338 results on '"Kopito, Ron R."'
Search Results
102. Cytosolic pH regulatesGClthrough control of phosphorylation states of CFTR
- Author
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Reddy, M. M., primary, Kopito, Ron R., additional, and Quinton, P. M., additional
- Published
- 1998
- Full Text
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103. Cotranslational Ubiquitination of Cystic Fibrosis Transmembrane Conductance Regulator in Vitro
- Author
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Sato, Sachiko, primary, Ward, Cristina L., additional, and Kopito, Ron R., additional
- Published
- 1998
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104. Retraction
- Author
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Kobayashi, Sumire, primary and Kopito, Ron R, additional
- Published
- 1997
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105. Cytosolic Degradation of T-cell Receptor α Chains by the Proteasome
- Author
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Yu, Helen, primary, Kaung, Geoffrey, additional, Kobayashi, Sumire, additional, and Kopito, Ron R., additional
- Published
- 1997
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106. Glycerol Reverses the Misfolding Phenotype of the Most Common Cystic Fibrosis Mutation
- Author
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Sato, Sachiko, primary, Ward, Cristina L., additional, Krouse, Mauri E., additional, Wine, Jeffrey J., additional, and Kopito, Ron R., additional
- Published
- 1996
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107. A Conserved Glutamate Is Responsible for Ion Selectivity and pH Dependence of the Mammalian Anion Exchangers AE1 and AE2
- Author
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Sekler, Israel, primary, Lo, Roger S., additional, and Kopito, Ron R., additional
- Published
- 1995
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108. The Role of Cysteine Residues in the Erythrocyte Plasma Membrane Anion Exchange Protein, AE1
- Author
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Casey, Joseph R., primary, Ding, Yue, additional, and Kopito, Ron R., additional
- Published
- 1995
- Full Text
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109. Cl−/HCO3− exchange function differs in adult and fetal rat hippocampal neurons
- Author
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Raley-Susman, Kathleen M., primary, Sapolsky, Robert M., additional, and Kopito, Ron R., additional
- Published
- 1993
- Full Text
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110. Perturbation of the Hematopoietic System during Embryonic Liver Development Due to Disruption of Polyubiquitin Gene Ubc in Mice.
- Author
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Kwon-Yul Ryu, Hyejin Park, Rossi, Derrick J., Weissman, Irving L., and Kopito, Ron R.
- Subjects
HEMATOPOIETIC system ,UBIQUITIN ,LIVER cells ,BONE marrow cells ,CELL proliferation ,ANIMAL models in research - Abstract
Disruption of the polyubiquitin gene Ubc leads to a defect in fetal liver development, which can be partially rescued by increasing the amount of ubiquitin. However, it is still not known why Ubc is required for fetal liver development and the nature of the defective cell types responsible for embryonic lethality have not been characterized. In this study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We found that Ubc was highly expressed in the embryonic liver, and the proliferation capacity of fetal liver cells was reduced in Ubc
-/- embryos. Specifically, Ubc was most highly expressed in hematopoietic cells, and the proliferation capacity of hematopoietic cells was significantly impaired in Ubc-/- embryos. While hematopoietic cell and hematopoietic stem cell (HSC) frequency was maintained in Ubc-/- embryos, the absolute number of these cells was diminished because of reduced total liver cell number in Ubc-/- embryos. Transplantations of fetal liver cells into lethally irradiated recipient mice by non-competitive and competitive reconstitution methods indicated that disruption of Ubc does not significantly impair the intrinsic function of fetal liver HSCs. These findings suggest that disruption of Ubc reduces the absolute number of HSCs in embryonic livers, but has no significant effect on the autonomous function of HSCs. Thus, the lethality of Ubc-/- embryos is not the result of intrinsic HSC failure. [ABSTRACT FROM AUTHOR]- Published
- 2012
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- View/download PDF
111. Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant ?-1 antitrypsin from the endoplasmic reticulum.
- Author
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Greenblatt, Ethan J, Olzmann, James A, and Kopito, Ron R
- Subjects
TRYPSIN inhibitors ,ENDOPLASMIC reticulum ,PROTEOLYTIC enzymes ,CYTOPLASM ,PROTEOLYSIS - Abstract
The degradation of misfolded secretory proteins is ultimately mediated by the ubiquitin-proteasome system in the cytoplasm, therefore endoplasmic reticulum-associated degradation (ERAD) substrates must be dislocated across the ER membrane through a process driven by the AAA ATPase p97/VCP. Derlins recruit p97/VCP and have been proposed to be part of the dislocation machinery. Here we report that Derlins are inactive members of the rhomboid family of intramembrane proteases and bind p97/VCP through C-terminal SHP boxes. Human Derlin-1 harboring mutations within the rhomboid domain stabilized mutant ?-1 antitrypsin (NHK) at the cytosolic face of the ER membrane without disrupting the p97/VCP interaction. We propose that substrate interaction and p97/VCP recruitment are separate functions that are essential for dislocation and can be assigned respectively to the rhomboid domain and the C terminus of Derlin-1. These data suggest that intramembrane proteolysis and protein dislocation share unexpected mechanistic features. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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112. The polyubiquitin Ubc gene modulates histone H2A monoubiquitylation in the R6/2 mouse model of Huntington's disease.
