RESUMO
During cellular stress, monoubiquitin is in demand due to the accumulation of misfolded proteins that require proteasomal degradation. Kimura et al. (2009) now show in yeast that monoubiquitin levels are bolstered during stress conditions by downregulation of the protein Rfu1, an inhibitor of the deubiquitinating enzyme Doa4.
Assuntos
Endopeptidases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Alostérica , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Humanos , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina Tiolesterase , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), localizing the enzyme to stalled replication forks (RFs) to promote their restart. Although phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (â¼15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial dynamics in the C-terminal domain (which contains T592), which could be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , DNA de Cadeia Simples , Guanosina Trifosfato/metabolismo , Cinética , Proteínas Monoméricas de Ligação ao GTP/genética , Fosforilação , Proteína 1 com Domínio SAM e Domínio HD/químicaRESUMO
The AAA+ ATPase p97 regulates ubiquitin-dependent protein homeostasis and has been pursued as a cancer drug target. The ATP-competitive inhibitor CB-5083 and allosteric inhibitor NMS-873 are the most advanced p97 inhibitors described to date. Previous studies have reported that their cytotoxicity can be readily overcome and involves single p97 mutations in the linker between the D1 and D2 ATPase domains and within D2. We report here that the proline 472 to leucine (P472L) mutation, in the D1-D2 linker and identified in CB-5083-resistant cells, desensitizes p97 to both inhibitor classes. This mutation does not disrupt the distinct D2-binding sites of the inhibitors. Instead, P472L changes ATPase domain communication within the p97 hexamer. P472L enhances cooperative D2 ATP binding and hydrolysis. This mechanism alters the function of the D1-D2 linker in the control of D2 activity involving the ATP-bound state of D1. Although increased D2 activity is sufficient to desensitize the P472L mutant to NMS-873, the mutant's desensitization to CB-5083 also requires D1 ATPase domain function. Our study highlights the remarkable adaptability of p97 ATPase domain communication that enables escape from mechanistically distinct classes of cytotoxic p97 inhibitors.
Assuntos
Adenosina Trifosfatases , Indóis/farmacologia , Mutação de Sentido Incorreto , Pirimidinas/farmacologia , Proteína com Valosina , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Substituição de Aminoácidos , Células HCT116 , Humanos , Domínios Proteicos , Proteína com Valosina/antagonistas & inibidores , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein's exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1's structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin-RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control.
Assuntos
Microscopia Eletrônica/métodos , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Proteínas Culina/química , Proteínas Culina/metabolismo , Proteínas Culina/ultraestrutura , Humanos , Imageamento Tridimensional , Modelos Moleculares , Tamanho da Partícula , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina/ultraestrutura , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The ATPase associated with various cellular activities p97 has a critical function in the cytoplasmic degradation of proteins misfolded in the ER (endoplasmic reticulum) through a mechanism known as ERAD (ER-associated degradation). During this process, p97 binds polyubiquitinated ERAD substrates and couples ATP hydrolysis to their dislocation from the ER as a prerequisite to destruction by the proteasome. The ubiquitin signals important for this process are not fully understood. In the present paper we report that p97 interacts with Lys11- and Lys48-linked ubiquitin polymers, but not those containing Lys63 linkages. Disruption of p97 through siRNA-mediated depletion, dominant-negative overexpression or chemical inhibition results in the accumulation of Lys11 and Lys48 ubiquitin chains predominantly at the ER membrane, and is associated with ER stress induction. We show that a catalytically inactive deubiquitinating enzyme and p97 cofactor YOD1 enhances the accumulation of Lys11- and Lys48-linked polyubiquitin in the cytoplasm, at the ER membrane and bound to p97. In addition to general effects on p97-associated ubiquitin polymers, the ERAD substrate CD3δ is modified with both Lys11 and Lys48 ubiquitin chains prior to p97-dependent dislocation. Collectively, the results of the present study are consistent with a major role for p97 in the recognition of Lys11 and Lys48 polyubiquitinated proteins before their degradation by the proteasome.
Assuntos
Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Insetos , Ligação Proteica/fisiologiaRESUMO
The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Culina/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Proteínas de Bactérias/genética , Complexo do Signalossomo COP9 , Proteínas Culina/genética , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Glutamina/genética , Glutamina/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteína NEDD8 , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/genéticaRESUMO
More than half of the ~20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.
RESUMO
Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Endorribonucleases/metabolismo , Células HEK293 , Células HeLa , Humanos , Técnicas In Vitro , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transfecção , Ubiquitinação/fisiologiaRESUMO
The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia-inducible factor-1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post-translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin-mediated degradation mediated by the CUL4A-DDB1-ROC1-beta-TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3beta. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.
