Browsing by Author "Potts, Patrick Ryan"
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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 Downregulation of the Cytosolic Iron-Sulfur Assembly Pathway in Cancer by an E3 Ubiquitin Ligase(2017-06-19) Weon, Jenny Linda; Tu, Benjamin; Potts, Patrick Ryan; Minna, John D.; Liu, YiIron-sulfur (Fe-S) clusters are considered to be one of the oldest cofactors utilized by proteins and are essential for life from bacteria to mammals. Multiple processes in the cell require Fe-S cofactors, such as electron transfer in mitochondrial respiration, enzymatic reactions, and as structural components in DNA repair enzymes. We describe here the first post-translational mechanism to regulate Fe-S assembly and delivery through the ubiquitination and degradation of a key cytosolic iron-sulfur cluster assembly (CIA) pathway component by a MAGE-RING ligase (MRL). The MAGE protein family consists of ~40 members in humans that function in complex with E3 ubiquitin ligases to enhance ubiquitination activity, alter E3 subcellular localization, and/or specify E3 targets. Using biochemical and cellular approaches we have discovered that the MAGE-F1-NSE1 ligase disrupts Fe-S cluster delivery through ubiquitination and degradation of the CIA pathway protein MMS19. MMS19 is a substrate specifying, late-acting component of the CIA pathway that facilitates Fe-S transfer from the multi-component cascade of assembly proteins to specific recipient apoproteins. Notably, many MMS19 targets are enzymes involved in DNA repair. We found that MAGE-F1 directs the E3 ligase NSE1 to target MMS19 for ubiquitination and degradation. Knockdown of MAGE-F1 stabilized MMS19 and overexpression of MAGE-F1 decreased MMS19 levels without affecting MMS19 mRNA levels. We further confirmed MAGE-F1 inhibits Fe-S incorporation into known MMS19-dependent Fe-S proteins, such as FANCJ, POLD1, RTEL1, XPD, and DPYD, but not MMS19-independent Fe-S proteins, such as PPAT. Loss of Fe-S incorporation leads to decreased DNA repair capacity of cells, exemplified by decreased homologous recombination rates and altered sensitivity to DNA damaging agents. Importantly, numerous cancer types harbor copy-number amplification of MAGE-F1, including lung squamous carcinoma and head and neck squamous carcinoma. Consistent with MAGE-F1 inhibitory activity on Fe-S incorporation into key DNA repair enzymes, MAGE-F1-amplified tumors bear a significantly greater mutational burden than non-MAGE-F1-amplified cancers and the expression of MAGE-F1-NSE1 correlates with poor patient prognosis. In summary, we provide the first evidence for post-translational regulatory control of Fe-S cluster assembly and a novel mechanism by which a broad spectrum of DNA repair enzymes can be regulated and lead to genomic instability in cancer.Item Functional Analysis of the Human SMC5/6 Complex in Homologous Recombination and Telomere Maintenance(2008-05-13) Potts, Patrick Ryan; Yu, HongtaoDNA repair is required for the genomic stability and well-being of an organism. The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex promotes HR by localizing to DSBs where it holds sister chromatids in close proximity to allow HR-induced strand invasion and exchange. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this has been unclear. We have characterized the role of the human SMC5/6 complex in HRmediated DNA damage repair. The yeast SMC5/6 complex has been shown to be composed of the SMC5-SMC6 heterodimer and six non-SMC element (NSE) proteins. We show that the human homolog of one of these NSE proteins, MMS21/NSE2, is a ligase for small ubiquitin-like modifier (SUMO). Depletion of MMS21 by RNA interference (RNAi) sensitizes cells toward DNA damageinduced apoptosis. This hypersensitization of MMS21-RNAi cells is not due to a defect in DNA damage-induced cell cycle checkpoint, but rather in the kinetics of DNA damage repair. Since the yeast SMC5/6 complex has been implicated in HR-mediated DSB repair, we investigated the role of the human SMC5/6 complex in HR-mediated DSB repair. RNAi-mediated knockdown of the SMC5/6 complex components specifically decreases sister chromatid HR, but not non-homologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. We show that one potential mechanism by which the SMC5/6 complex specifically promotes sister chromatid HR is by facilitating the recruitment of the SMC1/3 cohesin complex to DSBs. We next