- Author
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Bett, John S., Benn, Caroline L., Kwon-Yul Ryu, Kopito, Ron R., and Bates, Gillian P.
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HISTONES ,HUNTINGTON disease ,GENETIC disorders ,UBIQUITIN ,PROTEINS - Abstract
Huntington's disease (HD) is an inherited neurodegenerative disease caused by the expansion of a polyglutamine tract in the protein huntingtin (htt). HD brains are characterized by the presence of ubiquitin-positive neuronal inclusion bodies, suggesting that disturbances in the distribution of cellular ubiquitin may contribute to disease pathology. The fact that several neurodegenerative diseases are caused by mutations in ubiquitin-processing enzymes and that the polyubiquitin genes are required for resistance to cellular stress led us to investigate the effect of perturbing the ubiquitin system in HD. We crossed R6/2 transgenic HD mice with heterozygous polyubiquitin Ubc knockout mice ( Ubc+/−) and assessed the effect on the R6/2 neurological phenotype. Although the R6/2 phenotype was largely unaffected, surprisingly we observed some subtle improvements in various behavioural activities correlating with heterozygous Ubc knockout. Interestingly, immunoblot analysis revealed that the levels of monoubiquitylated histone H2A (uH2A), a modification associated with gene repression, were significantly increased in the brains of R6/2 mice. Furthermore, the reduction of Ubc expression in R6/2; Ubc+/− mice largely prevented this increase in uH2A levels. However, we were not able to show by the use of a limited number of quantitative RT-PCR assays that changes in the amount of uH2A in the R6/2- Ubc mice had an effect on disease-associated transcriptional abnormalities. These results suggest that the expression of aggregation-prone mutant htt causes disturbances to the ubiquitin system, which may contribute to disease due to the diverse and important roles of ubiquitin. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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- View/download PDF
113. Hypothalamic neurodegeneration and adult-onset obesity in mice lacking the Ubb polyubiquitin gene.
- Author
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Kwon-Yul Ryu, Garza, Jacob C., Xin-Yun Lu, Barsh, Gregory S., and Kopito, Ron R.
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HYPOTHALAMUS ,NEURODEGENERATION ,OBESITY ,UBIQUITIN ,GENES ,MICE - Abstract
Nearly all neurodegenerative diseases are associated with abnormal accumulation of ubiquitin (Ub) conjugates within neuronal inclusion bodies. To directly test the hypothesis that depletion of cellular Ub is sufficient to cause neurodegeneration, we have disrupted Ubb, one of four genes that supply Ub in the mouse. Here, we report that loss of Ubb led to a progressive degenerative disorder affecting neurons within the arcuate nucleus of the hypothalamus. This neurodegenerative cytopathology was accompanied by impaired hypothalamic control of energy balance and adult-onset obesity. Ubb was highly expressed in vulnerable hypothalamic neurons and total Ub levels were selectively reduced in the hypothalamus of Ubb-null mice. These findings demonstrate that maintenance of adequate supplies of cellular Ub is essential for neuronal survival and establish that decreased Ub availability is sufficient to cause neuronal dysfunction and death. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
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114. OS-9 and GRP94 deliver mutant α1-antitrypsin to the Hrd1?SEL1L ubiquitin ligase complex for ERAD.
- Author
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Christianson, John C., Shaler, Thomas A., Tyler, Ryan E., and Kopito, Ron R.
- Subjects
PROTEINS ,UBIQUITIN ,MANNOSE ,HOMOLOGY (Biology) ,PHOSPHATES - Abstract
Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant α
1 -antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells. [ABSTRACT FROM AUTHOR]- Published
- 2008
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115. The mouse polyubiquitin gene UbC is essential for fetal liver development, cell-cycle progression and stress tolerance.
- Author
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Kwon-Yul Ryu, Maehr, René, Gilchrist, Catherine A., Long, Michael A., Bouley, Donna M., Mueller, Britta, Ploegh, Hidde L., and Kopito, Ron R.
- Subjects
GENES ,MAMMALS ,UBIQUITIN ,PROTEINS ,CELL proliferation ,APOPTOSIS - Abstract
UbC is one of two stress-inducible polyubiquitin genes in mammals and is thought to supplement the constitutive UbA genes in maintaining cellular ubiquitin (Ub) levels during episodes of cellular stress. We have generated mice harboring a targeted disruption of the UbC gene. UbC
−/− embryos die between embryonic days 12.5 and 14.5 in utero, most likely owing to a severe defect in liver cell proliferation. Mouse embryonic fibroblasts from UbC−/− embryos exhibit reduced growth rates, premature senescence, increased apoptosis and delayed cell-cycle progression, with slightly, but significantly, decreased steady-state Ub levels. UbC−/− fibroblasts are hypersensitive to proteasome inhibitors and heat shock, and unable to adequately increase Ub levels in response to these cellular stresses. Most, but not all of the UbC−/− phenotypes can be rescued by providing additional Ub from a poly hemagglutinin-tagged Ub minigene expressed from the Hprt locus. We propose that UbC is regulated by a process that senses Ub pool dynamics. These data establish that UbC constitutes an essential source of Ub during cell proliferation and stress that cannot be compensated by other Ub genes. [ABSTRACT FROM AUTHOR]- Published
- 2007
- Full Text
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116. Sialoproteinaemia: lack of correlation with inhibition of <em>in vitro</em> lymphoblastosis induced by phytohaemagglutinin or alloantigen.