Assuntos
Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases/metabolismo , Fatores de Transcrição/fisiologia , Proteínas de Transporte/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Immunoblotting , Fosforilação , Estabilidade Proteica , Inibidores da Síntese de Proteínas/farmacologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
The ubiquitin system of protein modification has emerged as a crucial mechanism involved in the regulation of a wide array of cellular processes. As our knowledge of the pathways in this system has grown, so have the ties between the protein ubiquitin and human disease. The power of the ubiquitin system for therapeutic benefit blossomed with the approval of the proteasome inhibitor Velcade in 2003 by the FDA. Current drug discovery activities in the ubiquitin system seek to (i) expand the development of new proteasome inhibitors with distinct mechanisms of action and improved bioavailability, and (ii) validate new targets. This review summarizes our current understanding of the role of the ubiquitin system in various human diseases ranging from cancer, viral infection and neurodegenerative disorders to muscle wasting, diabetes and inflammation. I provide an introduction to the ubiquitin system, highlight some emerging relationships between the ubiquitin system and disease, and discuss current and future efforts to harness aspects of this potentially powerful system for improving human health. PUBLICATION HISTORY : Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).
Assuntos
Inibidores de Proteassoma , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina/metabolismo , Animais , Inibidores de Cisteína Proteinase/uso terapêutico , Descoberta de Drogas , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Doenças Musculoesqueléticas/tratamento farmacológico , Doenças Musculoesqueléticas/metabolismo , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The AAA ATPase p97/VCP regulates protein homeostasis using a diverse repertoire of cofactors to fulfill its biological functions. Here we use the allosteric p97 inhibitor NMS-873 to analyze its effects on enzyme composition and the ability of cells to adapt to its cytotoxicity. We found that p97 inhibition changes steady state cofactor-p97 composition, leading to the enrichment of a subset of its cofactors and polyubiquitin bound to p97. We isolated cells specifically insensitive to NMS-873 and identified a new mutation (A530T) in p97. A530T is sufficient to overcome the cytotoxicity of NMS-873 and alleviates p97 composition changes caused by the molecule but not other p97 inhibitors. This mutation does not affect NMS-873 binding but increases p97 catalytic efficiency through altered ATP and ADP binding. Collectively, these findings identify cofactor-p97 interactions sensitive to p97 inhibition and reveal a new on-target mechanism to suppress the cytotoxicity of NMS-873.
Assuntos
Acetanilidas/farmacologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Benzotiazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Acetanilidas/química , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Regulação Alostérica/efeitos dos fármacos , Benzotiazóis/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Células HCT116 , Humanos , Modelos Moleculares , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
The development of in vitro systems to monitor ubiquitin ligase activity with highly purified proteins has allowed for new insights into the mechanisms of protein ubiquitination to be uncovered. This chapter describes the methodologies employed to reconstitute ubiquitination of the budding yeast cyclin-dependent kinase inhibitor Sic1 by the evolutionarily conserved ubiquitin ligase SCF(Cdc4) and its ubiquitin-conjugating enzyme Cdc34. Based on our experience in reconstituting Sic1 ubiquitination, we suggest some parameters to consider that should be generally applicable to the study of different SCF complexes and other ubiquitin ligases.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas Inibidoras de Quinase Dependente de Ciclina , Escherichia coli/genética , Proteínas F-Box/isolamento & purificação , Fosforilação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina , Complexos Ubiquitina-Proteína Ligase/biossíntese , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/isolamento & purificação , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/isolamento & purificaçãoRESUMO
N(6)-methyladenosine (m(6)A) has been identified as the most abundant internal modification of messenger RNA in eukaryotes. m(6)A modification is involved in cell fate determination in yeast and embryo development in plants. Its mammalian function remains unknown but thousands of mammalian mRNAs and long non-coding RNAs (lncRNAs) show m(6)A modification and m(6)A demethylases are required for mammalian energy homeostasis and fertility. We identify two proteins, the putative m(6)A MTase, methyltransferase-like 3 (Mettl3; ref. ), and a related but uncharacterized protein Mettl14, that function synergistically to control m(6)A formation in mammalian cells. Knockdown of Mettl3 and Mettl14 in mouse embryonic stem cells (mESCs) led to similar phenotypes, characterized by lack of m(6)A RNA methylation and lost self-renewal capability. A large number of transcripts, including many encoding developmental regulators, exhibit m(6)A methylation inversely correlated with mRNA stability and gene expression. The human antigen R (HuR) and microRNA pathways were linked to these effects. This gene regulatory mechanism operating in mESCs through m(6)A methylation is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.
