLD functions as a K63 deubiquitinase to attenuate NF-κB signaling.Item Ubiquitin, the Proteasome, and Dynamics at the Protein/DNA Interface(2006-05-16) Nalley, Kip A.; Kodadek, Thomas J.Recently it has been discovered that a mutant species of Gal4, that contains a three amino acid change in a surface loop of the DNA binding domain, does not occupy the GAL 1/10 promoter under Gal4 inducing conditions as measured by Chromatin Immunoprecipitation (ChIP) assays. However, this protein, Gap71, occupies the promoter similarly to Gal4 under non-inducing (poised) conditions. Additionally this protein was found to be poorly ubiquitylated in vitro under conditions where Gal4 is ubiquitylated. In order to determine the mechanisms involved in the protein destabilization I have examined the properties of the individual mutations that comprise Gap71. These experiments have revealed that serine 22 is a site of phosphorylation of the Gal4 DBD and that lysine 23 is essential for S22 phosphorylation, possibly acting as part of the kinase recognition site. Mutation of either residue blocks Gal4 DBD phosphorylation, its subsequent ubiquitylation and compromises the ability of the activator to bind promoter DNA in vivo. These data represent the first report of an essential phosphorylation event for this paradigmatic transcription factor. In addition, experiments were done to directly measure the dynamics of the Gal4 /DNA complex. To measure the dynamics I have exploited the system developed by Dr. D. Picard and others using the Gal4 DNA binding domain fused to the estrogen receptor ligand binding domain. Each of these constructs has been shown to be inactive until the addition of estradiol, when they are released and bind the Gal4 UAS. These constructs allow me to temporally control the appearance of a large quantity protein that is able to compete with the endogenous Gal4 for the UAS sites in the genome. Under non-inducing conditions, the results are consistent with a rapidly exchanging complex. However, upon induction, the Gal4-promoter complexes "lock in" and exhibit long half-lives of one hour or more. Furthermore, pharmacological inhibition of proteasome-mediated proteolysis had little or no effect of Gal4-mediated gene expression. These studies show that proteasome-mediated turnover is not a general requirement for transactivator function and, when considered in the context of previous studies, that different transactivator-promoter complexes can have widely different lifetimes.