- Author
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Gary, Bruce N., Kopito, Ron R., Anderson, Leonard L., Baralt, Oscar L., Connery, Cheryl K., and Watkins Jr., Elton
- Subjects
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IMMUNOLOGY , *BLOOD plasma , *SERUM , *MEDICAL sciences , *SIALIC acids , *BIOCHEMISTRY - Abstract
Elevation of serum-bound sialic acid concentration in different disease states fails to correlate significantly with suppressive serum actions in mixed allogeneic lymphocyte cultures or phytohaemagglutinin cultures. Heat-decomplemented serum from patients with abnormal levels of bound sialic acid was added to parallel cultures containing similar blood lymphocyte populations derived from normal humans. Wide fluctuations of the rate of incorporation of tritiated thymidine into nucleoprotein indicated presence of suppressive elements other than sialoprotein in the added serum components. Serum with rising sialyl concentration derived from patients with cancer showed slight tendency to augment mixed lymphocyte and phytohaemagglutinin responses. The findings suggest that the previously documented nonspecific suppressive action of serum sialoprotein on human host lymphoblastic response to neuraminidase-treated cancer cells represents a mechanism unique to that culture system rather than a manifestation of a general immunoregulatory function of serum sialoprotein. [ABSTRACT FROM AUTHOR]
- Published
- 1976
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117. Identification of a 185-kDa band 3-related polypeptide in oxyntic cells.
- Author
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THOMAS, HOLLY A., MACHEN, TERRY E., SMOLKA, ADAM, BARON, ROLAND, and KOPITO, RON R.
- Published
- 1989
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118. Structure of the murine anion exchange protein.
- Author
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Kopito, Ron R. and Lodish, Harvey F.
- Published
- 1985
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119. The Missing Linker: An Unexpected Role for a Histone Deacetylase
- Author
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Kopito, Ron R.
- Subjects
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CELLS , *PROTEINS , *MICROTUBULES - Abstract
Molecular motors are the long-haul carriers of eukaryotic cells, moving cargos bidirectionally along microtubule tracks. In the December 12th issue of Cell, report that HDAC6, a tubulin deacetylase, functions as an adaptor that links cargos of aggregated protein to the minus end-directed motor, cytoplasmic dynein. [Copyright &y& Elsevier]
- Published
- 2003
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- View/download PDF
120. Primary structure and transmembrane orientation of the murine anion exchange protein
- Author
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Kopito, Ron R. and Lodish, Harvey F.
- Abstract
The amino-acid sequence of murine band 3, deduced from the nucleotide sequence of a complementary DNA clone, confirms that this integral membrane glycoprotein is composed of two major structural domains which correlate with its dual functions as the anchor for the erythrocyte cytoskeleton and as a plasma membrane anion antiporter. This latter activity resides within a highly Hydrophobie domain that crosses the plasma membrane at least 12 times.
- Published
- 1985
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121. Generation of truncated brain AE3 isoforms by alternate mRNA processing
- Author
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Morgans, Catherine W. and Kopito, Ron R.
- Abstract
AE3 gene is a member of the AE anion exchanger gene family that is expressed primarily in brain and heart. The principal product of the AE3 gene in rodent brain, FL-AE3p, when expressed in heterologous cell lines, gives rise to chloride-dependent changes in intracellular pH consistent with its operation as an anion exchanger. We have identified two novel isoforms of mouse AE3 that are generated by tissue-specific alternate RNA pro-cessing. One of these isoforms encodes a polypeptide, 14-AE3p, that corresponds to a portion of the NH2-ter-minal cytoplasmic domain of AE3. 14-AE3p lacks the entire transmembrane domain that-in FL-AE3p-forms the anion exchange channel. Immunoblots with antibodies to the NH2- and COOH-termini confirm that FL-AE3 and 14-AE3 are expressed in rat brain as 160 kDa and 74 kDa polypeptides, respectively. Unlike FL-AE3p, however, 14-AE3p is insoluble in non-ionic deter-gent, suggesting a possible association with the cytoskeleton.
- Published
- 1993
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122. Assessment of the flux of mitochondrial acetyl-CoA in liver and kidney by using the differential production of 14CO2 from tracers of (1-14C)- and (2-14C)-labelled 4-methyl-2-oxovalerate
- Author
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Tomera, John F., Kopito, Ron R., and Brunengraber, Henri
- Abstract
A procedure is described to convert rates of 14CO2 production into rates of mitochondrial acetyl-CoA production from a 14C-labelled substrate. The principle is illustrated in perfused rat liver and kidney by the differential yield of 14CO2 from 4-methyl-2-oxo[1-14C]valerate and 4-methyl-2-oxo[2-14C]valerate.
