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Alfred L Goldberg - One of the best experts on this subject based on the ideXlab platform.
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ubiquitinated proteins promote the association of Proteasomes with the deubiquitinating enzyme usp14 and the ubiquitin ligase ube3c
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Alfred L GoldbergAbstract:In mammalian cells, the 26S Proteasomes vary in composition. In addition to the standard 28 subunits in the 20S core particle and 19 subunits in each 19S regulatory particle, a small fraction (about 10–20% in our preparations) also contains the deubiquitinating enzyme Usp14/Ubp6, which regulates Proteasome activity, and the ubiquitin ligase, Ube3c/Hul5, which enhances proteasomal processivity. When degradation of ubiquitinated proteins in cells was inhibited, levels of Usp14 and Ube3c on Proteasomes increased within minutes. Conversely, when protein ubiquitination was prevented, or when purified Proteasomes hydrolyzed the associated ubiquitin conjugates, Usp14 and Ube3c dissociated rapidly (unlike other 26S subunits), but the inhibitor ubiquitin aldehyde slowed their dissociation. Recombinant Usp14 associated with purified Proteasomes preferentially if they contained ubiquitin conjugates. In cells or extracts, adding Usp14 inhibitors (IU-1 or ubiquitin aldehyde) enhanced Usp14 and Ube3c binding further. Thus, in the substrate- or the inhibitor-bound conformations, Usp14 showed higher affinity for Proteasomes and surprisingly enhanced Ube3c binding. Moreover, adding ubiquitinated proteins to cell extracts stimulated Proteasome binding of both enzymes. Thus, Usp14 and Ube3c cycle together on and off Proteasomes, and the presence of ubiquitinated substrates promotes their association. This mechanism enables Proteasome activity to adapt to the supply of substrates.
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tau driven 26s Proteasome impairment and cognitive dysfunction can be prevented early in disease by activating camp pka signaling
Nature Medicine, 2016Co-Authors: Natura Myeku, Alfred L Goldberg, Catherine L Clelland, Sheina Emrani, Nikolay V Kukushkin, Karen DuffAbstract:The ubiquitin Proteasome system (UPS) degrades misfolded proteins including those implicated in neurodegenerative diseases. We investigated the effects of tau accumulation on Proteasome function in a mouse model of tauopathy and in a cross to a UPS reporter mouse (line Ub-G76V-GFP). Accumulation of insoluble tau was associated with a decrease in the peptidase activity of brain 26S Proteasomes, higher levels of ubiquitinated proteins and undegraded Ub-G76V-GFP. 26S Proteasomes from mice with tauopathy were physically associated with tau and were less active in hydrolyzing ubiquitinated proteins, small peptides and ATP. 26S Proteasomes from normal mice incubated with recombinant oligomers or fibrils also showed lower hydrolyzing capacity in the same assays, implicating tau as a proteotoxin. Administration of an agent that activates cAMP-protein kinase A (PKA) signaling led to attenuation of Proteasome dysfunction, probably through Proteasome subunit phosphorylation. In vivo, this led to lower levels of aggregated tau and improvements in cognitive performance.
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camp induced phosphorylation of 26s Proteasomes on rpn6 psmd11 enhances their activity and the degradation of misfolded proteins
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Sudarsanareddy Lokireddy, Nikolay V Kukushkin, Alfred L GoldbergAbstract:Although rates of protein degradation by the ubiquitin-Proteasome pathway (UPS) are determined by their rates of ubiquitination, we show here that the Proteasome’s capacity to degrade ubiquitinated proteins is also tightly regulated. We studied the effects of cAMP-dependent protein kinase (PKA) on proteolysis by the UPS in several mammalian cell lines. Various agents that raise intracellular cAMP and activate PKA (activators of adenylate cyclase or inhibitors of phosphodiesterase 4) promoted degradation of short-lived (but not long-lived) cell proteins generally, model UPS substrates having different degrons, and aggregation-prone proteins associated with major neurodegenerative diseases, including mutant FUS (Fused in sarcoma), SOD1 (superoxide dismutase 1), TDP43 (TAR DNA-binding protein 43), and tau. 26S Proteasomes purified from these treated cells or from control cells and treated with PKA degraded ubiquitinated proteins, small peptides, and ATP more rapidly than controls, but not when treated with protein phosphatase. Raising cAMP levels also increased amounts of doubly capped 26S Proteasomes. Activated PKA phosphorylates the 19S subunit, Rpn6/PSMD11 (regulatory particle non-ATPase 6/Proteasome subunit D11) at Ser14. Overexpression of a phosphomimetic Rpn6 mutant activated Proteasomes similarly, whereas a nonphosphorylatable mutant decreased activity. Thus, Proteasome function and protein degradation are regulated by cAMP through PKA and Rpn6, and activation of Proteasomes by this mechanism may be useful in treating proteotoxic diseases.
