Browsing by Subject "Ubiquitination"
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Item Biochemical Characterization of IpaH E3 Ubiquitin Ligase Effector Proteins and Their Host Substrates(August 2021) Hansen, Justin Mark; Orth, Kim; Sperandio, Vanessa; Reese, Michael L.; Alto, NealShigella flexneri is a gram negative pathogen that utilizes its type 3 secretion system (T3SS) to inject effector proteins in the cytoplasm of host cells to manipulate host cells processes. T3SS effectors are able to post translationally modify host proteins to reprogram intracellular signaling pathways, actin dynamics, membrane trafficking, and innate immune pathways. This allows Shigella to modify the intracellular environment to be conducive to bacterial replication and dissemination to neighboring cells. Shigella flexneri and other bacteria including Salmonella and Yersinia secreted E3 ubiquitin ligases into the host cell cytoplasm via the Type III secretion system (T3SS) apparatus. The invasion plasmid antigen Hs (IpaHs) are a novel family of bacterial E3 ubiquitin ligases that are secreted by Shigella, Salmonella, and Yersinia. These bacterial enzymes highjack the host ubiquitin conjugation machinery by binding to ubiquitin-charged E2 conjugating enzymes and facilitating direct transfer of ubiquitin onto host substrates. IpaH effectors induce polyubiquitination and subsequent proteasomal degradation of their substrates during bacterial infection. The effector substrate interaction of IpaH1.4/2/5 and HOIP was previously characterized. I went on to identify that IpaH2.5 is able to inhibit the in vitro catalytic activity of HOIP via mono-ubiquitination of catalytic lysine residues in the HOIP ring-between-ring domain (RBR-C). Subsequent to this Ubiquitin activated interactive trapping (UBAIT) screening was then utilized to identify the host substrate of IpaH7.8, Gasdermin B (GSDMB). GSDMB belongs to a large family of pore forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to inflammatory disease, its function in human physiology remains poorly understood. I investigated a previously unrecognized host-pathogen conflict between GSDMB and the IpaH7.8 effector protein encoded by Shigella flexneri. Through extensive biochemical and cellular characterization, I show that IpaH7.8 ubiquitinates and targets GSDMB for proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of Natural Killer cells by suppressing Granzyme-A mediated activation of GSDMB. In contrast to the canonical function of most Gasdermin-family members, GSDMB does not inhibit Shigella by lysing infected cells. Rather, GSDMB exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveals a mechanism employed by pathogens to counteract this host defense system.Item The Cancer Specific Ubiquitin Ligase MAGE-A3/6-TRIM28 Drives Tumorigenesis by Ubiquitination and Proteasomal Degradation of AMPK(2015-08-27) Pineda, Carlos Tyler; Yu, Hongtao; Levine, Beth; White, Michael A.; Potts, Patrick RyanThe genes MAGE-A3 and MAGE-A6 (MAGE-A3/6) have a unique expression pattern in which they are normally expressed in the adult testis but are aberrantly expressed in cancer. It is known that when expressed in cancer, MAGE-A3/6 is a negative prognostic indicator and cancer cells are dependent on it for survival. Using the knowledge that MAGE-A3/6 binds and regulates the E3 ubiquitin ligase TRIM28, I investigated its biochemical role in cancer. I used unbiased methods to identify 19 novel substrates of MAGE-A3/6-TRIM28, including the known tumor suppressor AMPK. Ubiquitination of AMPK by MAGE-A3/6-TRIM28 induces its proteasomal degradation, thereby enhancing mTOR signaling and inhibiting autophagy within cells. Through this modulation of AMPK, MAGE-A3/6 is also able to act as an oncogene, inducing anchorage independent growth and the growth of tumors in vivo. Understanding the mechanism by which MAGE-A3/6 acts as an oncogene has revealed potential avenues of therapeutic intervention. Treatment of MAGE-A3/6 expressing cells with AMPK agonists reverses oncogenic properties in vitro. Ultimately, these studies have revealed how a germline protein functions in cancer and the potential points for therapeutic intervention.Item Characterization of Ubiquitin Ligase Targeting by Anticancer Sulfonamides(2020-08-01T05:00:00.000Z) Ting, Tabitha Chung-Yan; De Martino, George; Nijhawan, Deepak; Yu, Hongtao; DeBose-Boyd, Russell A.Aryl sulfonamides are small molecules that are selectively toxic to a subset of human cancer cell lines. Clinical trials of the aryl sulfonamide indisulam have resulted in modest clinical activity against a subset of solid tumors. Recent work revealed that indisulam recruits the RNA binding protein RBM39 to DCAF15, a component of the CRL4-DCAF15 E3 ubiquitin ligase. This recruitment results in RBM39 ubiquitination and degradation, leading to splicing defects and cancer cell death (Han et al., 2017; Uehara et al., 2017). The mechanism of action of sulfonamides is similar to that of immunomodulatory drugs (IMiDs), which recruit substrates to the closely related CRL4-CRBN E3 ubiquitin ligase for ubiquitination. Known for their roles in inhibiting embryonic development and cancer cell growth, IMiDs exert their pleiotropic effects by targeting a variety of substrate proteins to the