- Published
- 1983
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123. Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger
- Author
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Kopito, Ron R., primary, Lee, Beth S., additional, Simmons, Donna M., additional, Lindsey, Ann E., additional, Morgans, Catherine W., additional, and Schneider, Karin, additional
- Published
- 1989
- Full Text
- View/download PDF
124. The Mouse Polyubiquitin Gene Ubb Is Essential for Meiotic Progression.
- Author
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Kwon-Yul Ryu, Sinnar, Shamim A., Reinholdt, Laura G., Vaccari, Sergio, Hall, Susan, Garcia, Manuel A., Zaitseva, Tatiana S., Bouley, Donna M., Boekelheide, Kim, Handel, Mary Ann, Conti, Marco, and Kopito, Ron R.
- Subjects
UBIQUITIN ,GENES ,GERM cells ,MEIOSIS ,HYPOGONADISM ,SCHIZOSACCHAROMYCES pombe ,EUKARYOTIC cells ,LABORATORY mice - Abstract
Ubiquitin is encoded in mice by two polyubiquitin genes, Ubb and Ubc, that are considered to be stress inducible and two constitutively expressed monoubiquitin (Uba) genes. Here we report that targeted disruption of Ubb results in male and female infertility due to failure of germ cells to progress through meiosis I and hypogonadism. In the absence of Ubb, spermatocytes and oocytes arrest during meiotic prophase, before metaphase of the first meiotic division. Although cellular ubiquitin levels are believed to be maintained by a combination of functional redundancy among the four ubiquitin genes, stress inducibility of the two polyubiquitin genes, and ubiquitin recycling by proteasome- associated isopeptidases, our results indicate that ubiquitin is required for and consumed during meiotic progression. The striking similarity of the meiotic phenotype in Ubb
-/- germ cells to the sporulation defect in fission yeast (Schizosaccharomyces pombe) lacking a polyubiquitin gene suggests that a meiotic role of the polyubiquitin gene has been conserved throughout eukaryotic evolution. [ABSTRACT FROM AUTHOR]- Published
- 2008
- Full Text
- View/download PDF
125. Application and Analysis of the GFPu Family of Ubiquitin-Proteasome System Reporters.
- Author
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Bence, Neil F., Bennett, Eric J., and Kopito, Ron R.
- Abstract
An abstract of the article "Application and Analysis of the GFP
u Family of Ubiquitin-Proteasome System Reporters," by Neil F. Bence and colleagues is presented.- Published
- 2005
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- View/download PDF
126. A non‐canonical scaffold‐type E3 ligase complex mediates protein UFMylation.
- Author
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Peter, Joshua J, Magnussen, Helge M, DaRosa, Paul A, Millrine, David, Matthews, Stephen P, Lamoliatte, Frederic, Sundaramoorthy, Ramasubramanian, Kopito, Ron R, and Kulathu, Yogesh
- Subjects
- *
UBIQUITIN ligases , *UBIQUITIN-conjugating enzymes , *POST-translational modification , *UBIQUITINATION , *ADAPTOR proteins , *PROTEINS , *RIBOSOMAL proteins - Abstract
Protein UFMylation, i.e., post‐translational modification with ubiquitin‐fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold‐type E3 ligase mechanism that activates the UFM1‐conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex. Synopsis: UFMylation is a ubiquitin‐like post‐translational modification important for endoplasmic reticulum homeostasis, but how UFM1 is conjugated to target proteins remains poorly understood. Here, in vitro reconstitutions reveal the molecular players, minimal requirements and biochemical principles of UFM1 attachment to substrates. UFL1 and UFBP1 together form an active E3 ligase for UFM1.The UFL1/UFBP1 complex is an atypical scaffold‐type ligase that activates aminolysis by the UFM1‐conjugating E2 enzyme.CDK5RAP3 regulates activity of the UFL1/UFBP1 E3 ligase complex and promotes mono‐UFMylation of ribosomes.Activity of the E2 UFC1 is regulated by an N‐terminal extension and a unique TAK motif. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
127. Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.
- Author
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Bersuker, Kirill, Brandeis, Michael, and Kopito, Ron R.
- Subjects
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CELLULAR inclusions , *CYTOPLASM , *UBIQUITIN , *NEURODEGENERATION , *AUTOPHAGY , *HUNTINGTON disease , *GENETICS - Abstract
Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
128. Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover.
- Author
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Olzmann, James A., Richter, Caleb M., and Kopito, Ron R.
- Subjects
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TRIGLYCERIDES , *ENDOPLASMIC reticulum , *CYTOPLASMIC filaments , *PROTEIN analysis , *HYDROLYSIS - Abstract
UBXD8 is a membrane-embedded recruitment factor for the p97/VCP segregase that has been previously linked to endoplasmic reticulum (ER)-associated degradation and to the control of triacylglycerol synthesis in the ER. UBXD8 also has been identified as a component of cytoplasmic lipid droplets (LDs), but neither the mechanisms that control its trafficking between the ER and LDs nor its functions in the latter organelle have been investigated previously. Here we report that association of UBXD8 with the ER-resident rhomboid pseudoprotease UBAC2 specifically restricts trafficking of UBXD8 to LDs, and that the steady-state partitioning of UBXD8 between the ER and LDs can be experimentally manipulated by controlling the relative expression of these two proteins. We exploit this interaction to show that UBXD8-mediated recruitment of p97/VCP to LDs increases LD size by inhibiting the activity of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis. Our findings show that UBXD8 binds directly to ATGL and promotes dissociation of its endogenous coactivator, CGI-58. These data indicate that UBXD8 and p97/VCP play central integrative roles in cellular energy homeostasis. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
129. Impairment of the Ubiquitin-Proteasome System by Protein Aggregation.
- Author
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Bence, Neil F., Sampat, Roopal M., and Kopito, Ron R.
