Electron Transport Chain ABSTRACT MATERIALS & METHODS SS31 Inhibits Cytochrome C Structure Change and Peroxidase Activity Induced by Cyt C/Cardiolipin Interaction Mitochondria are essential in the conversion of food energy into ATP, a form of energy usable by cells. Cytochrome c (cyt c) is a mitochondrial protein of principal importance in the production of ATP. However, upon binding with cardiolipin (CL), cyt c undergoes a conformational change that leads to CL peroxidation, increased reactive oxygen species (ROS) production, and unregulated apoptosis. SS31 is a synthetic, cell-permeable tetrapeptide that targets the inner mitochondrial membrane. In this investigation, the ability of SS31 to inhibit the conformational change that takes place in cyt c upon interaction with CL, and the peroxidase activity of the cyt c/CL complex was studied using spectroscopic techniques. The results indicated that SS31 is able to prevent both of these detrimental effects of cyt c/CL interaction. This study provides compelling evidence for the role of SS31 as a mitochondria-protective molecule.   Effect of SS31 on Cyt C Structure: Circular Dichroism (CD) was used to analyze the conformational change that takes place in cyt c structure as an effect of cyt c/CL interaction, and to test whether SS31 could inhibit this change. Effect of SS31 on Cyt C/CL Peroxidation: Peroxidase activity was studied by the peroxidation of fluorescent Invitrogen 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP), also known as amplex red. RESULTS CONCLUSION 2 SS31 is able to prevent the conformational change that takes place in cyt c upon interaction with CL SS31 dose-dependently inhibits the peroxidase activity induced by cyt c/CL interaction. 1 Electron Transport Chain C1 C3 C2 C4 Cyt C ATP Synthase Mitochondrion Inner Membrane Outer Membrane CL 2 Cyt C/CL Interaction Cyt C/CL Interaction ROS Initiation of Mitochondrial Apoptotic Pathway Oxidation of important molecules (e.g. DNA, protein) INTRODUCTION DISCUSSION Mitochondria play a vital role in energy production through the synthesis of Adenosine triphosphate (ATP) Energy production is tightly coupled with the proper functioning of the electron transport chain (ETC) in the inner mitochondrial membrane. Electron transport oftentimes results in reactive oxygen species (ROS) production which causes oxidative damage to the molecules of the cell. Cytochrome c (cyt c) is a redox protein that mediates electron transfer between Complex III and IV in the ETC Cardiolipin (CL) is a mitochondrial phospholipid found in abundance in the inner mitochondrial membrane Cyt c/CL interactions lead to cyt c structure change, which leads to dysfunctional cyt c behavior cyt c redox potential decreases cyt c may exit into the cytoplasm and initiate apoptosis structure change facilitates the access of small molecules to the heme site which stimulates peroxidase activity SS31(D-Arg-dimethyl Tyr-Lys-Phe-NH2) tetrapeptide (Dr. Hazel H. Szeto and Dr. Peter Schiller)  selectively targets and concentrates in the inner mitochondrial membrane possesses strong anti-oxidative properties has been shown to scavenge ROS and reduce mitochondrial ROS production. has been shown to prevent apoptosis and necrosis Research suggests that SS31 may exert its mitoprotective action by targeting cyt c, thus promoting electron transfer in the mitochondrial ETC It was previously demonstrated that SS31 and its analogs are able to block mitochondrial peroxidation in the presence of intracellular peroxide-generating compounds. The cyt c/CL complex is the strongest peroxidase in mitochondria which can result in the total peroxidation of mitochondria in the presence of hydrogen peroxide The goal was to determine whether SS31 could inhibit cyt c structure change induced by CL interaction inhibit cyt c/CL complex-promoted oxidative stress SS31 is able to prevent the conformational change that takes place in cyt c upon interaction with CL This could prevent uncontrolled apoptosis induced by cyt c structure change and subsequent transport to the cytoplasm This also promotes normal mitochondrial function as it inhibits the reduction in redox potential that takes place upon cyt c structure change SS31 dose-dependently inhibits the peroxidase activity induced by cyt c/CL interaction SS31 could prevent the oxidation of critical cellular molecules, thus promoting cell health and growth SS31 could prevent the development of elusive diseases such as Parkinson’s, autism and even cancer which are all linked to ROS production SS31 could delay the effects of aging which ROS has also been associated with Since healthy mitochondria are a requirement for a healthy body, it is crucial for the scientific community to continue targeting mitochondria and looking for ways to improve mitochondrial function. It is also crucial for the scientific community to start investigating the role mitochondria may play in the development of complex disease. Along the way, it may find that SS31 is an invaluable agent to the cell. Cyt c Structure Change Cyt C/CL Complex 4 3 CL promotes cytochrome c structure change in a dose-dependent manner. SS31 inhibits CL-induced cytochrome c structure change in a dose-dependent manner. 5 6 Hypothesized Mode of Action of SS31 ACKNOWLEDGEMENTS ATP SS31 I would like to thank everyone who made this project possible Dr. Alexander V. Birk for teaching me all the laboratory techniques used to carry out this study, for his ongoing guidance throughout my project, and for showing me how to think critically and analytically. Dr. Hazel H. Szeto for allowing me to intern in her laboratory and conduct this research, and for transmitting her enthusiasm of mitochondrial research to me. Mr. Richard Lee for his constructive criticism throughout the writing of my paper. My parents for allowing me to participate in this internship and for their constant support and understanding. Dr. JoAnn Gensert for her guidance in making this board ROS QUESTION & HYPOTHESIS Experimental Question: Can SS31 inhibit cyt c/CL complex-promoted oxidative stress and the structure change that takes place in cyt c upon interaction with CL? Hypothesis: SS31 will inhibit cyt c structure change and peroxidase activity induced by cytc/CL interaction. Cyt C/CL Complex SS31 promoted normal mitochondrial function by preventing cytc/CL interaction. SS31 inhibits peroxidase activity of the cyt c/CL complex in a dose-dependent manner.