RESUMEN
Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.
Asunto(s)
Proteína Huntingtina , Lisosomas , Mitocondrias , Proteínas de Unión al ARN , Ubiquitina , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Lisosomas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Humanos , Ubiquitina/metabolismo , Mitocondrias/metabolismo , Autofagia , Animales , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Ratones , Unión Proteica , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Péptidos/metabolismoRESUMEN
A surprising complexity of ubiquitin signaling has emerged with identification of different ubiquitin chain topologies. However, mechanisms of how the diverse ubiquitin codes control biological processes remain poorly understood. Here, we use quantitative whole-proteome mass spectrometry to identify yeast proteins that are regulated by lysine 11 (K11)-linked ubiquitin chains. The entire Met4 pathway, which links cell proliferation with sulfur amino acid metabolism, was significantly affected by K11 chains and selected for mechanistic studies. Previously, we demonstrated that a K48-linked ubiquitin chain represses the transcription factor Met4. Here, we show that efficient Met4 activation requires a K11-linked topology. Mechanistically, our results propose that the K48 chain binds to a topology-selective tandem ubiquitin binding region in Met4 and competes with binding of the basal transcription machinery to the same region. The change to K11-enriched chain architecture releases this competition and permits binding of the basal transcription complex to activate transcription.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Activación Transcripcional , Ubiquitinación , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Sitios de Unión , Unión Competitiva , Cromatografía Liquida , Regulación Fúngica de la Expresión Génica , Lisina , Mutación , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-Actividad , Espectrometría de Masas en TándemRESUMEN
Deregulation of ubiquitin conjugation or deconjugation has been implicated in the pathogenesis of many human diseases including cancer. The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1), in association with UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been shown as a promising anticancer target. To further evaluate USP1/UAF1 as a therapeutic target, we conducted a quantitative high throughput screen of >400000 compounds and subsequent medicinal chemistry optimization of small molecules that inhibit the deubiquitinating activity of USP1/UAF1. Ultimately, these efforts led to the identification of ML323 (70) and related N-benzyl-2-phenylpyrimidin-4-amine derivatives, which possess nanomolar USP1/UAF1 inhibitory potency. Moreover, we demonstrate a strong correlation between compound IC50 values for USP1/UAF1 inhibition and activity in nonsmall cell lung cancer cells, specifically increased monoubiquitinated PCNA (Ub-PCNA) levels and decreased cell survival. Our results establish the druggability of the USP1/UAF1 deubiquitinase complex and its potential as a molecular target for anticancer therapies.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas de Arabidopsis/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Pirimidinas/síntesis química , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Pirimidinas/farmacología , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proteínas de Arabidopsis/antagonistas & inhibidores , Daño del ADN/fisiología , Proteínas Nucleares/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Ubiquitinación/efectos de los fármacos , Algoritmos , Butiratos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Ensayo de Unidades Formadoras de Colonias , Daño del ADN/genética , ADN de Neoplasias/antagonistas & inhibidores , ADN de Neoplasias/biosíntesis , Resistencia a Antineoplásicos , Electroforesis en Gel de Poliacrilamida , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Humanos , Indicadores y Reactivos , Compuestos de Fenilurea/farmacología , Pimozida/farmacología , Antígeno Nuclear de Célula en Proliferación/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Interferente Pequeño/genética , Proteínas Recombinantes/química , Recombinación Genética/efectos de los fármacos , Intercambio de Cromátides Hermanas/efectos de los fármacosRESUMEN
Ubiquitination has emerged as an essential signaling mechanism in eukaryotes. Deubiquitinases (DUBs) counteract the activities of the ubiquitination machinery and provide another level of control in cellular ubiquitination. Not surprisingly, DUBs are subjected to stringent regulations. Besides regulation by the noncatalytic domains present in the DUB sequences, DUB-interacting proteins are increasingly realized as essential regulators for DUB activity and function. This review focuses on DUBs that are associated with WD40-repeat proteins. Many human ubiquitin-specific proteases (USPs) were found to interact with WD40-repeat proteins, but little is known as to how this interaction regulates the activity and function of USPs. In recent years, significant progress has been made in understanding a prototypical WD40-repeat protein-containing DUB complex that comprises USP1 and USP1-associated factor 1 (UAF1). It has been shown that UAF1 activates USP1 through a potential active-site modulation, and the complex formation between USP1 and UAF1 is regulated by serine phosphorylation. Recently, human USPs have been recognized as a promising target class for inhibitor discovery. Small molecule inhibitors targeting several human USPs have been reported. USP1 is involved in two major DNA damage response pathways, DNA translesion synthesis and the Fanconi anemia pathway. Inhibiting the USP1/UAF1 deubiquitinase complex represents a new strategy to potentiate cancer cells to DNA-crosslinking agents and to overcome resistance that has plagued clinical cancer chemotherapy. The progress in inhibitor discovery against USPs and the WD40-repeat protein-containing USP complex will be discussed.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Proteínas de Arabidopsis/metabolismo , Biocatálisis , Reparación del ADN , Humanos , Proteínas Nucleares/metabolismo , Unión Proteica , Ubiquitina/metabolismo , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/clasificaciónRESUMEN
