From the Evasion of Degradation to Ubiquitin-Dependent Protein Stabilization

A hallmark of cancer is dysregulated protein turnover (proteostasis), which involves pathologic ubiquitin-dependent degradation of tumor suppressor proteins, as well as increased oncoprotein stabilization. The latter is due, in part, to mutation within sequences, termed degrons, which are required for oncoprotein recognition by the substrate-recognition enzyme, E3 ubiquitin ligase. Stabilization may also result from the inactivation of the enzymatic machinery that mediates the degradation of oncoproteins. Importantly, inactivation in cancer of E3 enzymes that regulates the physiological degradation of oncoproteins, results in tumor cells that accumulate multiple active oncoproteins with prolonged half-lives, leading to the development of “degradation-resistant” cancer cells. In addition, specific sequences may enable ubiquitinated proteins to evade degradation at the 26S proteasome. While the ubiquitin-proteasome pathway was originally discovered as central for protein degradation, in cancer cells a ubiquitin-dependent protein stabilization pathway actively translates transient mitogenic signals into long-lasting protein stabilization and enhances the activity of key oncoproteins. A central enzyme in this pathway is the ubiquitin ligase RNF4. An intimate link connects protein stabilization with tumorigenesis in experimental models as well as in the clinic, suggesting that pharmacological inhibition of protein stabilization has potential for personalized medicine in cancer. In this review, we highlight old observations and recent advances in our knowledge regarding protein stabilization.

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HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.23.16.5790-5802.2003 ◽

2003 ◽

Vol 23 (16) ◽

pp. 5790-5802 ◽

Cited By ~ 225

Author(s):

Arnaud Parcellier ◽

Elise Schmitt ◽

Sandeep Gurbuxani ◽

Daphné Seigneurin-Berny ◽

Alena Pance ◽

...

Keyword(s):

Direct Interaction ◽

Cell Types ◽

26S Proteasome ◽

Necrosis Factor Alpha ◽

Polyubiquitin Chains ◽

Ubiquitinated Proteins ◽

Activation Of Transcription ◽

Ubiquitin Proteasome

ABSTRACT HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-α). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-κB by degrading its main inhibitor, I-κBα. HSP27 overexpression increases NF-κB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ΤNF-α, and interleukin 1β. HSP27 does not affect I-κBα phosphorylation but enhances the degradation of phosphorylated I-κBα by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-κBα. A protein complex that includes HSP27, phosphorylated I-κBα, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-κBα. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-κB activity.

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Calpain activity and muscle wasting in sepsis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90226.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. E762-E771 ◽

Cited By ~ 77

Author(s):

Ira J. Smith ◽

Stewart H. Lecker ◽

Per-Olof Hasselgren

Keyword(s):

Transcription Factors ◽

Muscle Wasting ◽

26S Proteasome ◽

Calpain Activation ◽

Ubiquitin Proteasome ◽

Dependent Protein ◽

Foxo Transcription Factors ◽

Gsk 3Β ◽

Calpain System

Muscle wasting in sepsis reflects activation of multiple proteolytic mechanisms, including lyosomal and ubiquitin-proteasome-dependent protein breakdown. Recent studies suggest that activation of the calpain system also plays an important role in sepsis-induced muscle wasting. Perhaps the most important consequence of calpain activation in skeletal muscle during sepsis is disruption of the sarcomere, allowing for the release of myofilaments (including actin and myosin) that are subsequently ubiquitinated and degraded by the 26S proteasome. Other important consequences of calpain activation that may contribute to muscle wasting during sepsis include degradation of certain transcription factors and nuclear cofactors, activation of the 26S proteasome, and inhibition of Akt activity, allowing for downstream activation of Foxo transcription factors and GSK-3β. The role of calpain activation in sepsis-induced muscle wasting suggests that the calpain system may be a therapeutic target in the prevention and treatment of muscle wasting during sepsis. Furthermore, because calpain activation may also be involved in muscle wasting caused by other conditions, including different muscular dystrophies and cancer, calpain inhibitors may be beneficial not only in the treatment of sepsis-induced muscle wasting but in other conditions causing muscle atrophy as well.