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why do cellular proteins linked to k63 polyubiquitin chains not associate with Proteasomes
The EMBO Journal, 2013Co-Authors: James A Nathan, Steven P Gygi, Hyoung Tae Kim, Lily Ting, Alfred L GoldbergAbstract:Although cellular proteins conjugated to K48-linked Ub chains are targeted to Proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S Proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by Proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with Proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to Proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63-ubiquitinated molecules to the Proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48-ubiquitinated proteins and to stimulate Proteasome binding. The specificities of these proteins for K48- or K63-ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal-lysosomal pathway.
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hrpn13 adrm1 gp110 is a novel Proteasome subunit that binds the deubiquitinating enzyme uch37
The EMBO Journal, 2006Co-Authors: Songying Ouyang, Chaojun Li, Shiying Miao, Linfang Wang, Alfred L GoldbergAbstract:The 26S Proteasome catalyzes the degradation of most proteins in mammalian cells. To better define its composition and associated regulatory proteins, we developed affinity methods to rapidly purify 26S Proteasomes from mammalian cells. By this approach, we discovered a novel 46-kDa (407 residues) subunit of its 19S regulatory complex (previously termed ADRM1 or GP110). As its N-terminal half can be incorporated into the 26S Proteasome and is homologous to Rpn13, a 156-residue subunit of the 19S complex in budding yeast, we renamed it human Rpn13 (hRpn13). The C-terminal half of hRpn13 binds directly to the Proteasome-associated deubiquitinating enzyme, UCH37, and enhances its isopeptidase activity. Knockdown of hRpn13 in 293T cells increases the cellular levels of ubiquitin conjugates and decreases the degradation of short-lived proteins. Surprisingly, an overproduction of hRpn13 also reduced their degradation. Furthermore, transfection of the C-terminal half of hRpn13 slows proteolysis and induces cell death, probably by acting as a dominant-negative form. Thus in human 26S Proteasomes, hRpn13 appears to be important for the binding of UCH37 to the 19S complex and for efficient proteolysis.
Do Hee Lee - One of the best experts on this subject based on the ideXlab platform.
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Proteasome inhibitors valuable new tools for cell biologists
Trends in Cell Biology, 1998Co-Authors: Do Hee Lee, Alfred L GoldbergAbstract:Proteasomes are major sites for protein degradation in eukaryotic cells. The recent identification of selective Proteasome inhibitors has allowed a definition of the roles of the ubiquitin-Proteasome pathway in various cellular processes, such as antigen presentation and the degradation of regulatory or membrane proteins. This review describes the actions of these inhibitors, how they can be used to investigate cellular responses, the functions of the Proteasome demonstrated by such studies and their potential applications in the future.