- Subjects
- *
UBIQUITIN , *NEURODEGENERATION , *CELL death - Abstract
Investigates the impairment of the ubiquitin-proteasome system by protein aggregation. Cytopathological feature of neurodegenerative disorders; Factors contributing to the complete inhibition of the ubiquitin-proteasome system; Linkage of protein aggregation, cellular disregulation and cell death.
- Published
- 2001
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- View/download PDF
130. Ribosomal protein RPL26 is the principal target of UFMylation.
- Author
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Walczak, Christopher P., Leto, Dara E., Lichao Zhang, Riepe, Celeste, Muller, Ryan Y., DaRosa, Paul A., Ingolia, Nicholas T., Elias, Joshua E., and Kopito, Ron R.
- Subjects
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RIBOSOMAL proteins , *GENETIC mutation , *ENDOPLASMIC reticulum , *CYTOPLASM , *PROTEIN structure - Abstract
Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to endoplasmic reticulum (ER) stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here, we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
131. Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.
- Author
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Roberts, Melissa A., Deol, Kirandeep K., Mathiowetz, Alyssa J., Lange, Mike, Leto, Dara E., Stevenson, Julian, Hashemi, Sayed Hadi, Morgens, David W., Easter, Emilee, Heydari, Kartoosh, Nalls, Mike A., Bassik, Michael C., Kampmann, Martin, Kopito, Ron R., Faghri, Faraz, and Olzmann, James A.
- Subjects
- *
UBIQUITINATION , *GENETIC testing , *CRISPRS , *UBIQUITIN ligases , *LIPIDS , *FUNCTIONAL genomics - Abstract
Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism. [Display omitted] • Generation of genetically edited PLIN2 fluorescent reporter cell lines • CRISPR screens identify PLIN2 regulators in different cell lines and conditions • ERAD E3 ligase MARCH6 regulates triacylglycerol biosynthesis and lipid droplets • Development of CRISPRlipid as a data commons for lipid-related genetic screens Roberts et al. perform multiple CRISPR screens in genetically edited fluorescent PLIN2 reporter cell lines to identify regulators of PLIN2, revealing the ERAD E3 ligase MARCH6 as a regulator of triacylglycerol biosynthesis and lipid droplets. To promote exploration of lipid-related genetic screen data, the authors develop CRISPRlipid as an online data commons. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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- View/download PDF
132. Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial α-helical membrane anchors.
- Author
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Pataki, Camille I., Rodrigues, João, Lichao Zhang, Qian, Junyang, Efron, Bradley, Hastie, Trevor, Elias, Joshua E., Levitt, Michael, and Kopito, Ron R.
- Subjects
- *
BILAYER lipid membranes , *MOLECULAR dynamics , *MEMBRANE proteins , *MONOMOLECULAR films , *ORGANELLES - Abstract
Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayerintegrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic α-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent-membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide highthroughput methods to identify and study MIPs. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
133. Characterization of protein complexes of the endoplasmic reticulum-associated degradation E3 ubiquitin ligase Hrd1.
- Author
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Jiwon Hwang, Walczak, Christopher P., Shaler, Thomas A., Olzmann, James A., Lichao Zhang, Elias, Joshua E., and Kopito, Ron R.
- Subjects
- *
GLYCOPROTEINS , *HETEROGENEITY , *PROTEOLYSIS , *CHROMATOGRAPHIC analysis , *PROTEIN expression - Abstract
Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
134. Indirect inhibition of 265 proteasome activity in a model of Huntington's disease.
- Author
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Hipp, Mark S., Patel, Chetan N., Bersuker, Kirill, Riley, Brigit E., Kaiser, Stephen E., Shaler, Thomas A., Brandeis, Michael, and Kopito, Ron R.
- Subjects
- *
UBIQUITIN , *NEURODEGENERATION , *HUNTINGTIN protein , *HUNTINGTON disease , *SOLUBILITY - Abstract
Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington's disease, suggests that highly aggregated proteins interfere with 265 proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 265 function. We show that ubiquitinated N-htt--whether aggregated or not-did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 265 substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
135. Defining human ERAD networks through an integrative mapping strategy.
- Author
-
Christianson, John C., Olzmann, James A., Shaler, Thomas A., Sowa, Mathew E., Bennett, Eric J., Richter, Caleb M., Tyler, Ryan E., Greenblatt, Ethan J., Wade Harper, J., and Kopito, Ron R.
- Subjects
- *
ENDOPLASMIC reticulum , *UBIQUITIN , *PROTEOMICS , *FUNCTIONAL genomics , *SACCHAROMYCES cerevisiae - Abstract
Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organized into a functional network that coordinates recognition, ubiquitylation and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organization of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics and the transcriptional response to ER stress. This analysis supports an adaptive organization for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
136. Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation.
- Author
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Iwata, Atsushi, Christianson, John C., Bucci, Mirella, Ellerby, Lisa M., Nukina, Nobuyuki, Forno, Lysia S., and Kopito, Ron R.
