RESUMO
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.
Assuntos
Células Matadoras Naturais/metabolismo , Ativação Linfocitária/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Sistema y+L de Transporte de Aminoácidos/metabolismo , Animais , Células Matadoras Naturais/imunologia , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-cbl/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Transdução de Sinais/imunologiaRESUMO
Parkinson's disease (PD) is the most common neurodegenerative movement disorder. There are no available therapeutics that slow or halt the progressive loss of dopamine-producing neurons, which underlies the primary clinical symptoms. Currently approved PD drugs can provide symptomatic relief by increasing brain dopamine content or activity; however, the alleviation is temporary, and the effectiveness diminishes with the inevitable progression of neurodegeneration. Discovery and development of disease-modifying neuroprotective therapies has been hampered by insufficient understanding of the root cause of PD-related neurodegeneration. The etiology of PD involves a combination of genetic and environmental factors. Although a single cause has yet to emerge, genetic, cell biological and neuropathological evidence implicates mitochondrial dysfunction and protein aggregation. Postmortem PD brains show pathognomonic Lewy body intraneuronal inclusions composed of aggregated α-synuclein, indicative of failure to degrade misfolded protein. Mutations in the genes that code for α-synuclein, as well as the E3 ubiquitin ligase Parkin, cause rare inherited forms of PD. While many ubiquitin ligases label proteins with ubiquitin chains to mark proteins for degradation by the proteasome, Parkin has been shown to mark dysfunctional mitochondria for degradation by mitophagy. The ubiquitin proteasome system participates in several aspects of the cell's response to mitochondrial damage, affording numerous therapeutic opportunities to augment mitophagy and potentially stop PD progression. This review examines the role and therapeutic potential of such UPS modulators, exemplified by both ubiquitinating and deubiquitinating enzymes.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Doença de Parkinson/genética , alfa-Sinucleína/genética , Complexo de Endopeptidases do Proteassoma , Ubiquitina/metabolismo , Dopamina , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
COVID-19 , Hepatite C Crônica , Animais , Humanos , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/metabolismo , Antivirais/farmacologia , Antivirais/química , Inibidores de Proteases , Mamíferos/metabolismoRESUMO
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.
Assuntos
COVID-19 , Aprendizado Profundo , Preparações Farmacêuticas , Antivirais/farmacologia , Antivirais/uso terapêutico , Reposicionamento de Medicamentos , Humanos , Simulação de Acoplamento Molecular , SARS-CoV-2RESUMO
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.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmídeos/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Venenos de Aranha/metabolismo , Xilose/metabolismo , Aldose-Cetose Isomerases/genética , Clonagem Molecular , Vetores Genéticos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Venenos de Aranha/genética , Transformação GenéticaRESUMO
In eukaryotic cells, the reversible attachment of small ubiquitin-like modifier (SUMO) protein is a post-translational modification that has been demonstrated to play an important role in various cellular processes. Moreover, it has been found that SUMO as an N-terminal fusion partner enhances functional protein production in prokaryotic and eukaryotic expression systems, based upon significantly improved protein stability and solubility. Following the expression and purification of the fusion protein, the SUMO-tag can be cleaved by specific (SUMO) proteases via their endopeptidase activity in vitro to generate the desired N-terminus of the released protein partner. In addition to its physiological relevance in eukaryotes, SUMO can, thus, be used as a powerful biotechnological tool for protein expression in prokaryotic and eukaryotic cell systems.In this chapter, we will describe the construction of a fusion protein with the SUMO-tag, its expression in Escherichia coli, and its purification followed by the removal of the SUMO-tag by a SUMO-specific protease in vitro.
Assuntos
Técnicas de Laboratório Clínico , Células Eucarióticas/metabolismo , Células Procarióticas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteína SUMO-1/metabolismo , Algoritmos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular/métodos , Eficiência , Células Eucarióticas/química , Expressão Gênica , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Células Procarióticas/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/química , Proteína SUMO-1/genéticaRESUMO
Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.
