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Research of Gene Function Based on Protein-Protein Interction Network of Mitochondrial Intermembrane Space

Author: ZengZhiFeng
Tutor: YangXiaoMing
School: PLA Military Academy of Medical Sciences
Course: Biochemistry and Molecular Biology
Keywords: mitochondrial protein-protein interaction MCFP ATP Tim23 IMMT apoptosis
CLC: Q3
Type: Master's thesis
Year: 2009
Downloads: 98
Quote: 1
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Abstract

Mitochondria is an important intra-cellular organelle in eukaryotic cells, which. is the main venues of oxidative phosphorylation and ATP production in mammal cells. Mitochondria is made of four regions, including the out membrane, the inner membrane , the matrix and the intermembrane space. Mitochondrial intermembrane space(IMS) is the 6-8 nm width space between the out membrane and the inner membrane, which enriched with amorphous liquid , soluble enzymes, substrates and supporting factors. Although only 34 IMS proteins were confirmed to locate in mitochondria IMS, these proteins are involved into many critical physiological functions, such as cellular respiration, apoptosis and protein transport, and worked as a single protein or by the interaction of multiple proteins. Therefore , study of IMS protein and their interaction is very important to understand mechanism of mitochondrial functions and deseases .The interactions between single pair mitochondrial membrane space protein have been reported,but systematic research of protein-protein interaction network of mitochondrial intermembrane space still remains unreported. Therefore, by employing large-scale two yeast-hybrid screen assay, we found 49 pairs of protein-protein interaction in the IMS, and constructed the initial protein-protein interaction network in the IMS. Although the yeast two-hybrid technology is high-throughput, large-scale advantages, there are also false positive and false-negative outcomes. GST-pull down was used to verify the pre-screening results of the 12 pairs of interactions, 6 pairs of positive were confirmed. Taken together with 5 pairs positive results from total 6 pair interactions in previous screening, the interaction data obtained from GST-pull down showed that the positive rate is about 61% (11/18) . It suggested a better reliability of the yeast two-hybrid method .Study of protein-protein interaction network of proteins can reveal the function of unknown function or mechanism of clues. To investigate the construction of the network, we used a variety of experiments on two proteins which belong to two types of protein networks: SLC25A40 (referred to as MCFP) and IMMT (also known as mitofilin) to conduct a study of the function.MCFP is a new member of the mitochondrial transporter protein family. They are synthesized in the cytoplasm, then translocated to mitochondrial and located in the inner membrane by specific transport mechanism.These proteins are responsible for the transportation of small molecules across the mitochondrial inner membrane. They have a wide range of substrates, including ATP, H +, amino acids, folic acid, carnitine / acyl-carnitine, and so on, but the transfer substrates and physiological functions of MCFP remain unclear. By using blue native gel electrophoresis , RNAi and mass chromatographic analysis, we found that down-regulating expression of MCFP in HEK293 cell could lead to some molecules reduction, such as ATP synthase activity, ATP production, mitochondrial reactive oxygen species, SOD activity, and to decrease cell viability. Our results implied that MCFP was a member of mitochondrial function closely related proteins.Tim23 complex is one of the major mitochondrial protein transport complexs in the mitochondrial inner membrane. Most of the mitochondrial matrix proteins and part of the mitochondrial proteins are transported by the Tim23 complex. Tim23 is the core pore protein of the Tim23 complex, which is essential for mitochondrial protein transport. Study shows that Tim23 defection in Tim23 complex is closely related to certain neurodegenerative diseases. IMMT, which is an important mitochondrial inner membrane protein, plays a key role in maintaining the ridge of mitochondrial inner membrane. We proved the interaction between IMMT and Tim23 by GST-pull down, CO-IP and laser confocal microscopy. Using strategy of lentivirus infections, we succeeded in constructing Hela cells stably expressed IMMT. Based on it, we investigated that overexpression of IMMT affected apoptosis and change of Tim23 expression level after treatment of Hela cells within Epirubicin (1μg/ml) . Study showed that over-expression IMMT could effectively inhibit Tim23 degradation under Epirubicin , and retard the process of apoptosis. This study established the fundament for clariting the function of interaction between IMMT and Tim23, on another hand, it provided new strategies for treatment of Neurodegenerative disease associated with Tim23 defection .

Research of Gene Function Based on Protein-Protein Interction Network of Mitochondrial Intermembrane Space

Abstract

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