- Subjects
- *
NEURONS , *AUTOPHAGY , *NERVOUS system , *LEAVENING agents , *HEREDITY , *TOXICOLOGY - Abstract
CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous aggregates in mouse models of polyglutamine disease, but the cellular mechanism underlying this phenomenon is unknown. Here, we show that autophagy is essential for the elimination of aggregated forms of mutant huntingtin and ataxin-1 from the cytoplasmic but not nuclear compartments. Human orthologs of yeast autophagy genes, molecular determinants of autophagic vacuole formation, are recruited to cytoplasmic but not nuclear inclusion bodies in vitro and in vivo. These data indicate that autophagy is a critical component of the cellular clearance of toxic protein aggregates and may help to explain why protein aggregates are more toxic when directed to the nucleus. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
- View/download PDF
137. Formation of morphologically similar globular aggregates from diverse aggregation-prone proteins in mammalian cells.
- Author
-
Mukai, Hideyuki, Isagawa, Takayuki, Goyama, Emiko, Tanaka, Shuhei, Bence, Neil F., Tamura, Atsuo, Ono, Yoshitaka, and Kopito, Ron R.
- Subjects
- *
NEURODEGENERATION , *DEGENERATION (Pathology) , *HUNTINGTON disease , *AMINO acids , *GLUTAMINE , *ELECTRON microscopy - Abstract
Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin (Htt) protein. N-terminal Htt peptides with polyglutamine tracts in the pathological range (51-122 glutamines) form high-molecular-weight protein aggregates with fibrillar morphology in vitro, and they form discrete inclusion bodies in a cell-culture model. However, in some studies, formation of discrete Htt inclusions does not correlate well with cell death. We coexpressed N-terminal Htt fragments containing 91 glutamines fused to dif- ferent affinity tags in HEK293 cells, and we isolated small aggregates by double sequential-affinity chromatography to assure the isolation of multimeric molecules. Transmission electron microscopy and atomic force microscopy revealed the isolated aggregates as globules or clusters of globules 4-50 nm in diameter without any detectable fibrillar species. Because small nonfibrillar oligomers, not mature fibrils, recently have been suggested to be the principal cytotoxic species in neurodegenerative disease, these Htt globular aggregates formed in cells may represent the pathogenic form of mutant Htt. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
- View/download PDF
138. Specificity in intracellular protein aggregation and inclusion body formation.
- Author
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Rajan, Rahul S., Illing, Michelle E., Bence, Neil F., and Kopito, Ron R.
- Subjects
- *
PROTEINS , *CELLS , *PLASMIDS - Abstract
Examines the intracellular protein aggregation and inclusion body formation. Factors affecting protein aggregation; Correlation between cells and plasmids; Role of mammalian cells in cytoplasmic microtubules in inclusion bodies.
- Published
- 2001
- Full Text
- View/download PDF
139. The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.
- Author
-
Wąchalska M, Riepe C, Ślusarz MJ, Graul M, Borowski LS, Qiao W, Foltyńska M, Carette JE, Bieńkowska-Szewczyk K, Szczesny RJ, Kopito RR, and Lipińska AD
- Subjects
- Antigen Presentation, Cytomegalovirus, Endoplasmic Reticulum-Associated Degradation, Membrane Transport Proteins, Peptides, Ubiquitin-Protein Ligases genetics, ATP-Binding Cassette Transporters, Degrons, Herpesviridae physiology
- Abstract
The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2
KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation., Competing Interests: Competing interests statement:The authors declare no competing interest.- Published
- 2024
- Full Text
- View/download PDF
140. UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.
- Author
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DaRosa PA, Penchev I, Gumbin SC, Scavone F, Wąchalska M, Paulo JA, Ordureau A, Peter JJ, Kulathu Y, Harper JW, Becker T, Beckmann R, and Kopito RR
- Subjects
- Binding Sites, Biocatalysis, Cryoelectron Microscopy, Intracellular Membranes chemistry, Intracellular Membranes metabolism, Intracellular Membranes ultrastructure, Peptidyl Transferases chemistry, Peptidyl Transferases metabolism, Peptidyl Transferases ultrastructure, Protein Binding, Ribosomal Proteins chemistry, Ribosomal Proteins metabolism, Ribosomal Proteins ultrastructure, RNA, Transfer metabolism, Substrate Specificity, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum ultrastructure, Protein Processing, Post-Translational, Ribosome Subunits, Large, Eukaryotic chemistry, Ribosome Subunits, Large, Eukaryotic metabolism, Ribosome Subunits, Large, Eukaryotic ultrastructure, Ubiquitin-Protein Ligases chemistry, Ubiquitin-Protein Ligases metabolism, Ubiquitin-Protein Ligases ultrastructure
- Abstract
Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour
1 . UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3 . UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4 . Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2024
- Full Text
- View/download PDF
141. The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.
- Author
-
Wąhalska M, Riepe C, Ślusarz MJ, Graul M, Borowski LS, Qiao W, Foltynska M, Carette JE, Bieńkowska-Szewczyk K, Szczesny RJ, Kopito RR, and Lipińska AD
- Abstract
The transporter associated with antigen processing (TAP) is a key player in the MHC class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 (BoHV-1) impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and promotes its proteasomal degradation. How UL49.5 promotes TAP degradation is unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2
KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal in human cells. We propose that the C-terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the CRL2 E3 in ER-associated degradation., Competing Interests: Declaration of interests The authors declare no competing interests.- Published
- 2023
- Full Text
- View/download PDF
142. Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states.