Assuntos
Baculoviridae/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/genética , Animais , Baculoviridae/metabolismo , Clonagem Molecular , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Camundongos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteína SUMO-1/metabolismo , Spodoptera/citologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/isolamento & purificação , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de UbiquitinaRESUMO
Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green fluorescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar KM values, but SUMO protease had a 25-fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.
Assuntos
Clonagem Molecular/métodos , Fusão Gênica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteína SUMO-1/biossíntese , Proteína SUMO-1/genética , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Endopeptidases/biossíntese , Endopeptidases/química , Endopeptidases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/química , Proteína SUMO-1/química , SolubilidadeRESUMO
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.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Histona Acetiltransferases/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transativadores/metabolismo , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Animais , Autoimunidade/genética , Autoimunidade/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Imunidade , Ativação Linfocitária/imunologia , Lisina Acetiltransferase 5 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neoplasias/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Carga Tumoral , Peptidase 7 Específica de Ubiquitina , Proteases Específicas de Ubiquitina/genéticaRESUMO
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.
Assuntos
Cromossomos Artificiais de Levedura , Enzimas/genética , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/genética , Transformação Genética , Xilose/metabolismo , Café , Meios de Cultura/química , Etanol/metabolismo , Fermentação , Expressão Gênica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Zea maysRESUMO
The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges including protein misfolding, protein degradation, and low solubility. A novel SUMO fusion technology appears to enhance protein expression and solubility ( http://www.lifesensors.com ). Efficient removal of the SUMO tag by SUMO protease in vitro facilitates the generation of target protein with a native N-terminus. In addition to its physiological relevance in eukaryotes, SUMO can be used as a powerful biotechnology tool for enhanced functional protein expression in prokaryotes and eukaryotes.
Assuntos
Escherichia coli/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteína SUMO-1/biossíntese , Animais , Células Eucarióticas/metabolismo , Exopeptidases/genética , Exopeptidases/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteína SUMO-1/genéticaRESUMO
The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly clone and express heterologous gene open reading frames in bacteria and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving microbial strains for biofuel production. The process involves the production of full-length complementary DNA libraries as a source of plasmid-based clones to express the desired proteins in active form for determination of their functions. Proteins that were identified by high-throughput screening as having desired characteristics are overexpressed in microbes to enable them to perform functions that will allow more cost-effective and sustainable production of biofuels. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. This review describes the design and implementation of an automated integrated programmable robotic workcell capable of producing complementary DNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. These operations will allow tailoring microbial strains to use renewable feedstocks for production of biofuels, bioderived chemicals, fertilizers, and other coproducts for profitable and sustainable biorefineries.
Assuntos
Ensaios de Triagem em Larga Escala/instrumentação , Biologia Molecular/instrumentação , Proteômica/instrumentação , Robótica/instrumentação , Biologia Sintética/instrumentação , Bioengenharia/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Biologia Molecular/métodos , Proteômica/métodos , Biologia Sintética/métodosRESUMO
Biological proteins are known to fold into specific 3D conformations. However, the fundamental question has remained: Do they fold because they are biological, and evolution has selected sequences which fold? Or is folding a common trait, widespread throughout sequence space? To address this question arbitrary, unevolved, random-sequence proteins were examined for structural features found in folded, biological proteins. Libraries of long (71 residue), random-sequence polypeptides, with ensemble amino acid composition near the mean for natural globular proteins, were expressed as cleavable fusions with ubiquitin. The structural properties of both the purified pools and individual isolates were then probed using circular dichroism, fluorescence emission, and fluorescence quenching techniques. Despite this necessarily sparse "sampling" of sequence space, structural properties that define globular biological proteins, namely collapsed conformations, secondary structure, and cooperative unfolding, were found to be prevalent among unevolved sequences. Thus, for polypeptides the size of small proteins, natural selection is not necessary to account for the compact and cooperative folded states observed in nature.
RESUMO
SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases.