CRL4-CRBN E3. Despite major advances in our understanding of aryl sulfonamides, it is unclear whether sulfonamides also target multiple substrates or modulate the endogenous function of the CRL4-DCAF15 E3 ligase. This dissertation describes our efforts to define the requirements for RBM39 ubiquitination, identify other substrates that are recruited to the CRL4-DCAF15 E3 ligase, and further our understanding of the cellular consequences of indisulam treatment. In Chapters 2 and 3, we define the components required for RBM39 ubiquitination using a combination of in vitro and in vivo techniques. In Chapters 4 and 5, we identify putative endogenous substrates and a previously undescribed neo-substrate recruited to the CRL4-DCAF15 for ubiquitination. In Chapter 6, we characterize the cellular consequences of indisulam treatment and neo-substrate degradation. In aggregate, this work aims to contribute to our understanding of the sulfonamide mechanism of action and the field of targeted protein degradation.Item Novel Activities of Kinase-Fold Enzymes from Legionella pneumophila(2020-08-01T05:00:00.000Z) Black, Miles; Cobb, Melanie H.; Tagliabracci, Vincent S.; Mendell, Joshua T.; Olson, Eric N.Protein kinases are fundamental mediators of cell signaling that transfer phosphate from ATP to their substrates. The protein kinase superfamily encompasses a vast and diverse trove of enzymes from all domains of life, including remote members that are barely recognizable by their primary amino acid sequence. SidJ (Substrate of Icm/Dot J) is a distant protein kinase homolog from the human pathogen Legionella pneumophila. Contamination of water supplies with Legionella bacteria is a frequent cause of deadly pneumonia outbreaks (Legionnaire's disease). SidJ is a secreted Legionella virulence factor required for bacterial intracellular replication, but it is unknown how SidJ contributes to pathogenesis of Legionnaire's disease, or if SidJ has maintained the kinase fold or catalytic activity. In this work, I determine that SidJ is a calmodulin-binding protein which adopts a protein kinase fold. However, instead of phosphorylation, it catalyzes protein polyglutamylation. SidJ utilizes ATP to form an isopeptide bond between the amino group of free glutamate and the 𝛾-carboxyl group of a glutamate of its substrate. During infection, SidJ polyglutamylates and inactivates a family of Legionella "all-in-one" ubiquitin ligases. Polyglutamylation is crucial step in the intracellular lifecycle of the bacterium and is required for full Legionella virulence in a eukaryotic host. SidJ reveals the unexpected catalytic versatility of the protein kinase fold, and highlights a unique strategy that pathogenic bacteria use to thrive within host cells. Interestingly, SidJ lacks key catalytic residues believed to be required for kinase activity. The discovery that SidJ is a polyglutamylating enzyme suggests that catalytically incompetent or 'pseudo' enzymes may lack activity only when assayed for the wrong reaction.Item Role of c-Cbl in Invasion of Mammalian Cells by Rickettsia Conorii(2007-08-08) Ravikumar, Sai P.; Seemann, Joachim[SPECIFIC AIMS] Rickettsia conorii is an intracellular bacterium that causes Mediterranean spotted fever. R. conorii is transmitted from ticks to humans and invades vascular endothelial cells. A recent publication (Martinez et al., 2005) has identified Ku70 as a host cell receptor that binds to rOmpB, an R. conorii surface ligand. The engagement of Ku70 by rOmpB leads to a rapid ubiquitination of Ku70 by c-Cbl, followed by the entry of R. conorii into the host cell. However, the role of c-Cbl in ubiquitinating Ku70 and communicating with the invading bacterium remain to be clarified. Based on the report of Martinez et al and other groups on the roles played by R. conorii surface ligands rOmpA and rOmpB, we propose the following hypothesis: [HYPOTHESIS] c-Cbl ubiquitinates Ku70 in response to signals from a Rickettsia conorii cell surface protein called rOmpA. This ubiquitination event enables Ku70 and the attached bacteria to be endocytosed into the cell. [SPECIFIC AIMS] 1. To analyse the effect of c-Cbl - mediated ubiquitination on Ku70 localisation. We will investigate if ubiquitination of Ku70 by c-Cbl is necessary for Ku70 to localize to the bacterial entry foci. We will culture c-Cbl deficient cells and observe the effects of R. conorii infection on them. 2. To map the putative signal(s) involved in the communication between R. conorii rOmpA outside the cell and c-Cbl inside the cell. We will identify the putative rOmpA receptor on host cells and protein(s) that may act as bridges of communication between rOmpA and c-Cbl. 3. To investigate the mechanism of how ubiquitination of Ku70 enables R. conorii to enter a host cell. We will determine if any c-Cbl interacting proteins and/or endocytic proteins are recruited to the cell surface by ubiquitinated Ku70 to form a phagosome. If endocytic proteins are recruited, we will proceed.Item Ubiquitin Conjugase UBE2K Is Essential for Normal Rat Development and Spermatogenesis(2016-04-14) Chaudhary, Jaideep; Repa, Joyce J.; Mendelson, Carole R.; Wan, Yihong; Hamra, F. KentAnimal models allow us to investigate questions about human physiology using in vivo systems. Following the advent of