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1.
Cell ; 178(4): 949-963.e18, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31353221

RESUMO

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.


Assuntos
Neoplasias da Mama/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Cinamatos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Receptor de Estrogênio/uso terapêutico , Feminino , Fulvestranto/uso terapêutico , Células HEK293 , Xenoenxertos , Humanos , Indazóis/farmacologia , Ligantes , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Polimorfismo de Nucleotídeo Único , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
2.
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
3.
Nat Chem Biol ; 19(1): 55-63, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36577875

RESUMO

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.


Assuntos
Proteínas Nucleares , Fatores de Transcrição , Proteínas Nucleares/metabolismo , Microscopia Crioeletrônica , Fatores de Transcrição/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ligases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
4.
J Am Chem Soc ; 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38848252

RESUMO

ß-catenin (CTNNB1) is an oncogenic transcription factor that is important in cell-cell adhesion and transcription of cell proliferation and survival genes that drive the pathogenesis of many different types of cancers. However, direct pharmacological targeting of CTNNB1 has remained challenging. Here, we have performed a screen with a library of cysteine-reactive covalent ligands to identify the monovalent degrader EN83 that depletes CTNNB1 in a ubiquitin-proteasome-dependent manner. We show that EN83 directly and covalently targets CTNNB1 three cysteines C466, C520, and C619, leading to destabilization and degradation of CTNNB1. Through structural optimization, we generate a highly potent and relatively selective destabilizing degrader that acts through the targeting of only C619 on CTNNB1. Our results show that chemoproteomic approaches can be used to covalently target and degrade challenging transcription factors like CTNNB1 through destabilization-mediated degradation.

5.
Mol Cell ; 63(1): 4-6, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27392144

RESUMO

Abdul Rehman and colleagues identify a sixth family of deubiquitinase enzymes that are highly conserved throughout eukaryotes and show a remarkable selectivity for cleaving extended Lys-48-linked polyubiquitin chains.


Assuntos
Eucariotos , Poliubiquitina , Enzimas Desubiquitinantes , Humanos
6.
Am J Hematol ; 98(3): 449-463, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36594167

RESUMO

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.


Assuntos
Imunoconjugados , Linfoma não Hodgkin , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/patologia , Rituximab/uso terapêutico , Imunoconjugados/uso terapêutico
7.
Nat Rev Mol Cell Biol ; 12(7): 439-52, 2011 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-21697901

RESUMO

The proper regulation of apoptosis is essential for the survival of multicellular organisms. Furthermore, excessive apoptosis can contribute to neurodegenerative diseases, anaemia and graft rejection, and diminished apoptosis can lead to autoimmune diseases and cancer. It has become clear that the post-translational modification of apoptotic proteins by ubiquitylation regulates key components in cell death signalling cascades. For example, ubiquitin E3 ligases, such as MDM2 (which ubiquitylates p53) and inhibitor of apoptosis (IAP) proteins, and deubiquitinases, such as A20 and ubiquitin-specific protease 9X (USP9X) (which regulate the ubiquitylation and degradation of receptor-interacting protein 1 (RIP1) and myeloid leukaemia cell differentiation 1 (MCL1), respectively), have important roles in apoptosis. Therapeutic agents that target apoptotic regulatory proteins, including those that are part of the ubiquitin-proteasome system, might afford clinical benefits.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Ubiquitinação
8.
Cell ; 134(4): 668-78, 2008 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-18724939

RESUMO

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.


Assuntos
Anticorpos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo , Animais , Linhagem Celular , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Proteínas Formadoras de Poros Nucleares/química , Biblioteca de Peptídeos , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiae , Schizosaccharomyces , Ubiquitina/química , Ubiquitinação
9.
Nature ; 550(7677): 534-538, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29045385

RESUMO

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.


Assuntos
Aminopiridinas/química , Aminopiridinas/farmacologia , Indazóis/química , Indazóis/farmacologia , Fenóis/química , Fenóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Ubiquitina/metabolismo , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Especificidade por Substrato , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/deficiência , Peptidase 7 Específica de Ubiquitina/metabolismo
10.
J Am Chem Soc ; 143(28): 10571-10575, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34236858

RESUMO

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.


Assuntos
Aminas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Aminas/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
11.
Nature ; 528(7582): 370-5, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26649818

RESUMO

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.


Assuntos
Cisteína Endopeptidases/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Animais , Morte Celular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Feminino , Inflamação/genética , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Poliubiquitina/química , Poliubiquitina/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
12.
Bioorg Med Chem Lett ; 30(4): 126907, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31902710

RESUMO

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.