Assuntos
Fosfolipases A2 do Grupo X/metabolismo , Fosfolipases A2/metabolismo , Engenharia de Proteínas/métodos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Fosfolipases A2 do Grupo X/genética , Histidina/química , Humanos , Rim/citologia , Camundongos , Fosfolipases A2/genética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , SolubilidadeRESUMO
Dysregulation of the UPS (ubiquitin-proteasome system) has been implicated in a wide range of pathologies including cancer, neurodegeneration and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; yet toxicity with this target remains high. DUBs (deubiquitinating enzymes) represent an alternative target in the UPS with low predicted toxicity. Currently, there are no DUB inhibitors that have been used clinically. To address this situation, Progenra has developed a novel assay to measure the proteolytic cleavage of Ub (ubiquitin) or UBL (Ub-like protein) conjugates such as SUMO (small Ub-related modifier), NEDD8 (neural-precursor-cell-expressed, developmentally down-regulated 8) or ISG15 (interferon-stimulated gene 15) by isopeptidases. In this review, current platforms for detecting DUB inhibitors are discussed and the advantages and disadvantages of the approaches are underlined.
Assuntos
Bioensaio/métodos , Endopeptidases/metabolismo , Inibidores Enzimáticos/metabolismo , Ubiquitina/metabolismo , Doença , Endopeptidases/genética , Inibidores Enzimáticos/uso terapêutico , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/genéticaRESUMO
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.
Assuntos
Endopeptidases/metabolismo , Ubiquitina/metabolismo , Animais , Endopeptidases/isolamento & purificação , Camundongos , Inibidores de Proteases/farmacologiaRESUMO
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.
Assuntos
Etanol/metabolismo , Inseticidas/metabolismo , Saccharomyces cerevisiae/metabolismo , Venenos de Aranha/metabolismo , Aminoácidos/genética , Western Blotting , Microscopia Eletrônica de Varredura , Modelos Genéticos , Mutagênese , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Venenos de Aranha/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Tagging proteins with mono- or poly-ubiquitin is now recognized as a multifaceted and universal means of regulating cell growth and physiology. It does so by controlling the cellular lifetime of nearly all eukaryotic proteins and the cellular localization of many critical proteins. Enzymes of the ubiquitin pathway add (ligases) or remove (deubiquitinases [DUBs]) ubiquitin tags to or from their target proteins in a selective fashion. Similarly to the kinases and their corresponding phosphatases, ubiquitin ligases and DUBs have become actively studied molecular oncology targets for drug discovery. Approximately 79 functional DUBs exist in the human proteome, suggesting that selective intervention is a reasonable therapeutic objective, with the goal of downregulating or ablating oncogene products or, alternatively, upregulating or sparing tumor suppressors. In the following review, this fascinating class of regulatory enzymes will be described, and specific examples of DUBs that are viable targets for anticancer therapy will be considered.
Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Ubiquitina/efeitos dos fármacos , Ubiquitina/metabolismo , Humanos , Proteína NEDD8 , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/efeitos dos fármacos , Ubiquitina Tiolesterase/efeitos dos fármacos , Ubiquitinas/efeitos dos fármacosRESUMO
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.
Assuntos
Carbono-Nitrogênio Liases/análise , RNA Polimerases Dirigidas por DNA/metabolismo , Poliovirus/enzimologia , Proteína SUMO-1/análise , Ubiquitinas/análise , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The demands of structural and functional genomics for large quantities of soluble, properly folded proteins in heterologous hosts have been aided by advancements in the field of protein production and purification. Escherichia coli, the preferred host for recombinant protein expression, presents many challenges which must be surmounted in order to over-express heterologous proteins. These challenges include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and the necessity for good purification methodologies. Gene fusion technologies have been able to improve heterologous expression by overcoming many of these challenges. The ability of gene fusions to improve expression, solubility, purification, and decrease proteolytic degradation will be discussed in this review. The main disadvantage, cleaving the protein fusion, will also be addressed. Focus will be given to the newly described SUMO fusion system and the improvements that this technology has advanced over traditional gene fusion systems.