gene targeting by homologous recombination in mouse pluripotent embryonic stem cells during the early 1980's [Reviewd by (Hamra, 2010)], the laboratory mouse has emerged as the most widely published animal model in science (NCBI, PubMed). Interestingly, prior to the year 2000, annual publications in the laboratory rat outnumbered annual publications in the mouse by greater than 2-3 fold in almost every field of science spanning each decade in the 20th century (NCBI, PubMed). Popularity of the laboratory rat as a model organism in science derived from its many attributes for modeling human physiology and disease in a laboratory scale mammal. However, genetic tools to effectively manipulate the rat genome lagged behind that of the mouse for almost 4 decades up until ~2009 due to inabilities to maintain pluripotent rat cells in culture, and inefficient methods for micro-manipulating rat early embryos. Now, several laboratories have made large strides to advance technologies to genetically engineer rats (Geurts et al., 2009; Tong et al., 2011), including our laboratory, which succeeded in applying stem cell-based technologies to the rat's "genomic toolbox" by using donor stem spermatogonia as vectors to directly genetically modify the rat's germline (Chapman et al., 2015; Hamra et al., 2002; Izsvak et al., 2010). For this dissertation, I focused on phenotyping the growth and reproduction defects in the UBE2K knockout rat strain, which happens to represent the first knockout rat strain reported using genetically selected germline stem cells form culture (Izsvak et al., 2010). I reasoned that phenotyping UBE2K rats in more detail would allow me to formulate testable hypotheses on the cellular and molecular mechanisms by which UBE2K functioned to regulate rat body growth and reproduction. UBE2K is an ubiquitin ligase, and knocking its gene (Ube2k) out in rats resulted in stunted growth, compromised motor capacity of hind legs and infertility. I found infertility in male UBE2K-deficient rats to be caused by an arrest during meiosis-I that prevented the zygotene to pachytene spermatocyte transition. Based on known interactions of UBE2K, and the biochemical function of UBE2K as ubiquitin ligase, I hypothesized that UBE2K is a component of the PRC1 complex that ubiquitinates H2A to prevent transcription as a mechanism necessary for germ cells to undergo meiosis. I showed using immunostaining, that there is an inverse correlation between H2A ubiquitin staining and UBE2K expression that is disrupted in UBE2K-deficient rats, supporting my hypothesis. However, future work will need to address whether UBE2K directly or indirectly associates with the PRC1 complex. Based on my research presented in this Dissertation, I propose a novel function for the UBE2K ubiquitin conjugase in meiotic transcriptional control during spermatogenesis.Item Ubiquitination-Dependent Activation of IKK(2005-12-19) Ea, Chee-Kwee; Chen, Zhijian J.Ubiquitination plays two different roles in the nuclear factor κΒ (NF-κΒ ) pathway, the traditional K48-linked polyubiquitination-mediated IκΒ degradation and the non-traditional K63-linked polyubiquitination-mediated IKK activation. TRAF6 is a RING domain ubiquitin ligase that mediates the activation of protein kinases such as TAK1 and IKK by promoting the formation of a unique polyubiquitin chain linked through lysine-63 of ubiquitin. Previous studies have suggested that the ubiquitin ligase and signaling activity of TRAF6 may be regulated by its oligomerization. However, it is not known whether there is an endogenous "oligomerizer" that regulates TRAF6 activity. TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA, also known as T2BP) is one of such TRAF6 "oligomerizers". Recombinant TIFA protein, but not TRAF6-binding defective mutant protein, can activate IKK in crude cytosolic extracts. Furthermore, TIFA activates IKK in an in vitro reconstitution system consisting of purified proteins including TRAF6, the TAK1 kinase complex and the ubiquitin conjugating enzyme complex Ubc13/Uev1A. Interestingly, a fraction of recombinant TIFA protein exists as high molecular weight oligomers, and only these oligomeric forms of TIFA can activate IKK. Importantly, TIFA induces the oligomerization and polyubiquitination of TRAF6, which leads to the activation of TAK1 and IKK through a proteasome-independent mechanism. The receptor interacting protein kinase 1 (RIP1) is essential for the activation of NF-κΒ in response to tumor necrosis factor a (TNFa) stimulation. RIP1 undergoes TNF-induced polyubiquitination at Lysine 377 in the intermediate domain and the polyubiquitination of RIP1 is required for proper signal transduction. Furthermore, when introducing RIPK377R mutant into RIP-/- Jurkat cells, it fails to restore TNF-dependent IKK activation, and these RIPK377R cells are sensitive to TNFa-induced cell death. In addition, TAK1 and IKK kinase complexes are not recruited to TNFR1 followed TNFa stimulation in the absence of RIP1 polyubiquitination. Moreover, TAB2 and NEMO bind to K63-linked polyubiquitin chains and function as receptors that bind polyubiquitinated RIP1. These results indicate a unique interaction between a polyubiquitinated protein and a polyubiquitin binding protein can trigger the activation of TAK1 and IKK.