RESUMO
The murine leukaemia virus (MLV) Gag cleavage product, p12, is essential for both early and late steps in viral replication. The N-terminal domain of p12 binds directly to capsid (CA) and stabilises the mature viral core, whereas defects in the C-terminal domain (CTD) of p12 can be rescued by addition of heterologous chromatin binding sequences (CBSs). We and others hypothesised that p12 tethers the pre-integration complex (PIC) to host chromatin ready for integration. Using confocal microscopy, we have observed for the first time that CA localises to mitotic chromatin in infected cells in a p12-dependent manner. GST-tagged p12 alone, however, did not localise to chromatin and mass-spectrometry analysis of its interactions identified only proteins known to bind the p12 region of Gag. Surprisingly, the ability to interact with chromatin was conferred by a single amino acid change, M63I, in the p12 CTD. Interestingly, GST-p12_M63I showed increased phosphorylation in mitosis relative to interphase, which correlated with an increased interaction with mitotic chromatin. Mass-spectrometry analysis of GST-p12_M63I revealed nucleosomal histones as primary interactants. Direct binding of MLV p12_M63I peptides to histones was confirmed by biolayer-interferometry (BLI) assays using highly-avid recombinant poly-nucleosomal arrays. Excitingly, using this method, we also observed binding between MLV p12_WT and nucleosomes. Nucleosome binding was additionally detected with p12 orthologs from feline and gibbon ape leukemia viruses using both pull-down and BLI assays, indicating that this a common feature of gammaretroviral p12 proteins. Importantly, p12 peptides were able to block the binding of the prototypic foamy virus CBS to nucleosomes and vice versa, implying that their docking sites overlap and suggesting a conserved mode of chromatin tethering for different retroviral genera. We propose that p12 is acting in a similar capacity to CPSF6 in HIV-1 infection by facilitating initial chromatin targeting of CA-containing PICs prior to integration.
Assuntos
Capsídeo/metabolismo , Cromatina/metabolismo , Produtos do Gene gag/genética , Mitose , Nucleossomos/metabolismo , Vírion/genética , Integração Viral/fisiologia , Animais , Cromatina/química , Cromatina/virologia , Regulação da Expressão Gênica , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Mutação , Ligação Proteica , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Montagem de Vírus , Replicação ViralRESUMO
The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cristalografia por Raios X/métodos , Chaperonas Moleculares/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Células HEK293 , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ets/química , Proteínas Proto-Oncogênicas c-ets/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The use of synthetic peptide immunogens as a means to generate specific immunological reagents for a variety of purposes has increased markedly in recent years. In this chapter, we outline some of the salient factors to be considered when designing peptide immunogens and describe basic methodologies for the conjugation of short synthetic peptides to immunogenic carrier proteins.
Assuntos
Anticorpos/imunologia , Antígenos/química , Reagentes de Ligações Cruzadas/química , Hemocianinas/química , Peptídeos/química , Antígenos/imunologia , Hemocianinas/imunologia , Peptídeos/imunologiaRESUMO
The generation of polyclonal antibodies to an antigen of interest is an important technique applicable to many areas of biological research. In this chapter, we describe a basic immunization procedure designed to generate polyclonal antisera in rabbits and two methods that are commonly employed in the subsequent preliminary characterization of antipeptide antibodies raised in this way.