- Author
-
Riepe C, Wąchalska M, Deol KK, Amaya AK, Porteus MH, Olzmann JA, and Kopito RR
- Abstract
Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase, demonstrating that CFTR-F508del ERAD is highly buffered. Gene-drug interaction experiments demonstrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.
- Published
- 2023
- Full Text
- View/download PDF
143. RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum.
- Author
-
Scavone F, Gumbin SC, Da Rosa PA, and Kopito RR
- Subjects
- Ribosomal Proteins genetics, Ribosomal Proteins metabolism, Quality Control, Ubiquitins metabolism, Ribosomes metabolism, Endoplasmic Reticulum metabolism
- Abstract
Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs.
- Published
- 2023
- Full Text
- View/download PDF
144. PEX19 Coordinates Neutral Lipid Storage in Cells in a Peroxisome-Independent Fashion.
- Author
-
Lyschik S, Lauer AA, Roth T, Janitschke D, Hollander M, Will T, Hartmann T, Kopito RR, Helms V, Grimm MOW, and Schrul B
- Abstract
Cellular lipid metabolism is tightly regulated and requires a sophisticated interplay of multiple subcellular organelles to adapt to changing nutrient supply. PEX19 was originally described as an essential peroxisome biogenesis factor that selectively targets membrane proteins to peroxisomes. Metabolic aberrations that were associated with compromised PEX19 functions, were solely attributed to the absence of peroxisomes, which is also considered the underlying cause for Zellweger Spectrum Disorders. More recently, however, it was shown that PEX19 also mediates the targeting of the VCP/P97-recuitment factor UBXD8 to the ER from where it partitions to lipid droplets (LDs) but the physiological consequences remained elusive. Here, we addressed the intriguing possibility that PEX19 coordinates the functions of the major cellular sites of lipid metabolism. We exploited the farnesylation of PEX19 and deciphered the organelle-specific functions of PEX19 using systems level approaches. Non-farnesylated PEX19 is sufficient to fully restore the metabolic activity of peroxisomes, while farnesylated PEX19 controls lipid metabolism by a peroxisome-independent mechanism that can be attributed to sorting a specific protein subset to LDs. In the absence of this PEX19-dependent LD proteome, cells accumulate excess triacylglycerols and fail to fully deplete their neutral lipid stores under catabolic conditions, highlighting a hitherto unrecognized function of PEX19 in controlling neutral lipid storage and LD dynamics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lyschik, Lauer, Roth, Janitschke, Hollander, Will, Hartmann, Kopito, Helms, Grimm and Schrul.)
- Published
- 2022
- Full Text
- View/download PDF
145. Methods for genetic analysis of mammalian ER-associated degradation.
- Author
-
Leto DE and Kopito RR
- Subjects
- Animals, Cell Line, Gene Editing methods, Humans, RNA, Guide, CRISPR-Cas Systems genetics, Transduction, Genetic, CRISPR-Cas Systems, Endoplasmic Reticulum-Associated Degradation
- Abstract
Identification and degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is crucial for maintaining proteostasis, but only a handful of UPS components have been linked to the recognition of specific substrates. Studies in Saccharomyces cerevisiae using systematic perturbation of nonessential genes have uncovered UPS components that recognize and ubiquitylate model substrates of the UPS; however, similar analyses in metazoans have been limited. In this chapter, we describe methods for using CRISPR/Cas9 technology combined with genome-wide high complexity single guide (sgRNA) libraries and a transcriptional shutoff strategy for phenotypic selection based on kinetic measurements of protein turnover to identify the genes required to degrade model clients of the mammalian ER-associated degradation system. We also discuss considerations for screen design, execution, and interpretation., (© 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
146. Prion-Like Characteristics of Polyglutamine-Containing Proteins.
- Author
-
Pearce MMP and Kopito RR
- Subjects
- Animals, Humans, Neurodegenerative Diseases pathology, Prion Diseases pathology, Protein Folding, Proteostasis, Neurodegenerative Diseases metabolism, Peptides metabolism, Prion Diseases metabolism, Prion Proteins metabolism, Protein Aggregation, Pathological metabolism
- Abstract
Transmissible spongiform encephalopathies are infectious neurodegenerative diseases caused by the conversion of prion protein (PrP) into a self-replicating conformation that spreads via templated conversion of natively folded PrP molecules within or between cells. Recent studies provide compelling evidence that prion-like behavior is a general property of most protein aggregates associated with neurodegenerative diseases. Many of these disorders are associated with spontaneous protein aggregation, but genetic mutations can increase the aggregation propensity of specific proteins, including expansion of polyglutamine (polyQ) tracts, which is causative of nine inherited neurodegenerative diseases. Aggregates formed by polyQ-expanded huntingtin (Htt) in Huntington's disease can transfer between cells and seed the aggregation of cytoplasmic wild-type Htt in a prion-like manner. Additionally, prion-like properties of glutamine-rich proteins underlie nonpathological processes in yeast and higher eukaryotes. Here, we review current evidence supporting prion-like characteristics of polyQ and glutamine-rich proteins., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
