Browsing by Subject "Ubiquitin-Protein Ligases"
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Item Behavioral, Neurochemical, and Histological Characterization of Mice Deficient for Parkin, DJ-1, and Antioxidant Proteins(2011-08-10) Seamans, Katherine Webster; Goldberg, Matthew S.Parkinson’s disease is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra. The cause of Parkinson’s disease remains uncertain, however, evidence implicates mitochondrial dysfunction and oxidative stress with selective vulnerability of dopaminergic neurons. Although most cases of Parkinson’s disease are sporadic, 5-10% of cases are caused by mutations in a single gene. Loss-of-function mutations in parkin and DJ-1 were the first to be linked to recessively inherited parkinsonism. Surprisingly, mice bearing similar loss-of-function mutations in parkin and DJ-1 do not show age-dependent loss of nigral dopaminergic neurons or depletion of dopamine in the striatum. Although the normal cellular functions of Parkin and DJ-1 remain unclear, we hypothesized that Parkin and DJ-1 protect cells from oxidative stress and that loss-of-function mutations in these genes cause neurodegeneration in Parkinson’s disease by rendering cells more sensitive to mitochondrial dysfunction and oxidative stress. We crossed mice deficient for Parkin and DJ-1 with mice deficient for the major mitochondrial antioxidant protein Mn-superoxide dismutase or Cu/Zn-superoxide dismutase. Previous studies have shown that mice with reduced levels of Cu/Zn-superoxide dismutase or Mn-superoxide dismutase are more sensitive to dopaminergic neurotoxins whereas mice with increased levels of superoxide dismutase are more resistant to dopaminergic neurotoxins. We predicted that reducing levels of antioxidant proteins in parkin-/-DJ-1-/- mice would result in age-dependent nigral cell loss, striatal dopamine depletion or behavioral abnormalities. Characterization of these mice for behavioral abnormalities, neurotransmitter defects and neuropathology, revealed significant behavioral abnormalities in the mutant mice even in the absence of significant changes to dopamine levels in the striatum, dopamine receptor density, or dopaminergic neuron numbers. Aged parkin-/-DJ-1-/- and Mn-superoxide dismutase triple deficient mice have a surprising enhanced rotorod performance without the presence of an anxiety phenotype or hyperactivity. Cu/Zn-superoxide dismutase and Mn-superoxide dismutase triple deficient mice have elevated levels of dopamine in the striatum, however none of the mice present with nigral cell loss. Levels of D1-like and D2-like dopamine receptors in the striatum were unchanged. It is evident from our studies that on a parkin/DJ-1 null background, additional loss of major antioxidant proteins does not lead to a progressive loss of dopaminergic neurons in mice.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 Biochemical Dissection of Innate Immune Signaling Mechanisms Mediated by MAVS and Inflammasomes(2014-10-23) Chen, Jueqi; Tu, Benjamin; Chen, Zhijian J.; Olson, Eric N.; Buszczak, MichaelProject 1: MAVS Recruits Multiple Ubiquitin E3 Ligases to Activate Antiviral Signaling Cascades The RIG-I antiviral pathway plays a pivotal role in innate immune response against RNA viruses. Upon virus infection, viral RNA in the cytoplasm is detected by the RIG-I family of receptors, which activates an adaptor protein called MAVS (mitochondrial antiviral signaling). MAVS in turn leads to the activation of two transcription factors, NF-κB and IRF3, which coordinately induce type-I interferons and proinflammatory cytokines that are critical in eliminating viral infection. However, the exact mechanism of NF-κB and IRF3 activation by MAVS is still largely unknown. In this study we show that activated MAVS recruits three ubiquitin E3 ligases TRAF6, TRAF2 and TRAF5 to the mitochondria through distinct TRAF-binding motifs upon virus infection. Mutations that disrupt these motifs in MAVS abrogated the recruitment of these E3 ligases, and abolished the ability of MAVS to activate the downstream signaling. Interestingly, virus-induced prion-like aggregation of MAVS is essential for its interaction with these TRAF proteins as well as the downstream activation. Genetic evidence has shown that these E3 ligases function redundantly to activate NF-κB and IRF3, so that they not only amplify the antiviral signal, but also serve as backup systems against specific viruses that can degrade components in the antiviral pathway. Identifying these key players in MAVS pathway will help us explore novel therapeutic targets for infectious diseases caused by RNA viruses. Project 2: Dissecting