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1.
Cell ; 171(6): 1326-1339.e14, 2017 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-29103612

RESUMO

SCF (Skp1-Cullin-F-box) ubiquitin ligases comprise several dozen modular enzymes that have diverse roles in biological regulation. SCF enzymes share a common catalytic core containing Cul1⋅Rbx1, which is directed toward different substrates by a variable substrate receptor (SR) module comprising 1 of 69 F-box proteins bound to Skp1. Despite the broad cellular impact of SCF enzymes, important questions remain about the architecture and regulation of the SCF repertoire, including whether SRs compete for Cul1 and, if so, how this competition is managed. Here, we devise methods that preserve the in vivo assemblages of SCF complexes and apply quantitative mass spectrometry to perform a census of these complexes (the "SCFome") in various states. We show that Nedd8 conjugation and the SR exchange factor Cand1 have a profound effect on shaping the SCFome. Together, these factors enable rapid remodeling of SCF complexes to promote biased assembly of SR modules bound to substrate.


Assuntos
Proteínas Ligases SKP Culina F-Box/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Cromatografia de Afinidade , Proteínas Culina/metabolismo , Humanos , Espectrometria de Massas , Proteína NEDD8/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo
2.
Cell ; 153(1): 206-15, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23453757

RESUMO

The modular SCF (Skp1, cullin, and F box) ubiquitin ligases feature a large family of F box protein substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCF(Fbxw7) is extraordinarily stable, but, in the Nedd8-deconjugated state, the cullin-binding protein Cand1 augments its dissociation by one-million-fold. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1-Rbx1 equilibrates with multiple F box protein-Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.


Assuntos
Proteínas Ligases SKP Culina F-Box/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Proteínas Culina/metabolismo , Escherichia coli/genética , Proteínas F-Box/metabolismo , Humanos , Espectrometria de Massas , Proteínas Ligases SKP Culina F-Box/química
3.
Mol Cell ; 77(3): 446-460, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004468

RESUMO

Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients.


Assuntos
Engenharia Genética/métodos , Engenharia Genética/tendências , Engenharia de Proteínas/métodos , Engenharia de Proteínas/tendências , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Humanos , Morfolinos/genética , Transporte Proteico , Proteólise
4.
Mol Cell ; 77(5): 1092-1106.e9, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31973889

RESUMO

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.


Assuntos
Cromatografia Líquida de Alta Pressão , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Células HEK293 , Humanos , Cinética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Mapas de Interação de Proteínas , Proteólise , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
5.
Proc Natl Acad Sci U S A ; 121(18): e2318619121, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38657050

RESUMO

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.


Assuntos
Aciltransferases , Complexo de Golgi , Gotículas Lipídicas , Fosfolipases A2 Independentes de Cálcio , Humanos , Aciltransferases/metabolismo , Complexo de Golgi/metabolismo , Lipase/metabolismo , Lipase/genética , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Fosfolipases A2 Independentes de Cálcio/metabolismo
6.
Nature ; 580(7803): 329-338, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296187

RESUMO

The modern biopharmaceutical industry traces its roots to the dawn of the twentieth century, coincident with marketing of aspirin-a signature event in the history of modern drug development. Although the archetypal discovery process did not change markedly in the first seven decades of the industry, the past fifty years have seen two successive waves of transformative innovation in the development of drug molecules: the rise of 'rational drug discovery' methodology in the 1970s, followed by the invention of recombinant protein-based therapeutic agents in the 1980s. An incipient fourth wave is the advent of multispecific drugs. The successful development of prospectively designed multispecific drugs has the potential to reconfigure our ideas of how target-based therapeutic molecules can work, and what it is possible to achieve with them. Here I review the two major classes of multispecific drugs: those that enrich a therapeutic agent at a particular site of action and those that link a therapeutic target to a biological effector. The latter class-being freed from the constraint of having to directly modulate the target upon binding-may enable access to components of the proteome that currently cannot be targeted by drugs.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Descoberta de Drogas , Animais , Produtos Biológicos/metabolismo , Indústria Farmacêutica , Humanos , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
7.
Mol Cell ; 69(5): 773-786.e6, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29499133

