Complementation studies with human ClpP in Bacillus subtilis.
Biochim Biophys Acta Mol Cell Res
; 1867(9): 118744, 2020 09.
Article
em En
| MEDLINE
| ID: mdl-32442436
ABSTRACT
ATP-dependent intracellular proteolysis is essential for all living organisms. ClpP, the proteolytic subunit of the ATP-dependent Clp proteases, shares 56% protein identity between B. subtilis and man. The aim of this study was to verify, whether human ClpP (HClpP) is able to substitute the bacterial pendant, BClpP, irrespectively of the huge evolutionary distance. For this reason hclpP was expressed from the natural B. subtilis promoters at the original chromosomal site. Growth at 37 °C as well as sporulation in the presence of hclpP depict an intermediate phenotype between wild type and clpP mutant suggesting a partial functional substitution of BClpP by HClpP. Northern as well as Western blot analyses show a similar induction pattern of both, bclpP and hclpP during heat stress on the mRNA as well as on the protein levels. Co-immunoprecipitation experiments imply specific interaction of HClpP with bacterial ClpC, ClpX and ClpE during control as well as heat stress conditions. Radioactive pulse-chase labeling and immunoprecipitation revealed that a ClpXP substrate, the short-living regulatory protein MgsR, is degraded by HClpP, although with an extremely slower rate in comparison to BClpP. The occurrence of an exceptional thickened cell wall of a clpP mutant can be almost fully reversed by the complementation with HClpP. The utilization of the HClpP expressing strain as a test system for new biological or synthetic active substances targeting BClpP is discussed.
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Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bacillus subtilis
/
Endopeptidase Clp
/
Expressão Ectópica do Gene
Limite:
Humans
Idioma:
En
Revista:
Biochim Biophys Acta Mol Cell Res
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Alemanha