RESUMO
Viruses have evolved in tandem with the organisms that they infect. Afflictions of the plant and animal kingdoms with viral infections have forced the host organism to evolve new or exploit existing systems to develop the countermeasures needed to offset viral insults. As one example, nonsense-mediated mRNA decay, a cellular quality-control mechanism ensuring the translational fidelity of mRNA transcripts, has been used to restrict virus replication in both plants and animals. In response, viruses have developed a slew of means to disrupt or become insensitive to NMD, providing researchers with potential new reagents that can be used to more fully understand the NMD mechanism.
Assuntos
Interações Hospedeiro-Patógeno , Degradação do RNAm Mediada por Códon sem Sentido , Vírus/metabolismo , Animais , Humanos , Plantas/virologia , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Virais/genética , Fenômenos Fisiológicos Virais , Vírus/classificação , Vírus/genética , Vírus/crescimento & desenvolvimentoRESUMO
Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control.
Assuntos
Regulação da Expressão Gênica , Mamíferos/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Animais , Núcleo Celular/metabolismo , Códon sem Sentido/genética , Éxons/genética , Previsões , Regulação Viral da Expressão Gênica , Doenças Genéticas Inatas/genética , Humanos , Modelos Genéticos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ubiquitinação , Proteínas Virais/genéticaRESUMO
Secretory proteins destined for the lumen of the endoplasmic reticulum are key regulators of cellular functions and are thus subject to several levels of quality control. A recent study finds that the earliest step in secretory protein biogenesis--binding of the signal recognition particle to the signal sequence of the nascent peptide--is subject to a quality control process termed RAPP for 'regulation of aberrant protein production'. This process involves AGO2 and mRNA degradation.
Assuntos
Sinais Direcionadores de Proteínas , Proteínas/genética , Proteínas/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Humanos , Biossíntese de Proteínas , Proteínas/química , Proteólise , RNA Mensageiro/metabolismoRESUMO
The influenza virus uses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Neuraminidase/metabolismo , Orthomyxoviridae/fisiologia , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Linhagem Celular , Cães , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Recombinação Genética , Coloração e Rotulagem/métodos , Vírion/enzimologiaRESUMO
Long non-coding RNAs (lncRNAs), once relegated to junk products of the genome, are becoming better appreciated for the myriad functions they play in cellular processes. It is clear that for most of the cases studied, lncRNAs carry out their functions at least in part through interactions with proteins. Here we present two complementary biochemical methods for the analysis of lncRNA-containing ribonucleoprotein complexes, hereafter referred to as RNPs. The first strategy offers users the ability to purify RNPs based on a protein component and to analyze the spectrum of lncRNAs, other proteins, and, if present, other types of RNAs that are bound to it. The second makes use of a bacteriophage MS2 binding-site affinity-handle grafted onto an lncRNA of interest to investigate the proteins and RNAs that co-purify with the tagged RNA.
Assuntos
RNA Longo não Codificante , Ribonucleoproteínas , Sítios de Ligação , Levivirus/genética , RNA Longo não Codificante/análise , RNA Longo não Codificante/química , RNA Longo não Codificante/isolamento & purificação , Ribonucleoproteínas/análise , Ribonucleoproteínas/química , Ribonucleoproteínas/isolamento & purificaçãoRESUMO
Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Hemaglutininas Virais/análise , Proteínas Imobilizadas/análise , Vírus da Influenza A/química , Análise Serial de Proteínas/instrumentação , Proteínas Virais/análise , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Desenho de Equipamento , Escherichia coli/genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Humanos , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Sortases are a class of bacterial enzymes that possess transpeptidase activity. It is their ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile that makes sortase an attractive tool for protein engineering. This technique has been adopted for a range of applications, from chemistry-based to cell biology and technology. In this Minireview we provide a brief overview of the biology of sortase enzymes and current applications in protein engineering. We identify areas that lend themselves to further innovation and that suggest new applications.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Engenharia de Proteínas , Motivos de Aminoácidos , Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Staphylococcus aureus/enzimologiaRESUMO
The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives. The generality of N-terminal labeling with SrtA(staph) is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Corantes Fluorescentes/química , Peptídeos/química , Alanina , Sequência de Aminoácidos , Sítios de Ligação , Staphylococcus aureus/enzimologia , Streptococcus pyogenes/enzimologia , Especificidade por SubstratoRESUMO
Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Aminoaciltransferases/biossíntese , Proteínas de Bactérias/biossíntese , Cisteína Endopeptidases/biossíntese , Escherichia coli , Sondas Moleculares/síntese química , Sondas Moleculares/isolamento & purificação , Oligopeptídeos/síntese química , Oligopeptídeos/isolamento & purificação , Engenharia de Proteínas , Técnicas de Síntese em Fase Sólida , Coloração e RotulagemRESUMO
Mammalian-cell messenger RNAs (mRNAs) are generated in the nucleus from precursor RNAs (pre-mRNAs, which often contain one or more introns) that are complexed with an array of incompletely inventoried proteins. During their biogenesis, pre-mRNAs and their derivative mRNAs are subject to extensive cis-modifications. These modifications promote the binding of distinct polypeptides that mediate a diverse array of functions needed for mRNA metabolism, including nuclear export, inspection by the nonsense-mediated mRNA decay (NMD) quality-control machinery, and synthesis of the encoded protein product. Ribonucleoprotein complex (RNP) remodeling through the loss and gain of protein constituents before and after pre-mRNA splicing, during mRNA export, and within the cytoplasm facilitates NMD, ensuring integrity of the transcriptome. Here we review the mRNP rearrangements that culminate in detection and elimination of faulty transcripts by mammalian-cell NMD.
Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , Animais , Códon de Terminação , Humanos , Transporte Proteico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ribonucleoproteínas/metabolismoRESUMO
Creation of functional protein bioconjugates demands methods for attaching a diverse array of probes to target proteins with high specificity, under mild conditions. The sortase A transpeptidase enzyme from Staphylococcus aureus catalyzes the cleavage of a short 5-aa recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an oligoglycine peptide and the target protein. By functionalizing the oligoglycine peptide, it is possible to incorporate reporters into target proteins in a site-specific fashion. This reaction is applicable to proteins in solution and on the living cell surface. The method described in this unit only requires incubation of the target protein, which has been engineered to contain a sortase recognition site either at the C terminus or within solvent-accessible loops, with a purified sortase enzyme and a suitably functionalized oligoglycine peptide.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas , Sítios de Ligação , Peptídeos/metabolismo , Engenharia de Proteínas , Proteínas/análise , Proteínas/metabolismo , Staphylococcus aureus/enzimologia , Especificidade por SubstratoRESUMO
Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.