RESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , ADN de Cadena Simple , Guanosina Trifosfato/metabolismo , Cinética , Proteínas de Unión al GTP Monoméricas/genética , Fosforilación , Proteína 1 que Contiene Dominios SAM y HD/químicaRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas , Indoles/farmacología , Mutación Missense , Pirimidinas/farmacología , Proteína que Contiene Valosina , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Sustitución de Aminoácidos , Células HCT116 , Humanos , Dominios Proteicos , Proteína que Contiene Valosina/antagonistas & inhibidores , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismoRESUMEN
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.
Asunto(s)
Acetanilidas/farmacología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Benzotiazoles/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Acetanilidas/química , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Regulación Alostérica/efectos de los fármacos , Benzotiazoles/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Células HCT116 , Humanos , Modelos Moleculares , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Ciclopentanos/farmacología , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Leucemia/genética , Pirimidinas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Antineoplásicos/química , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Ciclopentanos/química , Análisis Mutacional de ADN , Inhibidores Enzimáticos/química , Genotipo , Humanos , Células K562 , Leucemia/metabolismo , Modelos Moleculares , Proteína NEDD8 , Mutación Puntual , Unión Proteica , Conformación Proteica , Pirimidinas/química , Relación Estructura-Actividad , Células U937 , Enzimas Activadoras de Ubiquitina/química , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
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.
Asunto(s)
Adenosina/análogos & derivados , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Adenosina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Células Madre Embrionarias/citología , Técnicas de Silenciamiento del Gen , Humanos , Metiltransferasas/química , Metiltransferasas/genética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , Insectos , Unión Proteica/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Cullin/metabolismo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Proteínas Bacterianas/genética , Complejo del Señalosoma COP9 , Proteínas Cullin/genética , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Glutamina/genética , Glutamina/metabolismo , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteína NEDD8 , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/genéticaRESUMEN
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.
Asunto(s)
Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteolisis , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Microscopía Electrónica/métodos , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/ultraestructura , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Proteínas Cullin/química , Proteínas Cullin/metabolismo , Proteínas Cullin/ultraestructura , Humanos , Imagenología Tridimensional , Modelos Moleculares , Tamaño de la Partícula , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina/ultraestructura , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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.
Asunto(s)
Ciclopentanos/farmacología , Pirimidinas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular Tumoral , Cromatografía Liquida , Proteínas Cullin/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Resistencia a Antineoplásicos/genética , Células HCT116 , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Alineación de Secuencia , Espectrometría de Masas en Tándem , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Retículo Endoplásmico/enzimología , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Endorribonucleasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Técnicas In Vitro , Proteínas de la Membrana/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transfección , Ubiquitinación/fisiologíaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Quinasas/metabolismo , Factores de Transcripción/fisiología , Proteínas Portadoras/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular , Línea Celular Tumoral , Cicloheximida/farmacología , Proteínas de Unión al ADN/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Immunoblotting , Fosforilación , Estabilidad Proteica , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
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.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulación Alostérica , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Humanos , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Pliegue de Proteína , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/fisiología , Proteínas Portadoras/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/genética , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Estrés Fisiológico/fisiología , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína LigasasRESUMEN
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).
Asunto(s)
Inhibidores de Proteasoma , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina/metabolismo , Animales , Inhibidores de Cisteína Proteinasa/uso terapéutico , Descubrimiento de Drogas , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Enfermedades Musculoesqueléticas/tratamiento farmacológico , Enfermedades Musculoesqueléticas/metabolismo , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Protein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys(63) of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys(63)-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys(63)-linked ubiquitin chain synthesis mechanism.
Asunto(s)
Lisina/química , Factores de Transcripción/fisiología , Enzimas Ubiquitina-Conjugadoras/fisiología , Animales , Escherichia coli/metabolismo , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Especificidad por Sustrato , Factor 6 Asociado a Receptor de TNF/genética , Factores de Transcripción/química , Ubiquitina/química , Enzimas Ubiquitina-Conjugadoras/químicaRESUMEN
Release of ubiquitin-charged Cdc34 from the SCF ubiquitin ligase followed by diffusion-driven collision with substrate has been proposed to underlie ubiquitination of the canonical SCF substrate Sic1. Cdc34 F72V, reported to be defective in dissociation from SCF, served as key validation. Here, we test predictions of this "hit-and-run" hypothesis. We find that Cdc34 F72V is generally defective in SCF-mediated activation but, contrary to expectation, does not compete with wild-type Cdc34 in vitro or in vivo and can fulfill the physiological role of Cdc34 with only moderate delay in Sic1 turnover. Whereas a hit-and-run mechanism might explain how Cdc34 can transfer ubiquitin to the ends of growing ubiquitin chains on SCF-bound substrates, molecular modeling suggests that an E2 docked to SCF can do so without dissociating. We propose that interactions between Cdc34 approximately Ub and SCF directly activate ubiquitin transfer within a substrate-SCF-Cdc34 approximately Ub ternary complex.