Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizusReportar como inadecuado




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BMC Biochemistry

, 16:21

Protein and enzyme biochemistry

Abstract

BackgroundMembers of Rhizopus species are the most common cause of mucormycosis, a rare but often fatal fungal infection. Host induced pathogen apoptosis and pathogen induced host cell apoptosis are often involved in fungal infections. In many organisms, the release of mitochondrial cytochrome c can trigger apoptosis by activating caspase proteases, but the role of fungal cytochrome c in apoptosis remains unknown.

ResultsDNA sequence encoding Rhizopus arrhizus cytochrome c was cloned and expressed in E. coli. Both native and recombinant cytochrome c were purified using ion exchange followed by gel filtration chromatography. The identities of purified proteins were confirmed by MALDI-MS and UV-Visible spectroscopy. For the first time, we demonstrated that Rhizopus arrhizus cytochrome c could activate human capspase-3 in HeLa cell extracts. We also found that Rhizopus arrhizus cytochrome c has redox potential, peroxidase activity, and spectral properties similar to human and horse cytochrome c proteins.

ConclusionsRhizopus arrhizus cytochrome c can activate human caspase-3 in HeLa cell extracts and it possesses similar physical and spectral properties as human and horse cytochrome c. This protein was found to have a previously unknown potential to activate human caspase-3, an important step in the apoptosis cascade.

AbbreviationsABTS2,2′-azino-bis3-ethylbenzothiazoline-6-sulphonic acid

Apaf-1Apoptotic protease activating factor 1

Cyt cCytochrome c

DTTDithiothreitol

MALDI MSMatrix-assisted laser desorption-ionization mass spectroscopy

SDS-PAGESodium dodecyl sulfate-polyacrylamide gel electrophoresis

Electronic supplementary materialThe online version of this article doi:10.1186-s12858-015-0050-9 contains supplementary material, which is available to authorized users.

An erratum to this article can be found at http:-dx.doi.org-10.1186-s12858-016-0059-8.

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Autor: Manoj Saxena - Rohit Kumar Sharma - Josell Ramirez-Paz - Arthur D. Tinoco - Kai Griebenow

Fuente: https://link.springer.com/







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