examined whether the SMC5/6 complex is also required for sister chromatid HR of telomeres. Specific types of cancer cells, known as alternative lengthening of telomeres (ALT) cells, rely on telomere recombination for telomere lengthening and unlimited replicative potential. We show that the SMC5/6 complex promotes telomere recombination and lengthening in ALT cells by MMS21-dependent sumoylation of telomere-binding proteins. Sumoylation of these telomere-binding proteins relocalizes telomeres to nuclear PML bodies where HR proteins facilitate telomere recombination. These studies identify the human SMC5/6 complex and SUMO modification as critical mediators of sister chromatid HR.Item Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance(2020-08-01T05:00:00.000Z) Lee, Anna Kunyoung; Chook, Yuh Min; Potts, Patrick Ryan; Conrad, Nicholas; Rice, Luke; Thomas, Philip J.Melanoma antigen (MAGE) genes are conserved in all eukaryotes and encode for proteins sharing a common MAGE homology domain. Although only a single MAGE gene exists in lower eukaryotes, the MAGE family rapidly expanded in eutherians and consists of more than 50 highly conserved genes in humans. A subset of MAGEs initially garnered interest as cancer biomarkers and immunotherapeutic targets due to their antigenic properties and unique expression pattern that is primary restricted to germ cells and aberrantly re-activated in various cancers. However, further investigation revealed that MAGEs not only drive tumorigenesis, but also regulate pathways essential for diverse cellular and developmental processes. Therefore, MAGEs are implicated in a broad range of diseases including neurodevelopmental, renal, and lung disorders, as well as cancer. Recent biochemical and biophysical studies indicate that MAGEs assemble with E3 RING ubiquitin ligases to form MAGE-RING ligases (MRLs) and act as regulators of ubiquitination by modulating ligase activity, substrate specification, and subcellular localization. Here, we present a comprehensive guide to MAGEs highlighting the molecular mechanisms of MRLs, their physiological roles in germ cell and neural development, oncogenic functions in cancer, and potential as therapeutic targets in disease. Stress granules (SG) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SG act as a protective mechanism to cope with acute stress, but persistent SG have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Importantly, knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermo-sensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.Item Yeast Ataxin-2 (Pbp1) Condensates Regulate TORC1 Activity and Autophagy in Response to Cellular Redox State(2018-11-26) Yang, Yu-San; O'Donnell, Kathryn A.; Tu, Benjamin; DeBerardinis, Ralph J.; Potts, Patrick RyanYeast ataxin-2, also known as Pbp1 (Poly(A) binding protein-binding protein 1), is an intrinsically disordered protein that has earlier been implicated in stress granule formation, RNA biology, and neurodegenerative disease. However, the normal endogenous function of Pbp1 and ataxin-2 remains poorly understood. In this dissertation, I identified Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Unlike the autophagy-deficient atg mutants that harbor severe growth defects, pbp1 null mutants exhibited significantly increased cell growth despite lack of autophagy. I discovered that Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region of Pbp1 forms reversible cross-β fibrils that facilitate phase transition of the protein into either liquid-like or gel-like states in vitro and enables self-association of full-length Pbp1 into pelletable assemblies in vivo. Sequence analysis revealed that Pbp1 LC region contains an unusually high frequency of methionine residues (24 methionines in 150 a.a.) compared to the rest of the yeast proteome. I showed that the phase separation of Pbp1 is mediated by these methionine residues, which are sensitive to H2O2-mediated oxidation and mitochondrial toxins in living cells. I also observed that the phase separation of Pbp1 mediated by its C-terminal LC region is responsive to the activity state of mitochondria and required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy in vivo. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling. These observations offer a mechanistic explanation describing how reversible formation of condensates formed from the LC region of Pbp1 has evolved as a sensor of cellular redox state.