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p62-containing, proteolytically active nuclear condensates, increase the efficiency of the ubiquitin–proteasome system

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2107321118 ◽

2021 ◽

Vol 118 (33) ◽

pp. e2107321118

Author(s):

Afu Fu ◽

Victoria Cohen-Kaplan ◽

Noa Avni ◽

Ido Livneh ◽

Aaron Ciechanover

Keyword(s):

Ubiquitin Ligase ◽

Protein Quality ◽

Ubiquitin Proteasome System ◽

Cellular Protein ◽

26S Proteasome ◽

Ubiquitin Chain ◽

Recognition Element ◽

Oxidative Stresses ◽

Ubiquitin Ligase E3 ◽

Ubiquitin Proteasome

Degradation of a protein by the ubiquitin–proteasome system (UPS) is a multistep process catalyzed by sequential reactions. Initially, ubiquitin is conjugated to the substrate in a process mediated by concerted activity of three enzymes; the last of them—a ubiquitin ligase (E3)—belongs to a family of several hundred members, each recognizing a few specific substrates. This is followed by repeated addition of ubiquitin moieties to the previously conjugated one to generate a ubiquitin chain that serves as a recognition element for the proteasome, which then degrades the substrate. Ubiquitin is recycled via the activity of deubiquitinating enzymes (DUBs). It stands to reason that efficiency of such a complex process would depend on colocalization of the different components in an assembly that allows the reactions to be carried out sequentially and processively. Here we describe nuclear condensates that are dynamic in their composition. They contain p62 as an essential component. These assemblies are generated by liquid–liquid phase separation (LLPS) and also contain ubiquitinated targets, 26S proteasome, the three conjugating enzymes, and DUBs. Under basal conditions, they serve as efficient centers for proteolysis of nuclear proteins (e.g., c-Myc) and unassembled subunits of the proteasome, suggesting they are involved in cellular protein quality control. Supporting this notion is the finding that such foci are also involved in degradation of misfolded proteins induced by heat and oxidative stresses, following recruitment of heat shock proteins and their associated ubiquitin ligase CHIP.

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Abstract 486: Down-regulation of type 1 cGMP-dependent Protein Kinase by the ubiquitin/proteasome pathway

Circulation ◽

10.1161/circ.116.suppl_16.ii_83-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

NUPUR DEY ◽

Jennifer L Busch ◽

Sharron H Francis ◽

Jackie D Corbin ◽

Thomas M Lincoln

Keyword(s):

Protein Kinase ◽

26S Proteasome ◽

Down Regulation ◽

Dependent Protein Kinase ◽

Ubiquitin Proteasome Pathway ◽

Cgmp Dependent Protein Kinase ◽

Ubiquitin Proteasome ◽

Dependent Protein ◽

Proteasome Pathway

Type 1 cGMP-dependent protein kinase (PKG-I) is a widely expressed serine/threonine protein kinase, and is a major mediator of nitric oxide (NO) signaling in vascular smooth muscle cells (VSMC). PKG-I level is highly variable in VSMC and several studies have shown that atherogenic inflammatory cytokines lower the steady-statel levels of PKG-I. The mechanism of action of down-regulation is not well defined, but induction of type II NO synthase (iNOS) and subsequent persistent elevation of cGMP appear to contribute to PKG-I down regulation. In the present study, we examined the role of the ubiquitin/proteasome pathway in PKG-Iα down-regulation in response to elevated cGMP. Incubation of cultured VSMC with 8-Br-cGMP for 6–12 hr lowered PKG-I expression as assessed by western blotting. To further examine the mechanism, Cos7 cells, which do not express PKG-I mRNA or protein, were transfected with PKG-Iα/pcDNA vector and incubated with 8-Br-cGMP. 8-Br-cGMP suppressed PKG-Iα protein level in Cos7 cells (half-maximal concentration = 250 μM). Pretreatment of these cells with the proteasome inhibitor, MG132, followed by 8-Br-cGMP treatment prevented the decline suggesting the involvement of the ubiquitin/26S proteasome pathway. Immunoprecipitation of PKG-I followed by immunoblotting with anti-ubiquitin revealed multiple ubiquitinated PKG bands in the 8-Br-cGMP treated samples but not in untreated samples. Ubiquitination and down-regulation were also inhibited by the specific PKG-I catalytic inhibitor DT-2, suggesting the possible involvement of PKG autophosphorylation in the 8-Br-cGMP induced down-regulation. Mutation of the PKG-Iα autophosphorylation sites to alanines was performed to identify the phosphorylated site responsible for cGMP-dependent ubiquitination. In contrast to wild type PKG-Iα, PKG-Iα S64A, but not the S50A mutant, was not down-regulated by 8-Br-cGMP suggesting that autophosphorylation of serine-64 is required for the ubiquitination and down-regulation of PKG-I. Autophosphorylation and cGMP-mediated down-regulation of PKG-I may be an important mechanism to control excess cGMP signaling in VSMC.

