Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus.
BACKGROUND
Members 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.


RESULTS
DNA 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.


CONCLUSIONS
Rhizopus 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.
Department of Environmental Sciences, University of Puerto Rico, Rio Piedras Campus, P.O. Box 70377, San Juan, PR, 00936-837, USA. mks131@gmail.com.
Manoj
M
Saxena
Department of Environmental Sciences, University of Puerto Rico, Rio Piedras Campus, P.O. Box 70377, San Juan, PR, 00936-837, USA. investigations7@gmail.com.
Rohit Kumar
RK
Sharma
Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, P.O. Box 70377, San Juan, PR, 00936-837, USA. josellyaima@hotmail.com.
Josell
J
Ramirez-Paz
Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, P.O. Box 70377, San Juan, PR, 00936-837, USA. atinoco9278@gmail.com.
Arthur D
AD
Tinoco
Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, P.O. Box 70377, San Juan, PR, 00936-837, USA. kai.griebenow@gmail.com.
Kai
K
Griebenow
D000595:Amino Acid Sequence; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006736:Horses; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012233:Rhizopus / Q000201:enzymology*
D045304:Cytochromes c; D053148:Caspase 3