RESUMO

Skp1⋅Cul1⋅F-box (SCF) ubiquitin ligase assembly is regulated by the interplay of substrate binding, reversible Nedd8 conjugation on Cul1, and the F-box protein (FBP) exchange factors Cand1 and Cand2. Detailed investigations into SCF assembly and function in reconstituted systems and Cand1/2 knockout cells informed the development of a mathematical model for how dynamical assembly of SCF complexes is controlled and how this cycle is coupled to degradation of an SCF substrate. Simulations predicted an unanticipated hypersensitivity of Cand1/2-deficient cells to FBP expression levels, which was experimentally validated. Together, these and prior observations lead us to propose the adaptive exchange hypothesis, which posits that regulation of the koff of an FBP from SCF by the actions of substrate, Nedd8, and Cand1 molds the cellular repertoire of SCF complexes and that the plasticity afforded by this exchange mechanism may enable large variations in FBP expression during development and in FBP gene number during evolution.


Assuntos
Proteínas F-Box , Regulação da Expressão Gênica , Modelos Biológicos , Modelos Químicos , Proteólise , Fatores de Transcrição , Animais , Proteínas Culina/química , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas F-Box/biossíntese , Proteínas F-Box/química , Proteínas F-Box/genética , Camundongos , Proteína NEDD8/química , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Proc Natl Acad Sci U S A ; 119(36): e2205608119, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36037385

RESUMO

Cop9 signalosome (CSN) regulates the function of cullin-RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCFFBXO5-APC/C-GMNN and CUL4DTL-SETD8, that contribute substantially to the toxicity of CSN inhibition. Our data highlight the importance of CSN-mediated NEDD8 deconjugation and adaptive exchange of CRL substrate receptors in sustaining CRL function and suggest approaches for leveraging CSN inhibition for the treatment of cancer.


Assuntos
Replicação do DNA , Ubiquitina-Proteína Ligases , Azepinas/metabolismo , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Sobrevivência Celular , Proteínas Culina/genética , Proteínas Culina/metabolismo , Imidazóis/metabolismo , Proteína NEDD8/metabolismo , Pirazóis/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Annu Rev Biochem ; 78: 399-434, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19489725

RESUMO

E3 ligases confer specificity to ubiquitination by recognizing target substrates and mediating transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to substrate. The activity of most E3s is specified by a RING domain, which binds to an E2 approximately ubiquitin thioester and activates discharge of its ubiquitin cargo. E2-E3 complexes can either monoubiquitinate a substrate lysine or synthesize polyubiquitin chains assembled via different lysine residues of ubiquitin. These modifications can have diverse effects on the substrate, ranging from proteasome-dependent proteolysis to modulation of protein function, structure, assembly, and/or localization. Not surprisingly, RING E3-mediated ubiquitination can be regulated in a number of ways. RING-based E3s are specified by over 600 human genes, surpassing the 518 protein kinase genes. Accordingly, RING E3s have been linked to the control of many cellular processes and to multiple human diseases. Despite their critical importance, our knowledge of the physiological partners, biological functions, substrates, and mechanism of action for most RING E3s remains at a rudimentary stage.


Assuntos
Ubiquitina-Proteína Ligases/química , Animais , Genoma Humano , Humanos , Estrutura Terciária de Proteína , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
10.
Cell ; 139(5): 957-68, 2009 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-19945379

RESUMO

Degradation by the ubiquitin-proteasome system requires assembly of a polyubiquitin chain upon substrate. However, the structural and mechanistic features that enable template-independent processive chain synthesis are unknown. We show that chain assembly by ubiquitin ligase SCF and ubiquitin-conjugating enzyme Cdc34 is facilitated by the unusual nature of Cdc34-SCF transactions: Cdc34 binds SCF with nanomolar affinity, nevertheless the complex is extremely dynamic. These properties are enabled by rapid association driven by electrostatic interactions between the acidic tail of Cdc34 and a basic 'canyon' in the Cul1 subunit of SCF. Ab initio docking between Cdc34 and Cul1 predicts intimate contact between the tail and the basic canyon, an arrangement confirmed by crosslinking and kinetic analysis of mutants. Basic canyon residues are conserved in both Cul1 paralogs and orthologs, suggesting that the same mechanism underlies processivity for all cullin-RING ubiquitin ligases. We discuss different strategies by which processive ubiquitin chain synthesis may be achieved.