the Mechanism of NLRP3 Inflammasome Activation Inflammasome is a multi-protein oligomer that serves as a platform for caspase-1-depedent activation of proinflammatory cytokines and the induction of a specific form of cell death termed pyroptosis. A plethora of pathogen-associated-molecular patterns (PAMPs) and damage-associated-molecular-patterns (DAMPs) have been found to activate inflammasome through NLRP3, a Nod-like receptor protein. As a result, the dysfunction of NLRP3 is closely associated with various health problems including autoimmune diseases, neurodegenerative diseases, susceptibility to pathogen infection, and metabolic disorders. Given the chemical and structural diversity of the stimuli, and the lack of evidence that NLRP3 directly interacts with these stimuli, it has been hypothesized that NLRP3 activation is triggered by a common cellular signal, the identity of which is still a mystery. We have reconstituted NLRP3 inflammasome pathway in HEK293T cell and used this system to establish a cell-free assay, in which NLRP3 from stimulated cells was added to cells containing Asc and caspase-1 to activate the downstream signaling. With this assay, we were able to obtain highly purified active NLRP3 for further characterization, and have found that dephosphorylation plays an important role in NLRP3 inflammasome activation. Moreover, both biochemistry and imaging data has suggested that NLRP3 is translocated to specific subcellular structures after stimulation, of which the significance is still under investigation. Our final goal is to dissect the detailed mechanism of NLRP3 activation, which can provide insight into the prevention and treatment of various related human diseases.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 FBXL5 Is Required for the Maintenance of Cellular Andsystemic Iron Homeostasis(2013-01-16) Ruiz, Julio Cesar Francisco; Bruick, Richard K.Iron is an essential element for most living organisms. Due to its chemical properties, iron plays an important role in many vital biochemical processes. Both iron excess and deficiency have detrimental effects in human health. Therefore, iron metabolism must be tight regulated. Maintenance of cellular iron homeostasis requires coordinate posttranscriptional regulation of iron metabolism genes by Iron Regulatory Proteins 1 and 2 (IRP1 and IRP2). IRP2 is targeted for proteasomal degradation in iron replete cells by the E3 ubiquitin ligase complex containing F-box and Leucine-rich Repeat Protein 5 (FBXL5). Depletion of FBXL5 leads to aberrant accumulation of IRP2 and misregulation of IRP2 under high iron conditions, underscoring FBXL5 importance in regulation of iron metabolism. Interestingly, FBXL5 is regulated in an inverse fashion to IRP2 as it is stabilized under iron-replete conditions and preferentially degraded when iron or oxygen becomes limiting. However, FBXL5Õs iron- and oxygen-dependent regulation and its role in the maintenance of systemic iron homeostasis are poorly understood. Biochemical and molecular biology assays revealed that FBXL5 features a hemerythrin-like domain that serves as a direct sensor of cellular iron as well as oxygen availability and subsequently governs FBXL5Õs own stability. Importantly, in vivo deletion of the ubiquitously-expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, though viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Fbxl5 heterozygous mice behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. IRP2Õs responsiveness to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the Divalent Metal Transporter-1. These results confirm FBXL5Õs role in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity.Item FBXL5: Sensor and Regulator of Mammalian Iron Homeostasis(2011-08-10) Salahudeen, Ameen Abdulla; Bruick, Richard K.While iron is an important cofactor for many proteins, the chemical properties of iron that favor its biological roles can lead to toxic side reactions that damage macromolecules. Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of gene products responsible for iron uptake, release, utilization, and storage. This process is mediated by Iron Regulatory Proteins (IRPs) that bind to Iron Responsive Elements (IREs) in the mRNAs of these genes. When iron bioavailability is low IRPs bind IREs within these mRNAs, affecting their subsequent translation or stability. When cellular free iron availability is high, IRPs are preferentially degraded by the proteasome. An SCF E3 ubiquitin ligase complex containing the FBXL5 protein regulates this process as a function of cellular iron and oxygen concentrations. This process occurs through the stability of FBXL5, which accumulates under iron and oxygen replete conditions and is targeted for degradation upon iron depletion. FBXL5 contains an iron- and oxygen -sensing hemerythrin domain that acts as a ligand-binding regulatory switch mediating its stability. As a result, FBXL5 directly senses iron and oxygen levels to serve as a regulator of cellular iron homeostasis.Item An Iron Sensing E3 Ubiquitin Ligase Regulates Iron Homeostasis(2011-08-10) Thompson, Joel William; Bruick, Richard K.Human iron homeostasis must be tightly regulated to provide sufficient iron for vital cellular processes while preventing the toxic accumulation of free iron. IRP2 plays a critical role in cellular iron homeostasis by coordinating the posttranscriptional regulation of a variety of genes involved in iron metabolism. Posttranslational regulation of IRP2 is essential for its ability to maintain cellular iron homeostasis. The protein is stabilized under iron deficient conditions but polyubiquitinated and degraded by the proteasome when iron is plentiful. However, the E3 ubiquitin ligase that targets IRP2 for degradation is unknown. Moreover, the mechanisms cells use to sense iron levels and correlate changes of this metabolite to differences in IRP2 stability remain poorly understood. To identify the E3 ubiquitin ligase responsible for IRP2 degradation, a high throughput RNAi screen was conducted. The top hit from the screen, FBXL5, interacts with and polyubiquitinates IRP2. Interestingly, FBXL5 is inversely regulated to IRP2. The protein is stabilized under conditions of excess iron and destabilized when iron is limiting. Deletion experiments identified the N terminus of FBXL5 as the region of the protein required for its iron dependent regulation. Bioinformatics predicted the N terminus encodes an iron binding hemerythrin domain. Consistent with this prediction X-ray crystallography demonstrated that the FBXL5 N-terminal domain adopts a hemerythrin fold with a diiron center. Mutation of iron ligating residues in the hemerythrin domain to abolish iron binding leads to constitutive destabilization of FBXL5. Collectively, these findings indicate that the hemerythrin domain acts as a ligand dependent regulatory switch controlling FBXL5Õs expression. Moreover, these data suggest that iron dependent regulation of FBXL5 exerts reciprocal effects on IRP2 stability. Thus, FBXL5 possesses an iron binding hemerythrin domain enabling cells to gauge bioavailable iron levels and control IRP2 expression accordingly, resulting in a tightly regulated circuit in the maintenance of iron homeostasis.Item Mechanistic Basis of Signal Amplitude Modulation by the Ras Effector IMP(2007-08-08) Chen, Chiyuan; White, Michael A.The RAF/MEK/MAP Kinase signal transduction cascade is the most extensively studied MAPK pathway that mediates diverse cellular responses to environmental cues, and makes a major contribution to Ras-dependent oncogenic transformation. The Ras effector and E3 ligase family member IMP (Impedes Mitogenic signal Propagation) acts as a steady-state resistor within the RAFMEK- ERK kinase module. IMP concentrations are regulated by Ras, through induction of autodegradation, and can modulate signal/response thresholds by directly limiting the assembly of functional KSR1-dependent RAF/MEK complexes. Here, we examine the mechanistic basis of signal amplitude modulation by the Ras effector IMP. We show that the capacity of IMP to inhibit signal propagation through RAF to MEK is a consequence of disrupting assembly of multivalent mitogenic complexes that are required for C-RAF kinase activation and functional coupling of active kinases to downstream substrates. We also study how Ras regulates IMP functions by isolating IMP mutants compromised for Ras interaction and how post-translational modifications such as sumoylation control IMP activities. Finally, we identify some candidate IMP binding proteins to further investigate how IMP impacts cell behaviors through protein-protein interactions and how IMP is modulated by other proteins.Item Molecular and Genetic Analysis of Parkin in Microglia Activation and Inflammation-Related Neurodegeneration(2010-05-14) Tran, Thi Anh; Tansey, Malú G.Parkinson’s disease (PD) is a progressive, neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Genetic mutations account for only 5-10% of PD cases. Oxidative stress and inflammation have both been linked to sporadic PD. Inflammation-induced injury to dopaminergic neurons can be significantly attenuated by impairment of microglial activation. In addition, previous studies from our lab reported that parkin-/- mice are more susceptible to inflammation-induced degeneration