RESUMO
Endoplasmic reticulum-localized DnaJ 4 (ERdj4) is an immunoglobulin-binding protein (BiP) cochaperone and component of the endoplasmic reticulum-associated degradation (ERAD) pathway that functions to remove unfolded/misfolded substrates from the ER lumen under conditions of ER stress. To elucidate the function of ERdj4 in vivo, we disrupted the ERdj4 locus using gene trap (GT) mutagenesis, leading to hypomorphic expression of ERdj4 in mice homozygous for the trapped allele (ERdj4(GT/GT)). Approximately half of ERdj4(GT/GT) mice died perinatally associated with fetal growth restriction, reduced hepatic glycogen stores, and hypoglycemia. Surviving adult mice exhibited evidence of constitutive ER stress in multiple cells/tissues, including fibroblasts, lung, kidney, salivary gland, and pancreas. Elevated ER stress in pancreatic ß cells of ERdj4(GT/GT) mice was associated with ß cell loss, hypoinsulinemia, and glucose intolerance. Collectively these results suggest an important role for ERdj4 in maintaining ER homeostasis during normal fetal growth and postnatal adaptation to metabolic stress.
Assuntos
Estresse do Retículo Endoplasmático/genética , Retardo do Crescimento Fetal/genética , Genes Essenciais , Proteínas de Choque Térmico/genética , Hipoglicemia/genética , Glicoproteínas de Membrana/genética , Animais , Glicemia/metabolismo , Cruzamentos Genéticos , Chaperona BiP do Retículo Endoplasmático , Feminino , Morte Fetal , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Feto , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Intolerância à Glucose , Glicogênio/deficiência , Proteínas de Choque Térmico/metabolismo , Homozigoto , Hipoglicemia/metabolismo , Hipoglicemia/patologia , Insulina/deficiência , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese , Transdução de SinaisRESUMO
We previously proposed a model of surfactant protein (SP)-C biosynthesis in which internalization of the proprotein from the limiting membrane of the multivesicular body to internal vesicles represents a key step in the processing and secretion of SP-C. To test this hypothesis, alanine mutagenesis of the N-terminal propeptide of SP-C was performed. Adenoviruses encoding mutant proproteins were infected into type II cells isolated from Sftpc(-/-) mice, and media analyzed for secreted SP-C 24 hours after infection. Mutation of S(12)PPDYS(17) completely blocked secretion of SP-C. PPDY (PY motif) has previously been shown to bind WW domains of neural precursor cell-expressed developmentally down-regulated (Nedd) 4-like E3 ubiquitin ligases. Purified recombinant glutathione S-transferase-SP-C propeptide (residues 1-35) bound recombinant Nedd4-2 strongly, and Nedd4 weakly; the S(12)PPDYS(17)mutation abrogated binding of SP-C to Nedd4-2. Immobilized recombinant Nedd4-2 WW domain captured SP-C proprotein from mouse type II cell lysates; in the reverse pulldown, endogenous SP-C in type II cells was captured by recombinant Nedd4-2. To determine if the interaction of Nedd4-2 and SP-C resulted in ubiquitination, the SP-C proprotein was immunoprecipitated from transiently transfected human embryonic kidney 293 cells, and analyzed by SDS-PAGE/Western blotting with ubiquitin antibody. Two ubiquitinated forms of SP-C were detected; ubiquitination was blocked by mutation of K6, but not K34, in the SP-C propeptide. Mutation of K6 also inhibited processing of SP-C proprotein to the mature peptide in human embryonic kidney 293 cells. Nedd4-2-mediated ubiquitination regulates lumenal relocation of SP-C, leading to processing and, ultimately, secretion of SP-C.