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Mitochondrial quality control by the ubiquitin–proteasome system

Biochemical Society Transactions ◽

10.1042/bst0391509 ◽

2011 ◽

Vol 39 (5) ◽

pp. 1509-1513 ◽

Cited By ~ 124

Author(s):

Eric B. Taylor ◽

Jared Rutter

Keyword(s):

Quality Control ◽

Ubiquitin Ligase ◽

Protein Quality ◽

Mitochondrial Protein ◽

Ubiquitin Proteasome System ◽

Degradation Pathway ◽

Outer Mitochondrial Membrane ◽

Mitochondrial Quality Control ◽

Ubiquitinated Proteins ◽

Ubiquitin Proteasome

Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondria-associated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin–proteasome system) with the OMM. In yeast, Vms1 [VCP (valosin-containing protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability.

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A role of Arabidopsis COP9 signalosome in multifaceted developmental processes revealed by the characterization of its subunit 3

Development ◽

10.1242/dev.128.21.4277 ◽

2001 ◽

Vol 128 (21) ◽

pp. 4277-4288 ◽

Cited By ~ 1

Author(s):

Zhaohua Peng ◽

Giovanna Serino ◽

Xing-Wang Deng

Keyword(s):

Protein Degradation ◽

26S Proteasome ◽

Cop9 Signalosome ◽

Developmental Processes ◽

Developmental Defects ◽

E3 Ligases ◽

Ubiquitinated Proteins ◽

Transgenic Lines ◽

Ubiquitin Proteasome

The COP9 signalosome is a highly conserved eight-subunit protein complex initially defined as a repressor of photomorphogenic development in Arabidopsis. It has recently been suggested that the COP9 signalosome directly interacts and regulates SCF type E3 ligases, implying a key role in ubiquitin-proteasome mediated protein degradation. We report that Arabidopsis FUS11 gene encodes the subunit 3 of the COP9 signalosome (CSN3). The fus11 mutant is defective in the COP9 signalosome and accumulates significant amount of multi-ubiquitinated proteins. The same mutant is specifically impaired in the 26S proteasome-mediated degradation of HY5 but not PHYA, indicating a selective involvement in protein degradation. Reduction-of-function transgenic lines of CSN3 produced through gene co-suppression also accumulate multi-ubiquitinated proteins and exhibit diverse developmental defects. This result substantiates a hypothesis that the COP9 signalosome is involved in multifaceted developmental processes through regulating proteasome-mediated protein degradation.

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A SUMO-Ubiquitin Relay Recruits Proteasomes to Chromosome Axes to Regulate Meiotic Recombination

10.1101/095711 ◽

2016 ◽

Cited By ~ 1

Author(s):

H.B.D. Prasada Rao ◽

Huanyu Qiao ◽

Shubhang K. Bhatt ◽

Logan R.J. Bailey ◽

Hung D. Tran ◽

...

Keyword(s):

Meiotic Recombination ◽

Meiotic Prophase ◽

Meiotic Chromosome ◽

Ubiquitin Proteasome System ◽

Protein Stabilization ◽

E3 Ligases ◽

Sumo Modification ◽

Sumo Conjugation ◽

Ubiquitin Proteasome ◽

Dependent Protein

AbstractMeiosis produces haploid gametes through a succession of chromosomal events including pairing, synapsis and recombination. Mechanisms that orchestrate these events remain poorly understood. We found that the SUMO-modification and ubiquitin-proteasomes systems regulate the major events of meiotic prophase in mouse. Interdependent localization of SUMO, ubiquitin and proteasomes along chromosome axes was mediated largely by RNF212 and HEI10, two E3 ligases that are also essential for crossover recombination. RNF212-dependent SUMO conjugation effected a checkpoint-like process that stalls recombination by rendering the turnover of a subset of recombination factors dependent on HEI10-mediated ubiquitylation. We propose that SUMO conjugation establishes a precondition for designating crossover sites via selective protein stabilization. Thus, meiotic chromosome axes are hubs for regulated proteolysis via SUMO-dependent control of the ubiquitin-proteasome system.One Sentence SummaryChromosomal events of meiotic prophase in mouse are regulated by proteasome-dependent protein degradation.

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Regulation of Subtilase Cytotoxin-Induced Cell Death by an RNA-Dependent Protein Kinase-Like Endoplasmic Reticulum Kinase-Dependent Proteasome Pathway in HeLa Cells

Infection and Immunity ◽

10.1128/iai.06164-11 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1803-1814 ◽

Cited By ~ 13

Author(s):

Kinnosuke Yahiro ◽

Hiroyasu Tsutsuki ◽

Kohei Ogura ◽

Sayaka Nagasawa ◽

Joel Moss ◽

...