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selective inhibitors of the Proteasome dependent and vacuolar pathways of protein degradation in saccharomyces cerevisiae
Journal of Biological Chemistry, 1996Co-Authors: Do Hee Lee, Alfred L GoldbergAbstract:We have studied whether various agents that inhibit purified yeast and mammalian 26 S Proteasome can suppress the breakdown of different classes of proteins in Saccharomyces cerevisiae. The degradation of short-lived proteins was inhibited reversibly by peptide aldehyde inhibitors of Proteasomes, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) and carbobenzoxyl-leucinyl-leucinyl-norvalinal (MG115), in a yeast mutant with enhanced permeability, but not in wild-type strains. Lactacystin, an irreversible Proteasome inhibitor, had no effect, but the β-lactone derivative of lactacystin, which directly reacts with Proteasomes, inhibited the degradation of short-lived proteins. These inhibitors also blocked the rapid ubiquitin-dependent breakdown of a β-galactosidase fusion protein and caused accumulation of enzymatically active molecules in cells. The degradation of the bulk of cell proteins, which are long-lived molecules, was not blocked by Proteasome inhibitors, but could be blocked by phenylmethylsulfonyl fluoride. This agent, which inhibits multiple vacuolar proteases, did not affect the Proteasome or breakdown of short-lived proteins. These two classes of inhibitors can thus be used to distinguish the cytosolic and vacuolar proteolytic pathways and to increase the cellular content of short-lived proteins.
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selective inhibitors of the Proteasome dependent and vacuolar pathways of protein degradation in saccharomyces cerevisiae
Journal of Biological Chemistry, 1996Co-Authors: Do Hee Lee, Alfred L GoldbergAbstract:We have studied whether various agents that inhibit purified yeast and mammalian 26 S Proteasome can suppress the breakdown of different classes of proteins in Saccharomyces cerevisiae. The degradation of short-lived proteins was inhibited reversibly by peptide aldehyde inhibitors of Proteasomes, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) and carbobenzoxyl-leucinyl-leucinyl-norvalinal (MG115), in a yeast mutant with enhanced permeability, but not in wild-type strains. Lactacystin, an irreversible Proteasome inhibitor, had no effect, but the beta-lactone derivative of lactacystin, which directly reacts with Proteasomes, inhibited the degradation of short-lived proteins. These inhibitors also blocked the rapid ubiquitin-dependent breakdown of a beta-galactosidase fusion protein and caused accumulation of enzymatically active molecules in cells. The degradation of the bulk of cell proteins, which are long-lived molecules, was not blocked by Proteasome inhibitors, but could be blocked by phenylmethylsulfonyl fluoride. This agent, which inhibits multiple vacuolar proteases, did not affect the Proteasome or breakdown of short-lived proteins. These two classes of inhibitors can thus be used to distinguish the cytosolic and vacuolar proteolytic pathways and to increase the cellular content of short-lived proteins.
Martin Rechsteiner - One of the best experts on this subject based on the ideXlab platform.
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a protein interaction network for ecm29 links the 26 s Proteasome to molecular motors and endosomal components
Journal of Biological Chemistry, 2010Co-Authors: Carlos Gorbea, Gregory Pratt, Vicenca Ustrell, Russell Bell, Sudhir Sahasrabudhe, Robert E Hughes, Martin RechsteinerAbstract:Ecm29 is a 200-kDa HEAT repeat protein that binds the 26 S Proteasome. Genome-wide two-hybrid screens and mass spectrometry have identified molecular motors, endosomal components, and ubiquitin-Proteasome factors as Ecm29-interacting proteins. The C-terminal half of human Ecm29 binds myosins and kinesins; its N-terminal region binds the endocytic proteins, Vps11, Rab11-FIP4, and rabaptin. Whereas full-length FLAG-Ecm29, its C-terminal half, and a small central fragment of Ecm29 remain bound to glycerol-gradient-separated 26 S Proteasomes, the N-terminal half of Ecm29 does not. Confocal microscopy showed that Ecm-26 S Proteasomes are present on flotillin-positive endosomes, but they are virtually absent from caveolin- and clathrin-decorated endosomes. Expression of the small central fragment of Ecm29 markedly reduces Proteasome association with flotillin-positive endosomes. Identification of regions within Ecm29 capable of binding molecular motors, endosomal proteins, and the 26 S Proteasome supports the hypothesis that Ecm29 serves as an adaptor for coupling 26 S Proteasomes to specific cellular compartments.
