Cytochrome c: Pro-Apoptotic and Antioxidant Functions
Cytochrome c: Pro-Apoptotic and Antioxidant Functions
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- Moscow State University / A.N. Belozersky Institute of Physical and Chemical Biology, Russia, Moscow\nInstitute of Bioorganic Chemistry, Russia, Moscow
- University of California / Department of Chemistry and Biochemistry, USA, CA, Santa Cruz
Project summaryThe primary goal of this project is to determine the structural elements of cytochrome c (cyt c) responsible for its pro-apoptotic and antioxidant activities in order to understand mechanisms of these activities and to engineer cyt c-based polypeptides which can be used as potential pharmaceuticals. The recently discovered pro-apoptotic activity  is characteristic for cytochromes c from various vertebrate species but not from yeast, although these proteins are highly homologous. In 1998 we have found  that cyt c effectively inhibits production of superoxide radicals in the initial span of the respiratory chain of intact mitochondria; this antioxidant activity of the yeast protein was less pronounced than that of vertebrate proteins. The structural basis for these differences is not known. The proposed project includes: (i) comparative computer analysis of various cytochromes c in order to identify amino acids that may play a role in the noncanonical activities of cyt c; (ii) mutagenesis of yeast cyt c in order to graft the pro-apoptotic activity onto it and to enhance its antioxidant activity; (iii) mutagenesis of horse cyt c in order to eliminate/decrease the pro-apoptotic and antioxidant activities; (iv) detailed investigation of the obtained mutant proteins in order to reveal the key amino acids involved in the activities; (v) study of interaction of cyt c and/or its mutants with prothymosin alpha using physical and chemical techniques including nuclear magnetic resonance and analysis of biological role of this interaction; (vi) engineering (and investigation?) of cyt c-based polipeptides of biomedical interest, e.g., cyt c possessing respiratory and antioxidant functions, but lacking pro-apoptotic activity.
Our preliminary comparative analysis of cytochromes c from yeast and other (predominately mammalian) species showed that the most promising candidate for mutagenesis is the first a-helix. It contains a number of exposed amino acids which are conservative through mammalian species, but different in yeast cyt c, namely amino acids in positions 2, 4, 7, 8, 11 (numbering according to horse cyt c). These amino acids form an outer hydrophylic surface of the first a-helix, and are likely to play a role in intermolecular interactions of cyt c with receptors/cofactors participating in pro-apoptotic or antioxidant functions. The analysis also revealed several additional amino acids in other parts of the molecule which may be essential for these functions, but are not clustered like those in the first a-helix. The mutated cyt c genes will be engineered using oligonucleotide-directed
site-specific mutagenesis and mutant proteins will be produced in Esherichia coli in expression systems obtained by Pollock et al.  for yeast cyt c and by us  for horse cyt c and its mutants. Structure and stability of mutated proteins will be characterized as well as their functioning as electron carriers in the mitochondrial respiratory chain, as antioxidants inhibiting generation of oxygen radicals by mitochondria and as pro-apoptotic signal in vitro and in intact cells. The muteins with depressed pro-apoptotic activity but normal respiratory and antioxidant functions would be of special interest. The species that can incorporate as apocytochromes in mammalian mitochondria to form functional cyt c will be selected and introduced in cyt c deficient cell lines (prepared recently by several groups in USA). These transfectants would be extremely important for studies of cyt c - independent apoptotic pathways, which underly the major mechanisms of immunity (FAS-dependent apoptosis) and inflammation (TNF-dependent apoptosis).
The results of the project will provide a rational basis for the design of a new generation of antioxidants with the properties of cyt c-mimetics. Low molecular weight compounds and small peptides with high affinity to putative "cyt c receptor" on the outer surface of mitochondria will be selected by their ability to block ROS production in isolated mitochondria. These agents will be tested for antioxidant and protective effects in intact cells where excessive mitochondrial ROS production would be induced by organic hydroperoxides, by depletion of mitochondrial gluthatione or by tumor necrosis factor (TNF). These antioxidants are suggested to protect heart, brain and other tissues during various pathologycal oxidative stresses, such as hypohxia / reperfusion and stroke, where mitochondria generate a majority of damaging ROS.
The other family of cyt c-mimetics will be selected in cell-free model of apoptosis by their ability to stimulate activity of pro-apoptotic proteases (caspases). These agents are suggested to induce apoptosis with high efficiency in tumor cells by-passing different defence mechanisms. The most important anti-apoptotic mechanisms mediated by oncoproteins (such as Bcl-2) interfere with the suicide signalling upstream of cyt c release into cytosol so cyt c-mimetics could be the powerful anti-cancer chemotherapeutic drugs per se or in combination with the other cytotoxic agents.
One more output of the project would be development of prototipic drugs which selectively prevents interaction of cyt c with the partner in apoptotic signalling pathway (Apaf-1) so inhibiting the major stress-induced apoptotic mechanism and increasing resistence of the cell against various insults (radiation, UV, oxidative stress etc). Low molecular weight drugs and small peptides (fragments of cyt c expressed after transfection) will be tested in the cell models with high efficiency of cyt c - dependent apoptosis which allow to select the survivors with impaired cyt c/Apaf-1 interaction.
A set of modern research techniques are planned to be applied to resolve the problems set in the project, namely, computer molecular modeling, PCR-based mutagenesis and other methods of genetic engineering, gene expression in combined expression systems including cyt c and heme lyase, liquid chromatography techniques, NMR, general physical and chemical methods of protein investigation. Functional activities of the obtained proteins and polypeptides will be studied using specific biological assays, namely, measurements of respiration and ROS production in isolated mitochondria and in intact cells, and detection of different apoptotic events (activation of caspases, fragmentation of DNA etc.) in living cells and in vitro.
The project team has all mayor equipment and facilities necessary for the implementation of the planned research. The project implies tight co-operation between three Russian research groups, which include qualified investigators possessing complementary experience in the field of the project. Scientists from the IBC are highly experienced in molecular modelling, protein engineering and NMR of proteins, researchers from the IPCB have profound knowledge in investigation of noncanonic activities of cyt c, while scientists from the IIE are highly qualified in preparative gene expression and physico-chemical testing of proteins. The American collaborator, Prof. A.L.Fink has unique experience in protein folding . Contacts with his laboratory will give to Russian scientists the possibility to improve their scientific level and to apply the accumulated experience to peaceful fundamental and applied research. As a whole the project will provide to weapon scientists and engineers from both Russian Institutes the opportunity for civil scientific and research activity thus responding to the primary objectives of the ISTC.
The project will give results of theoretical as well as practical value. The theoretical value will be in clarification of the structural basis of non-canonical functions of cyt c, a better understanding of the mechanisms of apoptotic and antioxidant activities. Practical value of this project is that elucidation of molecular elements responsible for non-canonical activities may be useful for the design of antioxidants and anti-cancer chemotherapeutic drugs with the properties of cyt c-mimetics.
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