Keyword(s):

Er Stress ◽

Hela Cells ◽

Ubiquitin Proteasome System ◽

Early Event ◽

Caspase Activation ◽

Ubiquitinated Proteins ◽

Ubiquitin Proteasome ◽

Dependent Protein ◽

Activating Transcription Factor ◽

Subtilase Cytotoxin

ABSTRACTShiga-toxigenicEscherichia coli(STEC) produces subtilase cytotoxin (SubAB), which cleaves the molecular chaperone BiP in the endoplasmic reticulum (ER), leading to an ER stress response and then activation of apoptotic signaling pathways. Here, we show that an early event in SubAB-induced apoptosis in HeLa cells is mediated by RNA-dependent protein kinase (PKR)-like ER kinase (PERK), not activating transcription factor 6 (ATF6) or inositol-requiring enzyme 1(Ire1), two other ER stress sensors. PERK knockdown suppressed SubAB-induced eIF2α phosphorylation, activating transcription factor 4 (ATF4) expression, caspase activation, and cytotoxicity. Knockdown of eIF2α by small interfering RNA (siRNA) or inhibition of eIF2α dephosphorylation by Sal003 enhanced SubAB-induced caspase activation. Treatment with proteasome inhibitors (i.e., MG132 and lactacystin), but not a general caspase inhibitor (Z-VAD) or a lysosome inhibitor (chloroquine), suppressed SubAB-induced caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that the ubiquitin-proteasome system controls events leading to caspase activation, i.e., Bax/Bak conformational changes, followed by cytochromecrelease from mitochondria. Levels of ubiquitinated proteins in HeLa cells were significantly decreased by SubAB treatment. Further, in an early event, some antiapoptotic proteins, which normally turn over rapidly, have their synthesis inhibited, and show enhanced degradation via the proteasome, resulting in apoptosis. In PERK knockdown cells, SubAB-induced loss of ubiquitinated proteins was inhibited. Thus, SubAB-induced ER stress is caused by BiP cleavage, leading to PERK activation, not by accumulation of ubiquitinated proteins, which undergo PERK-dependent degradation via the ubiquitin-proteasome system.

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Unstructured Biology of Proteins from Ubiquitin-Proteasome System: Roles in Cancer and Neurodegenerative Diseases

Biomolecules ◽

10.3390/biom10050796 ◽

2020 ◽

Vol 10 (5) ◽

pp. 796 ◽

Cited By ~ 2

Author(s):

Kundlik Gadhave ◽

Prateek Kumar ◽

Shivani Kapuganti ◽

Vladimir Uversky ◽

Rajanish Giri

Keyword(s):

Intrinsically Disordered Protein ◽

Intrinsic Disorder ◽

Ubiquitin Proteasome System ◽

Cellular Protein ◽

26S Proteasome ◽

Deubiquitinating Enzymes ◽

Intrinsically Disordered ◽

Anti Cancer ◽

Ubiquitin Proteasome ◽

Dependent Protein

The 26S proteasome is a large (~2.5 MDa) protein complex consisting of at least 33 different subunits and many other components, which form the ubiquitin proteasomal system (UPS), an ATP-dependent protein degradation system in the cell. UPS serves as an essential component of the cellular protein surveillance machinery, and its dysfunction leads to cancer, neurodegenerative and immunological disorders. Importantly, the functions and regulations of proteins are governed by the combination of ordered regions, intrinsically disordered protein regions (IDPRs) and molecular recognition features (MoRFs). The structure–function relationships of UPS components have not been identified completely; therefore, in this study, we have carried out the functional intrinsic disorder and MoRF analysis for potential neurodegenerative disease and anti-cancer targets of this pathway. Our report represents the presence of significant intrinsic disorder and disorder-based binding regions in several UPS proteins, such as extraproteasomal polyubiquitin receptors (UBQLN1 and UBQLN2), proteasome-associated polyubiquitin receptors (ADRM1 and PSMD4), deubiquitinating enzymes (DUBs) (ATXN3 and USP14), and ubiquitinating enzymes (E2 (UBE2R2) and E3 (STUB1) enzyme). We believe this study will have implications for the conformation-specific roles of different regions of these proteins. This will lead to a better understanding of the molecular basis of UPS-associated diseases.

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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 ◽

10.1073/pnas.1522332112 ◽

2015 ◽

Vol 112 (52) ◽

pp. E7176-E7185 ◽

Cited By ~ 114

Author(s):

Sudarsanareddy Lokireddy ◽

Nikolay Vadimovich Kukushkin ◽

Alfred Lewis Goldberg

Keyword(s):

Protein Degradation ◽

Intracellular Camp ◽

Small Peptides ◽

Phosphodiesterase 4 ◽

Ubiquitinated Proteins ◽

Fused In Sarcoma ◽

Mammalian Cell Lines ◽

Ubiquitin Proteasome ◽

Dependent Protein ◽

Proteasome Pathway

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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