of nigral DA neurons. Therefore, inflammatory responses are a critical determinant of DA neuronal survival. Microglia support neuronal survival by providing trophic factors and phagocytosing debris. However, with chronic inflammation glia release chemical mediators which are toxic to surrounding neurons. Our data provide evidence that Parkin is a negative regulator of microglial activation. parkin-/- mice display increased cytokine expression in the midbrain and increased cytokines in the serum suggesting parkin-/- mice are basally inflamed. Parkin loss-of-function mutations are linked to autosomal recessive PD. The parkin gene encodes an E3 ubiquitin ligase linked to mitochondrial dysfunction. Most studies on Parkin concentrate on its role in neurons, however, we hypothesize that Parkin function in microglial activation and inflammatory signaling also affect DA neuron survival. Our biochemical analyses of primary wild type microglia show Parkin expression is negatively regulated by inflammatory stimuli. Pharmacological or genetic inhibition of NF-κB, a transcription factor activated by inflammatory stimulation, blocks the inflammation-induced decrease in Parkin levels. Additionally, our data suggests that NF-κB may bind the parkin promoter, further implicating Parkin function in the inflammatory activation pathway. These novel findings suggest that in a normal cell experiencing inflammation, the decreased expression of Parkin, which has been shown to antagonize apoptotic signaling cascades, may render the cell more susceptible to death. Additionally, sources of inflammation including environmental triggers, infection, or traumatic injury could cause a normal individual to have the same susceptibility to PD as an individual with an inherited mutation, because inflammation leads to Parkin loss of function.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 Studies on Cellular Nutrient Responses and Protein Degradation(2015-06-01) Ghosh, Anwesha; Goodman, Joel M.; Cobb, Melanie H.; Albanesi, Joseph P.; Sternweis, Paul C.I have worked on two projects. The first project investigates mechanisms involved in cellular responses to amino acids. Amino-acid abundance promotes protein synthesis and cell growth via activation of the protein kinase mTOR, while amino-acid deprivation promotes protein degradation by autophagy. The heterodimeric G protein coupled receptor (GPCR) T1R1-T1R3 can act as an extracellular sensor for amino acids, promoting mTOR activity while repressing autophagy in cells. Quantitative PCR analysis revealed that T1R3 depletion increases mRNA expression of amino acid transporters as a compensatory mechanism induced by perceived starvation. The arrestin proteins can bind GPCRs to mediate their internalization or to facilitate downstream signaling. I tested the hypothesis that β-arrestin 2 might participate in regulation of mTOR activity and autophagy by amino acids. siRNA-mediated β-arrestin 2 depletion decreased T1R1-T1R3 protein expression, reduced mTOR activity and increased autophagy in different cell types. β-arrestin 2 loss increased phosphorylation of the MAP kinase ERK1/2, which may play a role in promoting autophagy. Taken together, these findings demonstrate a role for β-arrestin 2 in promoting mTOR activity and suppressing autophagy. The second project examined the role of different protein degradation pathways and an E3 ubiquitin ligase UBR5 in regulating the stability of the protein kinase WNK1, a key regulator of cellular ion homeostasis. Mutations that increase WNK1 protein expression cause familial hypertension, highlighting the importance of understanding the regulation of WNK1 protein expression. Cycloheximide chase experiments revealed that WNK1 degradation may be complex, as it does not follow simple exponential decay kinetics. Pharmacological inhibition of different protein degradation pathways showed that autophagy and the calpain system of non-lysosomal cysteine proteases, but not the proteasome, can promote WNK1 degradation. Inhibition of the protein chaperone Hsp90 increased WNK1 protein levels, possibly through stabilization of WNK1 by Hps70. Immunoprecipitation experiments demonstrated that UBR5 can associate with WNK1. siRNA-mediated silencing of UBR5 increased WNK1 stability, decreased the ubiquitination of an overexpressed N terminal fragment of WNK1, and reduced the levels of KLHL3, an adaptor protein that recruits WNK1 to the Cullin3-RBX1 E3 ligase complex for ubiquitination and degradation. Taken together, these findings identify degradation pathways and molecular players that regulate WNK1 stability.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.