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a protein interaction network for ecm29 links the 26 s Proteasome to molecular motors and endosomal components
Journal of Biological Chemistry, 2010Co-Authors: Carlos Gorbea, Gregory Pratt, Vicenca Ustrell, Russell Bell, Sudhir Sahasrabudhe, Robert E Hughes, Martin RechsteinerAbstract:Ecm29 is a 200-kDa HEAT repeat protein that binds the 26 S Proteasome. Genome-wide two-hybrid screens and mass spectrometry have identified molecular motors, endosomal components, and ubiquitin-Proteasome factors as Ecm29-interacting proteins. The C-terminal half of human Ecm29 binds myosins and kinesins; its N-terminal region binds the endocytic proteins, Vps11, Rab11-FIP4, and rabaptin. Whereas full-length FLAG-Ecm29, its C-terminal half, and a small central fragment of Ecm29 remain bound to glycerol-gradient-separated 26 S Proteasomes, the N-terminal half of Ecm29 does not. Confocal microscopy showed that Ecm-26 S Proteasomes are present on flotillin-positive endosomes, but they are virtually absent from caveolin- and clathrin-decorated endosomes. Expression of the small central fragment of Ecm29 markedly reduces Proteasome association with flotillin-positive endosomes. Identification of regions within Ecm29 capable of binding molecular motors, endosomal proteins, and the 26 S Proteasome supports the hypothesis that Ecm29 serves as an adaptor for coupling 26 S Proteasomes to specific cellular compartments.
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mobilizing the proteolytic machine cell biological roles of Proteasome activators and inhibitors
Trends in Cell Biology, 2005Co-Authors: Martin Rechsteiner, Christopher P HillAbstract:Proteasomes perform the majority of proteolysis that occurs in the cytosol and nucleus of eukaryotic cells and, thereby, perform crucial roles in cellular regulation and homeostasis. Isolated Proteasomes are inactive because substrates cannot access the proteolytic sites. PA28 and PA200 are activators that bind to Proteasomes and stimulate the hydrolysis of peptides. Several protein inhibitors of the Proteasome have also been identified, and the properties of these activators and inhibitors have been characterized biochemically. By contrast, their physiological roles – which have been reported to include production of antigenic peptides, Proteasome assembly and DNA repair – are controversial. In this article, we briefly review the biochemical data and discuss the possible biological roles of PA28, PA200 and Proteasome inhibitors.
Kristian Klindt - One of the best experts on this subject based on the ideXlab platform.
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the inducible β5i Proteasome subunit contributes to proinsulin degradation in grp94 deficient β cells and is overexpressed in type 2 diabetes pancreatic islets
American Journal of Physiology-endocrinology and Metabolism, 2020Co-Authors: Muhammad Saad Khilji, Tina Dahlby, Celina Pihl, Sophie Emilie Bresson, Danielle Verstappen, Phillip Alexander Keller Andersen, Jette Bach Agergaard, Tenna Holgersen Bryde, Kristian KlindtAbstract:Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when β-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard Proteasome, while the roles of other Proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different Proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active Proteasome subunit, namely, the inducible β5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of β5i-containing intermediate Proteasomes was significantly increased in these cells, as was β5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon β5i small interfering RNA-mediated knockdown. Finally, the fraction of β-cells expressing the β5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that β5i is an inducible Proteasome subunit dedicated to the degradation of mishandled proinsulin.
Danielle Verstappen - One of the best experts on this subject based on the ideXlab platform.
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the inducible β5i Proteasome subunit contributes to proinsulin degradation in grp94 deficient β cells and is overexpressed in type 2 diabetes pancreatic islets
American Journal of Physiology-endocrinology and Metabolism, 2020Co-Authors: Muhammad Saad Khilji, Tina Dahlby, Celina Pihl, Sophie Emilie Bresson, Danielle Verstappen, Phillip Alexander Keller Andersen, Jette Bach Agergaard, Tenna Holgersen Bryde, Kristian KlindtAbstract:Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when β-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard Proteasome, while the roles of other Proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different Proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active Proteasome subunit, namely, the inducible β5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of β5i-containing intermediate Proteasomes was significantly increased in these cells, as was β5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon β5i small interfering RNA-mediated knockdown. Finally, the fraction of β-cells expressing the β5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that β5i is an inducible Proteasome subunit dedicated to the degradation of mishandled proinsulin.
