RESUMEN
Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M-1 s-1, achieves sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.
Asunto(s)
COVID-19 , Hepatitis C Crónica , Animales , Humanos , Papaína/metabolismo , Péptido Hidrolasas/metabolismo , SARS-CoV-2/metabolismo , Antivirales/farmacología , Antivirales/química , Inhibidores de Proteasas , Mamíferos/metabolismoRESUMEN
SARS-CoV-2 protease Nsp3 is a therapeutic target for developing anti-SARS-CoV-2 drugs. Nsp3 is a large multi-spanning membrane protein, and its characterization in vitro has been challenging. Here we describe an in vitro assay to characterize the biochemical activity of full-length Nsp3 isolated from cells. The assay can be used to evaluate Nsp3 inhibitors.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Proteasas 3C de Coronavirus , Ubiquitinas/metabolismo , Ubiquitina/metabolismoRESUMEN
Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibited PLpro with k inact /K I = 10,000 M - 1 s - 1 , achieved sub-µM EC 50 values against three SARS-CoV-2 variants in mammalian cell lines, and did not inhibit a panel of human deubiquitinases at > 30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.
RESUMEN
The ongoing pandemic of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health. Drug repurposing is a time-efficient approach to finding effective drugs against SARS-CoV-2 in this emergency. Here, we present a robust experimental design combining deep learning with molecular docking experiments to identify the most promising candidates from the list of FDA-approved drugs that can be repurposed to treat COVID-19. We have employed a deep learning-based Drug Target Interaction (DTI) model, called DeepDTA, with few improvements to predict drug-protein binding affinities, represented as KIBA scores, for 2440 FDA-approved and 8168 investigational drugs against 24 SARS-CoV-2 viral proteins. FDA-approved drugs with the highest KIBA scores were selected for molecular docking simulations. We ran around 50,000 docking simulations for 168 selected drugs against 285 total predicted and/or experimentally proven active sites of all 24 SARS-CoV-2 viral proteins. A list of 49 most promising FDA-approved drugs with the best consensus KIBA scores and binding affinity values against selected SARS-CoV-2 viral proteins was generated. Most importantly, 16 drugs including anidulafungin, velpatasvir, glecaprevir, rifapentine, flavin adenine dinucleotide (FAD), terlipressin, and selinexor demonstrated the highest predicted inhibitory potential against key SARS-CoV-2 viral proteins. We further measured the inhibitory activity of 5 compounds (rifapentine, velpatasvir, glecaprevir, anidulafungin, and FAD disodium) on SARS-CoV-2 PLpro using Ubiquitin-Rhodamine 110 Gly fluorescent intensity assay. The highest inhibition of PLpro activity was seen with rifapentine (IC50: 15.18 µM) and FAD disodium (IC50: 12.39 µM), the drugs with high predicted KIBA scores and binding affinities.
Asunto(s)
COVID-19 , Aprendizaje Profundo , Preparaciones Farmacéuticas , Antivirales/farmacología , Antivirales/uso terapéutico , Reposicionamiento de Medicamentos , Humanos , Simulación del Acoplamiento Molecular , SARS-CoV-2RESUMEN
Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibited PLpro with kinact/KI = 10,000 M- 1 s- 1, achieved sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and did not inhibit a panel of human deubiquitinases at > 30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.
RESUMEN
TAM receptors (Tyro3, Axl, and Mer) are receptor tyrosine kinases (RTKs) that are expressed by multiple immune cells including NK cells. Although RTKs typically enhance cellular functions, TAM receptor ligation blocks NK-cell activation. The mechanisms by which RTKs block NK-cell signaling downstream of activating receptors are unknown. In this report, we demonstrate that TAM receptors attenuate NK cell responses via the activity of E3 ubiquitin ligase Casitas B lineage lymphoma b (Cbl-b). Specifically, we show that Tyro3, Axl, and Mer phosphorylate Cbl-b, and Tyro3 ligation activates Cbl-b by phosphorylating tyrosine residues 133 and 363. Ligation of TAM receptors by their ligand Gas6 suppresses activating receptor-stimulated NK-cell functions such as IFN-γ production and degranulation, in a TAM receptor kinase- and Cbl-b-dependent manner. Moreover, Gas6 ligation induces the degradation of LAT1, a transmembrane adaptor protein required for NK cell activating receptor signaling, in WT but not in Cbl-b knock-out NK cells. Together, these results suggest that TAM receptors may attenuate NK-cell function by phosphorylating Cbl-b, which in turn dampens NK-cell activation signaling by promoting the degradation of LAT1. Our data therefore support a mechanism by which RTKs attenuate, rather than stimulate, signaling pathways via the activation of ubiquitin ligases.