Assuntos
Adenosina/análogos & derivados , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Técnicas de Silenciamento de Genes , Humanos , Metiltransferases/química , Metiltransferases/genética , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562(MLN), R-U937(MLN)) were selected. R-K562(MLN) and R-U937(MLN) cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme's affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562(MLN) cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.
Assuntos
Antineoplásicos/farmacologia , Ciclopentanos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Leucemia/genética , Pirimidinas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Proteínas Culina/metabolismo , Ciclopentanos/química , Análise Mutacional de DNA , Inibidores Enzimáticos/química , Genótipo , Humanos , Células K562 , Leucemia/metabolismo , Modelos Moleculares , Proteína NEDD8 , Mutação Puntual , Ligação Proteica , Conformação Proteica , Pirimidinas/química , Relação Estrutura-Atividade , Células U937 , Enzimas Ativadoras de Ubiquitina/química , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
The combinatorial architecture of cullin 1-RING ubiquitin ligases, in which multiple F-box containing substrate receptors compete for access to CUL1, poses special challenges to assembling cullin 1-RING ubiquitin ligase complexes through high affinity protein interactions while maintaining the flexibility to dynamically sample the entire F-box containing substrate receptor repertoire. Here, using highly quantitative mass spectrometry, we demonstrate that this problem is addressed by CAND1, a factor that controls the dynamics of the global cullin 1-RING ubiquitin ligase network by promoting the assembly of newly synthesized F-box containing substrate receptors with CUL1-RBX1 core complexes. Our studies of in vivo cullin 1-RING ubiquitin ligase dynamics and in vitro biochemical findings showing that CAND1 can displace F-box containing substrate receptors from Cul1p suggest that CAND1 functions in a cycle that serves to exchange F-box containing substrate receptors on CUL1 cores. We propose that this cycle assures comprehensive sampling of the entire F-box containing substrate receptor repertoire in order to maintain the cullin 1-RING ubiquitin ligase landscape, a function that we show to be critical for substrate degradation and normal physiology.
Assuntos
Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
Inhibition of NEDD8-activating enzyme (NAE) has emerged as a highly promising approach to treat cancer through the adenosine sulfamate analog MLN4924. Here, we show that selective pressure results in HCT116 colorectal carcinoma cells with decreased MLN4924 sensitivity and identify a single-nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3. This reduces the enzyme's affinity for MLN4924 and ATP while increasing NEDD8 activation at physiological ATP concentrations. Expression of UBA3 A171T is sufficient to decrease MLN4924 sensitivity of naive HCT116 cells, indicating that it is a dominant suppressor of MLN4924-mediated cell death. Our data suggest that the on-target potency of MLN4924 selects for a point mutation in NAE that overcomes the molecule's inhibitory effects, allowing cancer cell survival.
Assuntos
Ciclopentanos/farmacologia , Pirimidinas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular Tumoral , Cromatografia Líquida , Proteínas Culina/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células HCT116 , Humanos , Dados de Sequência Molecular , Mutação Puntual , Alinhamento de Sequência , Espectrometria de Massas em Tandem , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/fisiologiaAssuntos
Ciclo Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas Inibidoras de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Cisteína Endopeptidases/metabolismo , Fase G1/fisiologia , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma , Fase S/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Brownell et al. (2010) elucidate the mechanism of action of MLN4924, a NEDD8-activating enzyme inhibitor. MLN4924 requires the activity of the enzyme to generate a NEDD8-adenylate analog that potently and selectively shuts down this posttranslational modification system.
RESUMO
Clearance of misfolded proteins by endoplasmic reticulum (ER)-associated degradation (ERAD) requires concerted activity of chaperones, adaptor proteins, ubiquitin ligases, and proteasomes. RNF5 is a ubiquitin ligase anchored to the ER membrane implicated in ERAD via ubiquitination of misfolded proteins. Among RNF5-associated proteins is JNK-associated membrane protein (JAMP), a 7-transmembrane protein located within the ER membrane that facilitates degradation of misfolded proteins through recruitment of proteasomes and ERAD regulatory components. Here we demonstrate that RNF5 associates with JAMP in the ER membrane. This association results in Ubc13-dependent RNF5-mediated noncanonical ubiquitination of JAMP. This ubiquitination does not alter JAMP stability but rather inhibits its association with Rpt5 and p97. Consequently, clearance of misfolded proteins, such as CFTRDelta508 and T cell receptor alpha, is less efficient, resulting in their greater accumulation. Significantly, the RNF5 effect on JAMP is seen prior to and after ER stress response, thereby highlighting a novel mechanism to limit ERAD and proteasome assembly at the ER, to the actual ER stress response.