Assuntos
Proteínas Ligases SKP Culina F-Box/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Culina/química , Proteínas Culina/metabolismo , Humanos , Modelos Moleculares , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/química , Ubiquitinação , Leveduras/metabolismo
11.
Nature ; 557(7705): 446-451, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29632312

RESUMO

Ribosomal surveillance pathways scan for ribosomes that are transiently paused or terminally stalled owing to structural elements in mRNAs or nascent chain sequences1, 2. Some stalls in budding yeast are sensed by the GTPase Hbs1, which loads Dom34, a catalytically inactive member of the archaeo-eukaryotic release factor 1 superfamily. Hbs1-Dom34 and the ATPase Rli1 dissociate stalled ribosomes into 40S and 60S subunits. However, the 60S subunits retain the peptidyl-tRNA nascent chains, which recruit the ribosome quality control complex that consists of Rqc1-Rqc2-Ltn1-Cdc48-Ufd1-Npl4. Nascent chains ubiquitylated by the E3 ubiquitin ligase Ltn1 are extracted from the 60S subunit by the ATPase Cdc48-Ufd1-Npl4 and presented to the 26S proteasome for degradation3-9. Failure to degrade the nascent chains leads to protein aggregation and proteotoxic stress in yeast and neurodegeneration in mice10-14. Despite intensive investigations on the ribosome quality control pathway, it is not known how the tRNA is hydrolysed from the ubiquitylated nascent chain before its degradation. Here we show that the Cdc48 adaptor Vms1 is a peptidyl-tRNA hydrolase. Similar to classical eukaryotic release factor 1, Vms1 activity is dependent on a conserved catalytic glutamine. Evolutionary analysis indicates that yeast Vms1 is the founding member of a clade of eukaryotic release factor 1 homologues that we designate the Vms1-like release factor 1 clade.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Transporte/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Biocatálise , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Domínio Catalítico/genética , Glutamina/genética , Glutamina/metabolismo , Humanos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Mutação Puntual , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteína Estafilocócica A/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteína com Valosina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
12.
Mol Cell ; 61(6): 809-20, 2016 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-26990986

RESUMO

Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.


Assuntos
Glutamato-Amônia Ligase/metabolismo , Fatores Imunológicos/metabolismo , Peptídeo Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acetilação , Proteínas Adaptadoras de Transdução de Sinal , Glutamina/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Talidomida/metabolismo , Ubiquitinação
13.
Cell ; 134(5): 804-16, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18775313

RESUMO

p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Linhagem Celular , Humanos , Redes e Vias Metabólicas , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteoma , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina
14.
Nature ; 552(7684): 194-199, 2017 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-29211715

RESUMO

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.


Assuntos
Dissuasores de Álcool , Alcoolismo/tratamento farmacológico , Antineoplásicos , Dissulfiram/farmacologia , Dissulfiram/uso terapêutico , Reposicionamento de Medicamentos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/metabolismo , Adulto , Dissuasores de Álcool/farmacologia , Dissuasores de Álcool/uso terapêutico , Alcoolismo/epidemiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cobre/química , Dinamarca/epidemiologia , Dissulfiram/química , Feminino , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Proteínas Nucleares/química , Agregados Proteicos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos
15.
EMBO J ; 36(3): 260-273, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28007894

