Experimental Verification Of Mitochondrial Protein Interactions

Mitochondria are very important organelle in cells eukaryotic and play a crucial role throughout the entire life activity. In addition to participating in many body physiological and pathological processes, except for ATP synthesis, fatty acid metabolism, TCA cycle, electron transport and oxidative phosphorylation process, mitochondria take part in many other vital physiological functions, including of the generation of reactive oxygen species to regulate the cell redox potential and signal transduction, apoptosis and regulation of the expression of certain genes. According to the mapping of human genes, it is estimated that there is around 1 000～2 000 kinds of mitochondrial protein, so far about more than 600 kinds of mitochondrial protein have been identified. And structure and function changes of mitochondrial protein is human disease-related. Such as degenerative diseases, heart disease, aging cancer and so on. Therefore, in recent years, the interaction of mitochondrial protein research has become the focus of the study of mitochondrial problem in the post-genome period.Protein is the majority of life, it is not exist in separately, but in interaction with other proteins and nucleic acids, small molecules, which understanded the performance of its functions. Protein-protein interactions can alter the kinetics of intracellular protein features, such as substrate binding, catalytic activity; and generate a new binding site which can changes in protein substrate specificity; it also enable other protein inactivation which regulates the expression of other genes. Only protein-protein interactions go smoothly, cells in the normal course of life is guaranteed. Therefore, the study of protein interactions for the understanding of protein function and clarifying the law of life is significant.At present, the study methods of protein-protein interaction, including yeast two-hybrid (Yeast two-hybrid system), in vitro binding assay (In vitro binding), immunoprecipitation (Coimmunoprecipitation), and mass spectrometry of affinity chromatography, etc. which are costly and long-termed. Therefore, bioinformatics study of protein-protein interaction provides a new direction and an important experiment clue .The process of protein-protein interaction studyed by bioinformatics which is in the integration of the human transcriptome, proteomics, metabolomics and other high-throughput data based on the dynamics of protein-protein interaction network and combined with data mining and information integration, the mitochondrial protein function is analysised by information theory and dynamic network topology structure ;In accordance with the mitochondrial disease-related proteins in the network of relations, we can find new mitochondrial proteins related to diseases , explore and elaborate and molecular mechanisms of the mitochondria-related diseases and laid the foundation of science for mitochondrial diseases. The discovery of disease genes and pathogenic mechanism could provide the new ideas and new ways to a theoretical basis and experimental evidence.This topic is that the six pairs human mitochondrial protein predicted protein-protein interactions is very possible in bioinformatics analysis progress the experiments. If the experiment proved the existence of interaction between proteins ,it could be identified new features of known mitochondrial proteins and provides a new way for functions of the unknown annotation of mitochondrial proteins.Currently there are many experimental methods used for protein - protein interaction studies. Because of the complexity of organisms and the limited technical means, so far, there is not technology can effectively solve the problem. Therefore, the use of a mammalian subject of a joint two-hybrid system, co-localization immunofiuorescence and immunoprecipitation experiments, etc, with a view to obtain positive results more reliable.Mammalian two-hybrid system is developed on the basis of the classical yeast two-hybrid system, but avoid the yeast two-hybrid technology inherent deficiencies, such as many protein interactions dependent on post-translational processing like as glycosylation, disulfide bond formation, and so on,which can not be in the nucleus, that draw the false-negative contusion, .moreover, the correct folding and the function of some protein depend on other non-yeast protein auxiliary, which had limited the research of certain extracellular protein , the cell membrane acceptor and so on. Mammalian two-hybrid system has a physiological environment closer to system in which protein are in synthesis, processing and response, and by which be in favor of protein-protein interactions found initially.Immunofluorescence technique, also known as fluorescent antibody technique ,is that immunolabelling technic is firstly develop technology as a immunofluorescence technique. It is a technical built on the basis of in immunology, biochemistry and microscopy technologies. basic principle studied with an unknown protein subcellular localization by immunofluorescence is that the fluorescent pigment will not affect the antigen (protein) antibody activity markers on the antibodies, which have the corresponding antigen (protein) combination and showed a specific fluorescence reaction in the fluorescence microscope. According to the distribution of fluorescence staining and sub-cellular protein in cells we can determine the unknown protein location. Because protein subcellular localization and its function is directly related to, such as the majority receptor are located in the cell membrane, the transcription factor translocate to the nucleus to play a role in transcriptional regulation, we can know protein function through protein localization research. This issue is that the unknown (target) protein coding sequence cloned into the expression vector and then transfected cells. And then tagged the target protein with fluorescent-labeled antibodies, while colored mitochondria, we can depend on relationship between the target protein and the positioning of mitochondria to determine whether is the mitochondrial protein or not.Co-Immunoprecipitation is the study of classical methods based protein-protein interaction between the antibody and antigen-specific specifically role, is effective method to determine interaction of the two proteins in intact cells in physiological . The principle is that cells were cracking in the non-denaturing conditions, the a number of protein - protein interaction exist in the cells has been retained integratly. If protein X antibody immunoprecipitate X, then X-binding protein in the body can precipitate Y down. This method commonly used in the determination of whether the two target proteins combined in the cells; also can be used to identify a specific protein the new partner .In view of the above speculation, we successfully constructed carrier pM3-X and pVP16-Y which include goal gene in the mammal two hybrid system, and carrier with the fluorescein enzyme reported gene cotransfect the 3T3 cell, detected by the M5 Microplate Luminometer reader. We detected whether there are interaction in every action. then we fused HA and the X(Y) coded sequence to carrier pcDNA3 by two-step clone and constructed pcDNA3-X(Y)-HA carrier with the HA label, soon after,we transfected the Hela cell. 48 hours later, and fixed cell marked by AlexaFluor488 immunity fluorescence and mitochondria MitoFluor Red 589 dye, then observed the protein expression and the localization by the fluorescence microscope, At last,we fused HA and the X(Y) coded sequence to carrier pcDNA3 by two-step clone, constructed the pcDNA3-X(Y)-HA carrier with the HA label. Which is preparation for further immunoprecipitation experiments.Through the above-mentioned work, we concluded that:1. this topic had built platform detect protein interaction by the mammal two hybrid system, we got the experimental results interaction happened between CYP11A1 and CYP11B1 which provided a new research topic of study of cholesterol metabolism.2. we found the mitochondria protein(BINP3、PEA15、CYP11B1 and CYB5R3) localization situation that are consistent with the mitochondria positioning by the immunity fluorescence experiments, and obtained conclusion MTRR do not locate in the mitochondria in the physiological condition.3.The work had successful constructed pcDNA3-X(Y)-HA carriers with HA label and laid the foundation that detect protein interaction by the immunity co-precipitation examination. .This topic’s findings had enriched the mitochondria protein information.And it had provided the thread and the methods for the research of mitochondria protein and mitochondria protein interaction.