Deubiquitinating enzymes (DUBs) are important for the normal function of a number of cellular processes, including transcriptional regulation, cell cycle control, and DNA damage response. The enzymatic activity of DUB is regulated by different mechanisms. DUBs in several different families are post-translationally modified by phosphorylation. Large-scale phosphoproteomic studies of human DUBs revealed that a majority of ubiquitin-specific proteases (USPs) are phosphorylated. USP1 is a prototypical DUB that requires a specific interaction with a WD40-repeat protein, UAF1, for its catalytic activity. In this study, we show that Ser313 phosphorylation in USP1 is required for its interaction with UAF1 and for the stimulation of USP1's activity. In contrast, two other known USP1 serine phosphorylations (Ser42 and Ser67) are dispensable with respect to the activity of the USP1/UAF1 complex. An S313D phosphomimetic mutation in USP1 can substitute for Ser313 phosphorylation in promoting the formation of the USP1/UAF1 complex. We further demonstrated that CDK1 is responsible for Ser313 phosphorylation, and protein phosphatase treatment of USP1 can lead to inactivation of USP1/UAF1. An inserted domain in USP1 (amino acids 235-408) was found to interact with UAF1, and this interaction is mediated by Ser313 phosphorylation. Our findings revealed an intriguing mechanism of regulating USP1 activity that combines phosphorylation of a key serine residue in USP1 and the specific interaction of USP1 with a WD40-repeat protein UAF1. The pSer313-dependent formation of the USP1/UAF1 complex points to a new approach for inhibiting USP1 activity by disrupting the interaction between the UAF1's WD40-repeat domain and the Ser313-containing phosphopeptide in USP1.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Arabidopsis , Activación Enzimática , Humanos , Fosforilación , Serina/metabolismo , Proteasas Ubiquitina-EspecíficasRESUMEN
Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina/metabolismo , Proteínas de Arabidopsis , Línea Celular , Replicación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Endopeptidasas/metabolismo , Humanos , Estructura Terciaria de Proteína , Intercambio de Cromátides Hermanas , Ubiquitina-Proteína Ligasas/fisiología , Proteasas Ubiquitina-EspecíficasRESUMEN
Ubiquitin-specific proteases (USPs) constitute the largest family of the human deubiquitinating enzymes. USP1 belongs to the cysteine protease family and contains a catalytic triad comprised of C90, H593, and D751. Notably, the catalytic activity of USP1 is stimulated through the formation of a tight complex with a WD40 repeat protein UAF1 (USP1-associated factor 1). Our kinetic analyses revealed a general base catalysis in USP1/UAF1, in contrast to an ion-pair mechanism as demonstrated for papain and cathepsin. The pK(a) value of the catalytic cysteine was determined to be 8.67 ± 0.07 in a pH-dependent inactivation study of USP1/UAF1 by iodoacetamide. A normal solvent kinetic isotope effect of 2.8 for k(cat) and 3.0 for k(cat)/K(m) was observed in the USP1/UAF1-catalyzed hydrolysis of ubiquitin-AMC substrate. Moreover, proton inventory analysis supported the transfer of a single solvent-derived proton in the transition state. Our study also revealed the molecular basis for the activation of USP1 by UAF1. Although the pK(a) of the catalytic cysteine in USP1 and USP1/UAF1 was almost identical, the pK(a) of the catalytic histidine in USP1/UAF1 was 0.43 pH unit lower than that in USP1, which facilitates general base catalysis at a neutral pH and contributes to the elevated catalytic efficiency. We ruled out that the higher catalytic efficiency is due to a tighter binding of ubiquitin. Our results support a regulatory mechanism in which UAF1 activates USP1 by modulating its active site conformation. This finding has a general implication for the regulation of USPs that form complex with partner proteins.
Asunto(s)
Proteínas Nucleares/metabolismo , Biocatálisis , Dominio Catalítico , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Proteínas Nucleares/antagonistas & inhibidores , Cloruro de Sodio/química , Ubiquitina/farmacologíaRESUMEN
Ubiquitin-specific proteases (USPs) have in recent years emerged as a promising therapeutic target class. We identified selective small-molecule inhibitors against a deubiquitinase complex, the human USP1/UAF1, through quantitative high throughput screening (qHTS) of a collection of bioactive molecules. The top inhibitors, pimozide and GW7647, inhibited USP1/UAF1 noncompetitively with a K(i) of 0.5 and 0.7 µM, respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase, and cysteine proteases. The USP1/UAF1 inhibitors act synergistically with cisplatin in inhibiting cisplatin-resistant non-small cell lung cancer (NSCLC) cell proliferation. USP1/UAF1 represents a promising target for drug intervention because of its involvement in translesion synthesis and Fanconi anemia pathway important for normal DNA damage response. Our results support USP1/UAF1 as a potential therapeutic target and provide an example of targeting the USP/WD40 repeat protein complex for inhibitor discovery.