Asunto(s)
Células Asesinas Naturales/metabolismo , Activación de Linfocitos/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Sistema de Transporte de Aminoácidos y+L/metabolismo , Animales , Células Asesinas Naturales/inmunología , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-cbl/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Transducción de Señal/inmunologíaRESUMEN
USP7 is a deubiquitinating enzyme that plays a pivotal role in multiple oncogenic pathways and therefore is a desirable target for new anti-cancer therapies. However, the lack of structural information about the USP7-inhibitor interactions has been a critical gap in the development of potent inhibitors. USP7 is unique among USPs in that its active site is catalytically incompetent, and is postulated to rearrange into a productive conformation only upon binding to ubiquitin. Surprisingly, we found that ubiquitin alone does not induce an active conformation in solution. Using a combination of nuclear magnetic resonance, mass spectrometry, computational modeling, and cell-based assays, we found that DUB inhibitors P22077 and P50429 covalently modify the catalytic cysteine of USP7 and induce a conformational switch in the enzyme associated with active site rearrangement. This work represents the first experimental insights into USP7 activation and inhibition and provides a structural basis for rational development of potent anti-cancer therapeutics.
Asunto(s)
Inhibidores de Proteasas/farmacología , Tiofenos/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Dominio Catalítico , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/química , Relación Estructura-Actividad , Especificidad por Sustrato , Tiofenos/química , Ubiquitina/metabolismo , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
Foxp3+ T-regulatory (Treg) cells are known to suppress protective host immune responses to a wide variety of solid tumors, but their therapeutic targeting is largely restricted to their transient depletion or "secondary" modulation, e.g. using anti-CTLA-4 monoclonal antibody. Our ongoing studies of the post-translational modifications that regulate Foxp3 demonstrated that the histone/protein acetyltransferase, Tip60, plays a dominant role in promoting acetylation, dimerization and function in Treg cells. We now show that the ubiquitin-specific protease, Usp7, controls Treg function largely by stabilizing the expression and promoting the multimerization of Tip60 and Foxp3. Genetic or pharmacologic targeting of Usp7 impairs Foxp3+ Treg suppressive functions, while conventional T cell responses remain intact. As a result, pharmacologic inhibitors of Usp7 can limit tumor growth in immunocompetent mice, and promote the efficacy of antitumor vaccines and immune checkpoint therapy with anti-PD1 monoclonal antibody in murine models. Hence, pharmacologic therapy with Usp7 inhibitors may have an important role in future cancer immunotherapy.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Histona Acetiltransferasas/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transactivadores/metabolismo , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Animales , Autoinmunidad/genética , Autoinmunidad/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Inmunidad , Activación de Linfocitos/inmunología , Lisina Acetiltransferasa 5 , Ratones , Ratones Noqueados , Ratones Transgénicos , Neoplasias/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Carga Tumoral , Peptidasa Específica de Ubiquitina 7 , Proteasas Ubiquitina-Específicas/genéticaRESUMEN
Small cell carcinoma of the gall bladder (SCCGB) is a rare condition, with only 53 prior cases reported in the world literature when our case was first diagnosed. Our patient was found to have limited stage disease and was treated with sequential laparoscopic cholecystectomy, etoposide/carboplatin chemotherapy followed by consolidating loco-regional radiation therapy. She is alive and well without evidence of disease more than 132 months since diagnosis. We describe here our experience in the diagnosis, staging workup, treatment, and surveillance of a case of SCCGB and review the published literature. Treated aggressively with currently available methods, patients with limited stage SCCGB can have an excellent prognosis. The authors' intent is to provide a reasonable plan of treatment for other physicians facing such an unusual patient.
Asunto(s)
Carcinoma de Células Pequeñas/diagnóstico , Carcinoma de Células Pequeñas/terapia , Neoplasias de la Vesícula Biliar/diagnóstico , Neoplasias de la Vesícula Biliar/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Pequeñas/patología , Colecistectomía Laparoscópica , Terapia Combinada , Diagnóstico Diferencial , Diagnóstico por Imagen , Femenino , Neoplasias de la Vesícula Biliar/patología , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , RadioterapiaRESUMEN
A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.
Asunto(s)
Cromosomas Artificiales de Levadura , Enzimas/genética , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Saccharomyces cerevisiae/genética , Transformación Genética , Xilosa/metabolismo , Café , Medios de Cultivo/química , Etanol/metabolismo , Fermentación , Expresión Génica , Saccharomyces cerevisiae/crecimiento & desarrollo , Zea maysRESUMEN
The ubiquitin pathway regulates diverse functions including protein localization and stability. The complexity of the pathway involving nearly 40 identified E2 conjugating enzymes and over 600 E3 ligases raises the issue of specificity. With the E2s and E3s fitting into a limited number of classes based on bioinformatics, structures, and proven activities, there is not a clear picture as to what would determine which E2/E3 enzyme pair would be functional. There have been many reports of limited E2/E3 activity profiling with a small number of E2s and E3s. We have expanded on this to investigate the activity of ubiquitin E2s covering the majority of the reported classes/families in concert with a number of E3s implicated in a variety of diseases. Using an ELISA-based assay we screened 10 E3 ligases against a panel of 11 E2s to determine which E2/E3 pairs exhibited E3 autoubiquitylation activity. In addition, the ubiquitin chain linkage preference by certain E2/E3 pairs was investigated. Finally, substrate ubiquitylation was assayed for the E3 ligase MuRF1 using various E2/MuRF1 pairs. These studies demonstrate the utility of identifying the correct E2/E3 pair to monitor specific substrate ubiquitylation.
