RESUMO
The 2A proteinases (2A(pro)) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2A(pro) also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2A(pro) (extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2A(pro) substrate region). In terms of overall fold, it is similar to the crystal structure of the mature human rhinovirus serotype 2 (HRV2) 2A(pro), but the relatively low level (40%) of sequence identity leads to a substantially different surface. We show that differences in the cI-to-eI2 loop between HRV2 and CVB4 2A(pro) translate to differences in the mechanism of eIF4GI recognition. Additionally, the nuclear magnetic resonance relaxation properties of CVB4 2A(pro), particularly of residues G1 to S7, F64 to S67, and P107 to G111, reveal that the substrate region is exchanging in and out of a conformation in which it occupies the active site with association and dissociation rates in the range of 100 to 1,000 s(-1). This exchange influences the conformation of the active site and points to a mechanism for how self-processing can occur efficiently while product inhibition is avoided.
Assuntos
Cardiomiopatia Dilatada/etiologia , Cisteína Endopeptidases/química , Enterovirus Humano B/enzimologia , Proteínas Virais/química , Sequência de Aminoácidos , Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/virologia , Domínio Catalítico , Infecções por Coxsackievirus/complicações , Infecções por Coxsackievirus/enzimologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Enterovirus Humano B/genética , Enterovirus Humano B/patogenicidade , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Rhinovirus/enzimologia , Rhinovirus/genética , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Termodinâmica , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Truncated tau is of great interest because of its important role in neurofibrillary pathogenesis in Alzheimer's disease (AD). A major obstacle for characterization of detailed biochemical and biological properties of truncated tau species and their fragments has been the lack of reliable and quick purification methods. Uneven distribution of acidic and basic residues in tau determines that the N- and C-terminal tau fragments require entirely different purification conditions. Conventional methods take several days; they do not allow purification of the acidic N-terminal tau fragments and do not prevent aggregation during purification that makes purified truncated tau unusable in functional studies. To prevent these inherent problems, we have designed a two-step, highly efficient purification procedure yielding a fully functional, non-aggregated homogeneous population of truncated tau molecules. Various forms of tau produced in bacteria without the need for a heat pre-treatment step were subjected to anion- and cation-exchange chromatography. Conditions were developed that allowed effective separation and purification of acidic and/or basic tau species. Following the gel filtration step, up to 10mg of tau proteins with 96% purity was obtained within one working day. Purified truncated tau exhibited an unmodified immunoreactivity and allowed its functional activity analysis. Since many neurodegenerative diseases have implicated similar disordered proteins in their pathogenesis, our procedure will allow their detailed analysis and characterization.
Assuntos
Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Doenças Neurodegenerativas/fisiopatologia , Proteínas tau/isolamento & purificação , Western Blotting , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas tau/fisiologiaRESUMO
Infection of mammalian cells with picornaviruses like entero-, rhino-, and aphthoviruses leads to an inhibition of cap-dependent cellular protein synthesis by the cleavage of both translation initiation factors, eIF4GI and eIF4GII. In entero- and rhinovirus infection this cleavage process is mediated by the viral 2A proteinase (2A(pro)). In order to discriminate between a direct mode of eIF4G cleavage and an indirect cleavage via activation of a cellular proteinase, a thermosensitive 2A(pro) mutant (ts-2A(pro)) of human rhinovirus 2 was employed. Temperature shift experiments of cytoplasmic HeLa cell extracts incubated with ts-2A(pro) strongly support a direct mode of cleavage of eIF4GI and eIF4GII by the viral 2A(pro).