RESUMO
Mouse guanylate-binding proteins (mGBPs) are recruited to various invasive pathogens, thereby conferring cell-autonomous immunity against these pathogens. However, whether and how human GBPs (hGBPs) target M. tuberculosis (Mtb) and L. monocytogenes (Lm) remains unclear. Here, we describe hGBPs association with intracellular Mtb and Lm, which was dependent on the ability of bacteria to induce disruption of phagosomal membranes. hGBP1 formed puncta structures which were recruited to ruptured endolysosomes. Furthermore, both GTP-binding and isoprenylation of hGBP1 were required for its puncta formation. hGBP1 was required for the recovery of endolysosomal integrity. In vitro lipid-binding assays demonstrated direct binding of hGBP1 to PI4P. Upon endolysosomal damage, hGBP1 was targeted to PI4P and PI(3,4)P2-positive endolysosomes in cells. Finally, live-cell imaging demonstrated that hGBP1 was recruited to damaged endolysosomes, and consequently mediated endolysosomal repair. In summary, we uncover a novel interferon-inducible mechanism in which hGBP1 contributes to the repair of damaged phagosomes/endolysosomes.
Assuntos
Proteínas de Ligação ao GTP , Fagossomos , Humanos , Animais , Camundongos , Proteínas de Ligação ao GTP/metabolismo , Fagossomos/metabolismo , Interferons/metabolismo , Endossomos/metabolismoRESUMO
Alanine expansion mutations in poly(A)-binding protein nuclear 1 (PABPN1) cause muscle weakness in the late-onset disorder oculopharyngeal muscular dystrophy. In affected muscles, expanded PABPN1 forms nuclear aggregates, depleting levels of soluble PABPN1 and inducing a genome-wide shift from distal to proximal polyadenylation site usage. PABPN1 protein accumulation is regulated by the ubiquitin proteasome system, which is highly dysregulated in oculopharyngeal muscular dystrophy. We show that ARIH2 E3-ligase regulates PABPN1 protein accumulation and aggregation. Levels of ARIH2 mRNA are regulated by PABPN1 via proximal polyadenylation site usage. We demonstrate that masking the proximal polyadenylation site in ARIH2 3' untranslated region by antisense oligonucleotides elevates the expression of ARIH2 and PABPN1 and restores myogenic defects that are induced by ARIH2 or PABPN1 down-regulation in cell culture. In vivo ARIH2 mRNA levels significantly decrease from midlife in vastus lateralis muscles and highly correlate with muscle degeneration. We suggest that the expression of both genes is maintained by a feed-forward loop between mRNA stability regulated by PABPN1 and protein turnover regulated by ARIH2.
Assuntos
Envelhecimento/patologia , Músculo Esquelético/patologia , Distrofia Muscular Oculofaríngea/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Envelhecimento/fisiologia , Animais , Western Blotting , Linhagem Celular , Regulação da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Imunoprecipitação , Músculo Esquelético/metabolismo , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína I de Ligação a Poli(A)/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Ubiquitina-Proteína Ligases/genéticaRESUMO
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.
Assuntos
Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Proteínas Mutantes/metabolismo , Proteína II de Ligação a Poli(A)/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Desmina/genética , Modelos Animais de Doenças , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/metabolismo , Músculos/patologia , Distrofia Muscular Oculofaríngea/genética , Proteínas Mutantes/química , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais , Solubilidade , Transcriptoma , Transfecção , Expansão das Repetições de Trinucleotídeos/genética , UbiquitinaçãoRESUMO
The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1.