Asunto(s)
Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Especificidad por Sustrato , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Ume6p represses early meiotic gene transcription in Saccharomyces cerevisiae by recruiting the Rpd3p histone deacetylase and chromatin-remodeling proteins. Ume6p repression is relieved in a two-step destruction process mediated by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. The first step induces partial Ume6p degradation when vegetative cells shift from glucose- to acetate-based medium. Complete proteolysis happens only upon meiotic entry. Here we demonstrate that the first step in Ume6p destruction is controlled by its acetylation and deacetylation by the Gcn5p acetyltransferase and Rpd3p, respectively. Ume6p acetylation occurs in medium lacking dextrose and results in a partial destruction of the repressor. Preventing acetylation delays Ume6p meiotic destruction and retards both the transient transcription program and execution of the meiotic nuclear divisions. Conversely, mimicking acetylation induces partial destruction of Ume6p in dextrose medium and accelerates meiotic degradation by the APC/C. These studies reveal a new mechanism by which acetyltransferase activity induces gene expression through targeted destruction of a transcriptional repressor. These findings also demonstrate an important role for nonhistone acetylation in the transition between mitotic and meiotic cell division.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Meiosis/fisiología , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Meiosis/genética , Procesamiento Proteico-Postraduccional , Proteínas Represoras/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Musculares/antagonistas & inhibidores , Atrofia Muscular/prevención & control , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Biocatálisis/efectos de los fármacos , Western Blotting , Línea Celular , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Glucocorticoides/farmacología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas , Especificidad por Sustrato , Proteínas de Motivos Tripartitos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)-compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain's inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform's broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.
Asunto(s)
Enzimas Reparadoras del ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Descubrimiento de Drogas , Humanos , Luminiscencia , Proteínas del Tejido Nervioso/metabolismo , Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequeñas , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.
Asunto(s)
Isomerasas Aldosa-Cetosa/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Plásmidos/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Venenos de Araña/metabolismo , Xilosa/metabolismo , Isomerasas Aldosa-Cetosa/genética , Clonación Molecular , Vectores Genéticos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/ultraestructura , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Venenos de Araña/genética , Transformación GenéticaRESUMEN
New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin alpha-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction.
Asunto(s)
Etanol/metabolismo , Insecticidas/metabolismo , Saccharomyces cerevisiae/metabolismo , Venenos de Araña/metabolismo , Aminoácidos/genética , Western Blotting , Microscopía Electrónica de Rastreo , Modelos Genéticos , Mutagénesis , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Venenos de Araña/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A(2) (PLA(2)). Isopeptidase activity releases PLA(2), which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA(2) assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA(2) assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.
Asunto(s)
Endopeptidasas/metabolismo , Ubiquitina/metabolismo , Animales , Endopeptidasas/aislamiento & purificación , Ratones , Inhibidores de Proteasas/farmacologíaRESUMEN
Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been studied in Saccharomyces cerevisiae, histone modifications that are associated with transcriptional repression have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively regulates transcription in S. cerevisiae. We show that all four core histones are sumoylated and identify specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a potential block to these activating modifications. These results indicate that sumoylation is the first negative histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a general dynamic mark to oppose transcription.
Asunto(s)
Histonas/metabolismo , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Secuencia de Aminoácidos , Western Blotting , Inmunoprecipitación , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Telómero , Ubiquitina/metabolismoRESUMEN
Sumoylation, the process by which the ubiquitin-related SUMO protein is covalently attached to lysine side chains in other proteins, is involved in numerous processes in the eukaryotic cell, including transcriptional repression. In this study, we identify Gcn5, the histone-modifying subunit of the transcriptional regulatory complex SAGA, as a sumoylation substrate in yeast. In vitro, multiple sumoylation of recombinant Gcn5 alone or as a trimer with its interacting proteins Ada2 and Ada3 did not affect Gcn5's histone acetyltransferase (HAT) activity, suggesting that modification of Gcn5 with yeast SUMO (Smt3) may not directly regulate its HAT function. Through site-directed mutagenesis, the primary in vivo sumoylation site was identified as lysine-25, although an unsumoylatable K-to-R mutation of this residue led to no obvious in vivo effects. However, fusion of SUMO to the N-terminus of Gcn5 to mimic constitutive sumoylation resulted in defective growth on 3-aminotriazole media and reduced basal and activated transcription of the SAGA-dependent gene TRP3. Taken together with recent identification of multiple additional subunits of SAGA as sumoylated proteins in vivo, these data suggest that Gcn5 sumoylation may have an inhibitory role in transcriptional regulation.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Escherichia coli/genética , Histona Acetiltransferasas/genética , Lisina/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.