RESUMO
Before acquiring their mature state, cochlear hair cells undergo a series of changes in expression of ion channels. How this complex mechanism is achieved is not fully understood. Tmprss3, a type II serine protease expressed in hair cells, is required for their proper functioning at the onset of hearing. To unravel the role of Tmprss3 in the acquisition of mature K(+) currents, we compared their function by patch-clamp technique in wild-type Tmprss3(WT) and Tmprss3(Y260X)-mutant mice. Interestingly, only outward K(+) currents were altered in Tmprss3(Y260X)-mutant mice. To determine by which mechanism this occurred, we compared the protein network of Tmprss3(WT) and Tmprss3(Y260X)-mutant mice using proteomic analysis. This led to the identification of a pathway related to potassium Kcnma1 channels. This pathway was validated by immunohistochemistry, focusing on the most downregulated protein that was identified as a cochlear Kcnma1-associated protein, APOA1. Finally, we show that, in contrast to Tmprss3(WT), Kcnma1 channels were absent at the neck of inner hair cells (IHCs) in Tmprss3(Y260X)-mutant mice. In conclusion, our data suggest that lack of Tmprss3 leads to a decrease in Kcnma1 potassium channels expression in (IHCs).
Assuntos
Regulação para Baixo , Células Ciliadas Auditivas Internas/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Serina Proteases/genética , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Cóclea/citologia , Eletroforese em Gel Bidimensional , Expressão Gênica , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Potássio/metabolismo , Transporte Proteico , Proteoma/metabolismo , Serina Proteases/metabolismo , Espectrometria de Massas em TandemRESUMO
Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions.
Assuntos
Proteínas de Choque Térmico HSP27/genética , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/metabolismo , Células HeLa , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Transfecção , Células Tumorais CultivadasRESUMO
Mutations in the type II transmembrane serine protease 3 (TMPRSS3) gene cause non-syndromic autosomal recessive deafness (DFNB8/10), characterized by congenital or childhood onset bilateral profound hearing loss. In order to explore the physiopathology of TMPRSS3 related deafness, we have generated an ethyl-nitrosourea-induced mutant mouse carrying a protein-truncating nonsense mutation in Tmprss3 (Y260X) and characterized the functional and histological consequences of Tmprss3 deficiency. Auditory brainstem response revealed that wild type and heterozygous mice have normal hearing thresholds up to 5 months of age, whereas Tmprss3(Y260X) homozygous mutant mice exhibit severe deafness. Histological examination showed degeneration of the organ of Corti in adult mutant mice. Cochlear hair cell degeneration starts at the onset of hearing, postnatal day 12, in the basal turn and progresses very rapidly toward the apex, reaching completion within 2 days. Given that auditory and vestibular deficits often co-exist, we evaluated the balancing abilities of Tmprss3(Y260X) mice by using rotating rod and vestibular behavioral tests. Tmprss3(Y260X) mice effectively displayed mild vestibular syndrome that correlated histologically with a slow degeneration of saccular hair cells. In situ hybridization in the developing inner ear showed that Tmprss3 mRNA is localized in sensory hair cells in the cochlea and the vestibule. Our results show that Tmprss3 acts as a permissive factor for cochlear hair cells survival and activation at the onset of hearing and is required for saccular hair cell survival. This mouse model will certainly help to decipher the molecular mechanisms underlying DFNB8/10 deafness and cochlear function.
