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

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

Inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate

“…This result indicates that during the conjugation the ability of the Cyt c to induce caspase-3 activation was not compromised. This result is significant as many of the surface lysines are known to be involved in the Apaf-1 Cyt c interaction, which is important for the activation of the caspase-3 [ 41 , 42 ]. This result implies that conjugation does not necessarily adversely impact the Cyt c ability to induce apoptosis even though conjugation involves some of the lysines.…”

Cytochrome c: An extreme multifunctional protein with a key role in cell fate

“…The results obtained in this study provide a significant contribution to a better understanding of the key role played by apoptosis in fungal infections, where two opposing processes occur during mucormycosis infection: pathogen-induced host cell death and host-induced pathogen cell death. Interestingly, this study proves that both human and fungal cyt c play a major (and opposite) role in fungal infections, determining the development or clearing of mucormycosis in humans [67].…”

A Cytochrome c‐Chlorotoxin Hybrid Protein as a Possible Antiglioma Drug

“…Similarly, recombinant human Cytc was obtained from the unmodified pBTR1 vector. The protein expression method used here was adapted from a protocol we previously published in our laboratory [49] . Briefly, the bacteria growth media was prepared using 12 g of tryptone, 24 g of yeast extract, 8 mL glycerol, 2.3 g NaH 2 PO 4 and 12.5 g Na 2 HPO 4 and 100 mg of ampicillin for each liter.…”

“…The protein expression method used here was adapted from a protocol we previously published in our laboratory. [49] Briefly, the bacteria growth media was prepared using 12 g of tryptone, 24 g of yeast extract, 8 mL glycerol, 2.3 g NaH 2 PO 4 and 12.5 g Na 2 HPO 4 and 100 mg of ampicillin for each liter. To inoculate liter of the prepared media, 10 mL of bacteria solution were grown at 37°C under continuous shaking at rpm.…”