Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis

Institute of Biomedical Informatics, School of Medicine, Tsinghua University, Beijing 100084, China.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2006; 103(24):8965-70. DOI: 10.1073/pnas.0603327103
Source: PubMed


Hydrogen peroxide (H(2)O(2)) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H(2)O(2) in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H(2)O(2) generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H(2)O(2) instead of to cytochrome c oxidase. In our studies, we found that mouse testis-specific cytochrome c (T-Cc) can catalyze the reduction of H(2)O(2) three times faster than its counterpart in somatic cells (S-Cc) and that the T-Cc heme has the greater resistance to being degraded by H(2)O(2). Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H(2)O(2). Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis.

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Available from: Sheng Ye, Dec 17, 2013
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    • "It has been suggested that, under usual circumstances, the peroxidase activity of Fe 3+ cyt c is an antioxidant, protective enzyme helping to remove excess H 2 O 2 [47]. Various experimental results have shown that endogenous and exogenously added Fe 3+ cyt c mediates the removal of H 2 O 2 and protects mitochondria from oxidative damage [93] [94] [95] [96]. It has been suggested that the peroxidase activity of CL–cyt c may favorably compete with other mitochondrial and peroxisomal H 2 O 2 -scavenging enzymes to control H 2 O 2 levels at the expense of intracellular reductants [47]. "
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    • "The rate of Cyt c degradation upon exposure to oxidative stress was monitored as previously described [61,71]. In brief, 200 μl of 1.5 mM H2O2 was added to 1 ml of Cyt c and Cyt c glycoconjugates (0.2 mg/ml) in 20 mM PBS pH 7.4 at 21°C. "
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    ABSTRACT: Background Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability. Results Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers. Conclusion The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.
    BMC Biochemistry 08/2014; 15(1):16. DOI:10.1186/1471-2091-15-16 · 1.44 Impact Factor
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    • "The slight differences in amino acid composition between the isoforms affect its function. In comparison with the somatic isoform, testes Cytc shows a threefold increased activity to reduce hydrogen peroxide ; however, it also shows a fourfold increased ability to trigger apoptosis (Liu et al. 2006 ) . For COX, six subunit isoforms have been identi fi ed in mammals to date. "
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    ABSTRACT: The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.
    Advances in Experimental Medicine and Biology 06/2012; 748:237-64. DOI:10.1007/978-1-4614-3573-0_10 · 1.96 Impact Factor
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