Assuntos
Anticorpos Monoclonais/imunologia , Portadores de Fármacos/química , Receptor alfa de Estrogênio/imunologia , Anticorpos Monoclonais/química , Antineoplásicos/química , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Receptor alfa de Estrogênio/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Células MCF-7 , Proteólise/efeitos dos fármacos , Receptor ErbB-2/metabolismo
13.
Drug Discov Today Technol ; 31: 109-123, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31200854

RESUMO

The Ubiquitin/Proteasome System comprises an essential cellular mechanism for regulated protein degradation. Ubiquitination may also promote the assembly of protein complexes that initiate intracellular signaling cascades. Thus, proper regulation of substrate protein ubiquitination is essential for maintaining normal cellular physiology. Deubiquitinases are the class of enzymes responsible for removing ubiquitin modifications from target proteins and have been implicated in regulating human disease. As such, deubiquitinases are now recognized as emerging drug targets. Small molecule deubiquitinase inhibitors have been developed; among those, inhibitors for the deubiquitinases USP7 and USP14 are the best-characterized given that they are structurally validated. In this review we discuss the normal physiological roles of the USP7 and USP14 deubiquitinases as well as the pathological conditions associated with their dysfunction, with a focus on oncology and neurodegenerative diseases. We also review structural biology of USP7 and USP14 enzymes and the characterization of their respective inhibitors, highlighting the various molecular mechanisms by which these deubiquitinases may be functionally inhibited. Finally, we summarize the cellular and in vivo studies performed using the structurally-validated USP7 and USP14 inhibitors.


Assuntos
Pirróis/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Animais , Humanos , Estrutura Molecular , Pirróis/química , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo
15.
Chemistry ; 24(19): 4830-4834, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29493023

RESUMO

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.


Assuntos
Indóis/química , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Humanos , Imunoconjugados/administração & dosagem , Células MCF-7 , Camundongos
16.
Mol Cell ; 40(4): 548-57, 2010 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21095585

RESUMO

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger 4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.


Assuntos
NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Dedos de Zinco , Sítios de Ligação , Cristalografia por Raios X , Lisina/metabolismo , Modelos Moleculares , Mutação/genética , Proteínas Nucleares/química , Poliubiquitina/metabolismo , Ligação Proteica , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo
17.
Nature ; 474(7351): 403-6, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21572435

RESUMO

The proto-oncogenes ETV1, ETV4 and ETV5 encode transcription factors in the E26 transformation-specific (ETS) family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COP1 (also known as RFWD2) as a tumour suppressor that negatively regulates ETV1, ETV4 and ETV5. ETV1, which is mutated in prostate cancer more often, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs and was 50-fold more stable than wild-type ETV1. Almost all patient translocations render ETV1 insensitive to COP1, implying that this confers a selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. Combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein, and elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a tumour suppressor whose downregulation promotes prostatic epithelial cell proliferation and tumorigenesis.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Camundongos , Proteínas Nucleares/deficiência , PTEN Fosfo-Hidrolase/deficiência , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
18.
Nature ; 471(7336): 110-4, 2011 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-21368834

RESUMO

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Fibroblastos , Humanos , Camundongos , Mitose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Paclitaxel/farmacologia , Farmacogenética , Fosforilação/efeitos dos fármacos , Poliploidia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Vincristina/farmacologia
20.
Biochem J ; 465(1): 1-26, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25631680

RESUMO

The post-translational modification of proteins with ubiquitin represents a complex signalling system that co-ordinates essential cellular functions, including proteolysis, DNA repair, receptor signalling and cell communication. DUBs (deubiquitinases), the enzymes that disassemble ubiquitin chains and remove ubiquitin from proteins, are central to this system. Reflecting the complexity and versatility of ubiquitin signalling, DUB activity is controlled in multiple ways. Although several lines of evidence indicate that aberrant DUB function may promote human disease, the underlying molecular mechanisms are often unclear. Notwithstanding, considerable interest in DUBs as potential drug targets has emerged over the past years. The future success of DUB-based therapy development will require connecting the basic science of DUB function and enzymology with drug discovery. In the present review, we discuss new insights into DUB activity regulation and their links to disease, focusing on the role of DUBs as regulators of cell identity and differentiation, and discuss their potential as emerging drug targets.


Assuntos
Células/enzimologia , Células/patologia , Doença , Proteases Específicas de Ubiquitina/metabolismo , Animais , Humanos , Especificidade por Substrato
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