147. Characterization of protein complexes of the endoplasmic reticulum-associated degradation E3 ubiquitin ligase Hrd1.
- Author
-
Hwang J, Walczak CP, Shaler TA, Olzmann JA, Zhang L, Elias JE, and Kopito RR
- Subjects
- Endoplasmic Reticulum chemistry, Endoplasmic Reticulum genetics, HEK293 Cells, Humans, Multiprotein Complexes chemistry, Multiprotein Complexes genetics, Multiprotein Complexes isolation & purification, Ubiquitin-Protein Ligases chemistry, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases isolation & purification, Endoplasmic Reticulum metabolism, Gene Expression Regulation, Enzymologic physiology, Multiprotein Complexes metabolism, Proteolysis, Ubiquitin-Protein Ligases metabolism
- Abstract
Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
- Full Text
- View/download PDF
148. The mammalian endoplasmic reticulum-associated degradation system.
- Author
-
Olzmann JA, Kopito RR, and Christianson JC
- Subjects
- Animals, Mannose-Binding Lectins metabolism, Polysaccharides metabolism, Polyubiquitin metabolism, Endoplasmic Reticulum physiology, Endoplasmic Reticulum-Associated Degradation physiology, Mammals physiology, Models, Biological, Proteasome Endopeptidase Complex metabolism, Protein Folding
- Abstract
The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system. ERAD proceeds through four tightly coupled steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasomal degradation. The ERAD machinery shows a modular organization with central ER membrane-embedded ubiquitin ligases linking components responsible for recognition in the ER lumen to the ubiquitin-proteasome system in the cytoplasm. The core ERAD machinery is highly conserved among eukaryotes and much of our basic understanding of ERAD organization has been derived from genetic and biochemical studies of yeast. In this article we discuss how the core ERAD machinery is organized in mammalian cells.
- Published
- 2013
- Full Text
- View/download PDF
149. Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate.
- Author
-
Tyler RE, Pearce MM, Shaler TA, Olzmann JA, Greenblatt EJ, and Kopito RR
- Subjects
- Alkaloids pharmacology, Basigin genetics, Basigin metabolism, Binding Sites genetics, Enzyme Inhibitors pharmacology, Glycosylation, HEK293 Cells, Humans, Immunoblotting, Lectins genetics, Mass Spectrometry, Mutation, Neoplasm Proteins genetics, Polysaccharides metabolism, Proteasome Endopeptidase Complex metabolism, Protein Binding, Proteins genetics, Proteins metabolism, Proteolysis drug effects, RNA Interference, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Endoplasmic Reticulum-Associated Degradation, Lectins metabolism, Neoplasm Proteins metabolism
- Abstract
Degradation of folding- or assembly-defective proteins by the endoplasmic reticulum-associated degradation (ERAD) ubiquitin ligase, Hrd1, is facilitated by a process that involves recognition of demannosylated N-glycans by the lectin OS-9/XTP3-B via the adaptor protein SEL1L. Most of our knowledge of the machinery that commits proteins to this fate in metazoans comes from studies of overexpressed mutant proteins in heterologous cells. In this study, we used mass spectrometry to identify core-glycoslyated CD147 (CD147(CG)) as an endogenous substrate of the ERAD system that accumulates in a complex with OS-9 following SEL1L depletion. CD147 is an obligatory assembly factor for monocarboxylate transporters. The majority of newly synthesized endogenous CD147(CG) was degraded by the proteasome in a Hrd1-dependent manner. CD147(CG) turnover was blocked by kifunensine, and interaction of OS-9 and XTP3-B with CD147(CG) was inhibited by mutations to conserved residues in their lectin domains. These data establish unassembled CD147(CG) as an endogenous, constitutive ERAD substrate of the OS-9/SEL1L/Hrd1 pathway.
- Published
- 2012
- Full Text
- View/download PDF
150. Live-cell imaging of ubiquitin-proteasome system function.
- Author
-
Hipp MS, Bersuker K, and Kopito RR
- Subjects
- Endoplasmic Reticulum metabolism, Fluorescence, Genes, Reporter, Green Fluorescent Proteins biosynthesis, Green Fluorescent Proteins genetics, HEK293 Cells, Humans, Ubiquitin chemistry, Ubiquitin genetics, Ubiquitin metabolism, Flow Cytometry methods, Proteasome Endopeptidase Complex genetics, Proteasome Endopeptidase Complex metabolism
- Abstract
The role of the ubiquitin-proteasome system (UPS) in maintaining protein homeostasis has generated a demand for assays that quantify UPS function in the presence of chemical and protein UPS inhibitors. Here, we describe protocols that measure changes in UPS reporter levels in response to changes in the expression level, localization, or aggregation state of a second protein. We utilize cell lines stably expressing fluorescent UPS substrates that are transfected with a second protein tagged with a compatible fluorophore. We describe protocols to correlate levels of UPS substrates with changes in the levels or properties of the transfected protein.
- Published
- 2012
- Full Text
- View/download PDF
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