RESUMO

The F-box protein FBXW7 is the substrate-recruiting subunit of an SCF ubiquitin ligase and a major tumor-suppressor protein that is altered in several human malignancies. Loss of function of FBXW7 results in the stabilization of numerous proteins that orchestrate cell proliferation and survival. Little is known about proteins that directly regulate the function of this protein. In the current work, we have mapped the interactome of the enigmatic pseudophosphatase STYX We reasoned that a catalytically inactive phosphatase might have adopted novel mechanisms of action. The STYX interactome contained several F-box proteins, including FBXW7. We show that STYX binds to the F-box domain of FBXW7 and disables its recruitment into the SCF complex. Therefore, STYX acts as a direct inhibitor of FBXW7, affecting the cellular levels of its substrates. Furthermore, we find that levels of STYX and FBXW7 are anti-correlated in breast cancer patients, which affects disease prognosis. We propose the STYX-FBXW7 interaction as a promising drug target for future investigations.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas F-Box/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Proteína 7 com Repetições F-Box-WD , Humanos
16.
Nature ; 578(7795): 372-373, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32066917
17.
Adv Exp Med Biol ; 1217: 33-46, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31898220

RESUMO

Cullin-RING ubiquitin ligases (CRLs) determine the substrate specificity of ubiquitination reactions, and substrates are recruited to the cullin core through binding to their cognate substrate receptor modules. Because a family of substrate receptors compete for the same cullin core, the assembly and activity of CRLs are dynamically regulated to fulfill the needs of the cell to adapt to the changing pool of proteins demanding ubiquitination. Cullins are modified by NEDD8, a ubiquitin-like protein. This process, referred to as neddylation, promotes the E3 activity of CRLs by inducing conformational rearrangement in the Cullin-RING catalytic core. Cand1 is a cullin-associated protein whose binding is excluded by cullin neddylation. Although early biochemical studies suggested that Cand1 inhibits CRL activity, genetic studies revealed its positive role in ubiquitination. Emerging evidence from kinetic and quantitative proteomic studies demonstrated that Cand1 stimulates assembly of new Skp1-Cul1-F-box protein (SCF) complexes by exchanging the Skp1-F-box protein substrate receptor modules. Furthermore, aided by refined experimental design as well as computational simulation, an attractive model has been developed in which substrate, neddylation cycle and Cand1-mediated "adaptive exchange" collaborate to maintain the dynamics of the cellular SCF repertoire. Here, we review and discuss recent advances that have deepened our understanding of CRL regulation.


Assuntos
Proteínas Culina/química , Proteínas Culina/metabolismo , Animais , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Humanos , Proteômica , Especificidade por Substrato , Ubiquitina/metabolismo , Ubiquitinação
18.
Proc Natl Acad Sci U S A ; 114(22): E4380-E4388, 2017 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-28512218

RESUMO

p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate-Ub-GFP modified with K48-linked ubiquitin chains-for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.


Assuntos
Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/metabolismo , Proteínas Nucleares/metabolismo , Osteíte Deformante/genética , Osteíte Deformante/metabolismo , Proteínas/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Trifosfato de Adenosina/metabolismo , Demência Frontotemporal/etiologia , Humanos , Hidrólise , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Distrofia Muscular do Cíngulo dos Membros/etiologia , Mutação , Miosite de Corpos de Inclusão/etiologia , Osteíte Deformante/etiologia , Desdobramento de Proteína , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Ubiquitina/metabolismo , Proteína com Valosina/química
19.
Proc Natl Acad Sci U S A ; 114(14): 3565-3571, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28320958

RESUMO

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4CRBN and p97 pathways.


Assuntos
Glutamato-Amônia Ligase/metabolismo , Glutamina/farmacologia , Peptídeo Hidrolases/metabolismo , Proteólise , Proteína com Valosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Caseína Quinase I/metabolismo , Humanos , Fator de Transcrição Ikaros/metabolismo , Células MCF-7 , Fatores de Terminação de Peptídeos/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases , Ubiquitinação
20.
Nat Chem Biol ; 13(7): 709-714, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28459440

RESUMO

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.


Assuntos
Quelantes/farmacologia , Inibidores Enzimáticos/farmacologia , Metaloproteases/antagonistas & inibidores , Transativadores/antagonistas & inibidores , Zinco/química , Quelantes/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Células HeLa , Humanos , Metaloproteases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacologia , Relação Estrutura-Atividade , Transativadores/metabolismo
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