peptide-based-identification-functional-motifs-their-binding

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Morehouse School of Medicine

Institute for Systems Biology

Nef is secreted from infected cells in exosomes and is found in abundance in the sera of HIV-infected individuals. Secreted exosomal Nef (exNef) induces apoptosis in uninfected CD4âº T cells and may be a key component of HIV pathogenesis. The exosomal pathway has been implicated in HIV-1 virus release, suggesting a possible link between these two viral processes. However, the underlying mechanisms and cellular components of exNef secretion have not been elucidated. We have previously described a Nef motif, the secretion modification region (SMR; amino acids 66 to 70), that is required for exNef secretion. In silico modeling data suggest that this motif can form a putative binding pocket. We hypothesized that the Nef SMR binds a cellular protein involved in protein trafficking and that inhibition of this interaction would abrogate exNef secretion. By using tandem mass spectrometry and coimmunoprecipitation with a novel SMR-based peptide (SMRwt) that blocks exNef secretion and HIV-1 virus release, we identified mortalin as an SMR-specific cellular protein. A second set of coimmunoprecipitation experiments with full-length Nef confirmed that mortalin interacts with Nef via Nef's SMR motif and that this interaction is disrupted by the SMRwt peptide. Overexpression and microRNA knockdown of mortalin revealed a positive correlation between exNef secretion levels and mortalin protein expression. Using antibody inhibition we demonstrated that the Nef/mortalin interaction is necessary for exNef secretion. Taken together, this work constitutes a significant step in understanding the underlying mechanism of exNef secretion, identifies a novel host-pathogen interaction, and introduces an HIV-derived peptide with antiviral properties.

Secretion modification region-derived peptide disrupts HIV-1 Nef's interaction with mortalin and blocks virus and Nef exosome release.

Nef est s&eacute;cr&eacute;t&eacute;e par les cellules infect&eacute;es dans les exosomes et se trouve en abondance dans le s&eacute;rum des personnes infect&eacute;es par le VIH. S&eacute;cr&eacute;t&eacute;es exosomal Nef (exNef) induit l&#39;apoptose dans les cellules non infect&eacute;es de CD4&#x207A; T et peut &ecirc;tre un &eacute;l&eacute;ment cl&eacute; de la pathog&eacute;n&egrave;se du VIH. La voie d&#39;exosomal a &eacute;t&eacute; impliqu&eacute;e dans la lib&eacute;ration de virus VIH-1, ce qui sugg&egrave;re un lien possible entre ces deux processus virales. Cependant, les m&eacute;canismes sous-jacents et les composants cellulaires de la s&eacute;cr&eacute;tion d&#39;exNef n&#39;ont pas &eacute;t&eacute; &eacute;lucid&eacute;s. Nous avons d&eacute;j&agrave; d&eacute;crit un motif de la Nef, la r&eacute;gion de modification de s&eacute;cr&eacute;tion (SMR ; acides amin&eacute;s 66 &agrave; 70), qui est n&eacute;cessaire pour la s&eacute;cr&eacute;tion d&#39;exNef. In silico, mod&eacute;lisation des donn&eacute;es sugg&egrave;rent que ce motif peut se former une poche de liaison putative. Nous avons &eacute;mis l&#39;hypoth&egrave;se que la Nef SMR se lie &agrave; une prot&eacute;ine cellulaire impliqu&eacute;e dans le trafic de prot&eacute;ine et que l&#39;inhibition de cette interaction abolirait la s&eacute;cr&eacute;tion exNef. &Agrave; l&#39;aide de spectrom&eacute;trie de masse et de co-immunopr&eacute;cipitation avec un nouveau peptide ax&eacute;e sur le SMR (SMRwt) qui bloque la s&eacute;cr&eacute;tion exNef et lib&eacute;ration de virus VIH-1, nous avons identifi&eacute; mortalin comme une prot&eacute;ine cellulaire SMR sp&eacute;cifiques. Une deuxi&egrave;me valeur de co-immunopr&eacute;cipitation exp&eacute;rimente le Nef pleine longueur a confirm&eacute; que mortalin interagit avec la Nef par motif de SMR de la Nef et que cette interaction est perturb&eacute;e par le peptide SMRwt. Surexpression et microARN pr&eacute;cipitation de mortalin ont r&eacute;v&eacute;l&eacute; une corr&eacute;lation positive entre les niveaux de s&eacute;cr&eacute;tion d&#39;exNef et expression de la prot&eacute;ine mortalin. &Agrave; l&#39;aide d&#39;inhibition d&#39;anticorps, nous avons d&eacute;montr&eacute; que l&#39;interaction de la Nef/mortalin est n&eacute;cessaire pour la s&eacute;cr&eacute;tion d&#39;exNef. Pris ensemble, ce travail constitue une &eacute;tape importante pour comprendre le m&eacute;canisme sous-jacent de la s&eacute;cr&eacute;tion d&#39;exNef, identifie une interaction h&ocirc;te-pathog&egrave;ne nouveau et pr&eacute;sente un peptide d&eacute;riv&eacute; du VIH avec des propri&eacute;t&eacute;s antivirales.

Peptides de r&eacute;gion-d&eacute;riv&eacute;s s&eacute;cr&eacute;tion modification perturbe interaction VIH-1 de la Nef avec les virus mortalin et blocs et Nef exosome lib&eacute;ration.

NEF &egrave; secreta dalle cellule infette in fatto e si trova in abbondanza nel siero di individui HIV-positivi. Secreta exosomal Nef (exNef) induce apoptosi in cellule T CD4&#x207A; non infette e pu&ograve; essere una componente chiave della patogenesi dell&#39;HIV. Il percorso exosomal &egrave; stato implicato nel rilascio di virus HIV-1, suggerendo un possibile collegamento tra questi due processi virali. Tuttavia, i meccanismi sottostanti e componenti cellulari della secrezione exNef avere non state chiarite. In precedenza abbiamo descritto un motivo Nef, la regione modifica la secrezione (SMR; aminoacidi 66-70), che &egrave; necessario per la secrezione exNef. In silico di modellazione dati suggeriscono che questo motivo pu&ograve; formare una tasca associazione putativo. Abbiamo ipotizzato che il Nef SMR si lega una proteina cellulare coinvolta nel traffico di proteine e che l&#39;inibizione di questa interazione sarebbe abolire la secrezione exNef. Mediante spettrometria di massa tandem e coimmunoprecipitation con un peptide romanzo basato su SMR (SMRwt) che blocca la secrezione exNef e rilascio del virus HIV-1, abbiamo identificato mortalin come una proteina cellulare SMR-specifici. Un secondo set di esperimenti di coimmunoprecipitation con full-length Nef ha confermato che mortalin interagisce con Nef via motivo SMR Nef e che questa interazione &egrave; perturbata dal peptide SMRwt. Sovraespressione e microRNA atterramento di mortalin hanno rivelato una correlazione positiva tra livelli di secrezione di exNef e mortalin l&#39;espressione della proteina. Mediante inibizione dell&#39;anticorpo abbiamo dimostrato che l&#39;interazione Nef/mortalin &egrave; necessario per la secrezione exNef. Presi insieme, quest&#39;opera costituisce un passo significativo nella comprensione del meccanismo sottostante della secrezione exNef, identifica un&#39;interazione ospite-patogeno romanzo e introduce un peptide derivato da HIV con propriet&agrave; antivirali.

Peptide derivato da regione modifica di secrezione interferisce con l&#39;interazione di HIV-1 Nef con virus mortalin e blocchi e Nef exosome rilascio.

Martin N. Shelton

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