Identification Of The Interaction Between Sterol Carrier Protein 2 (SCP2) And Phytanoyl-CoA Hydroxylase (Phyh)

All kinds of life information in vivo are passed to corresponding proteins by transcription and translation of different genes and obtain their own biochemical characteristics and biological activity; but each protein in cells does not perform its own function independently, they complete specific functions in the particular time and space through forming large complexes with other interacting proteins in cells, moreover, only in the form of complexes can some proteins play their roles. 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 change substrate specificity of protein; it can also inactivate other proteins, which regulates the expression of other genes. Only protein-protein interactions go smoothly can the normal course of life in cells be guaranteed. Therefore, the study of protein interactions is significant for the understanding of protein functions and clarifying the law of life.The methods used to study protein-protein interactions currently include yeast two-hybrid, in vitro binding assay, coimmunoprecipitation, tandem affinity purification, mass spectrometry, protein chip, bioinformatics-based analysis methods and so on. Among them, bioinformatics analysis is often in no need of concrete practice, on the contrary, it acquires a large amount of information by study in the whole genome scale. Therefore, bioinformatics analysis offers a new direction for the study on protein interactions and provides important clues for experiments. Early study in our laboratory found 22 mitochondrial proteins in mice with endotoxin shock by differential in-gel electrophoresis (DIGE) and mass spectrometry, the amount of which changed. Then protein-protein interactions among them were predicted by means of bioinformatics analysis. At last a pair of protein-protein complexes was found:sterol carrier protein 2 (SCP2)/phytane acid oxidase (Phyh). It was found that after LPS stimulated mice for 1h, both were downregulated in the mitochondria, and the reduced multiple was the same. Therefore, we predicted that they may transport out of mitochondria through a certain path together with each other or in the form of complexes with other proteins, and then participate in stress reaction to endotoxin. In addition, through analysis on the two proteins we also found that they had some common places, for example, they are both localized in peroxisome and mitochondria and participate in fatty acid metabolism. Based on these, we determined this study, that is, identification of of interaction between SCP2 and Phyh by use of relevant experimental methods. If the experiments prove that the two proteins interact with each other, it can indicate that they are involved in fatty acid metabolism in the form of protein complexes, and can also provide a new direction for the study of some metabolic diseases related to the two proteins.Phyh is mainly localized in peroxisome and mitochondria. It is responsible for the first reaction of peroxisome catalytic a-oxidation pathway, that is, transferring phytane coenzyme A into 2-hydroxy phytane acid coenzyme A. Previous studies have found that Phyh gene mutation could cause phytane acid oxidation disorder, which can lead to a range of symptoms of kidneys and nervous system in the body. At present the study focuses on relations between many types of gene mutations and Refsum’s disease. So far, it has already been reported abroad that several Phyh mutations, such as Arg275Trp, which means that in the 275th amino acid tryptophan takes the place of arginine. Mutated Phyh loses phytane acid oxidation activity, which can lead to phytane acid metabolism disorders and a large number of phytane acid accumulation in various tissues and body fluids, and then result in Refsum’s disease, ceribro-hepato-renal syndrome, rhizomelic chondrodysplasia, neonatal adrenoleukodystrophy (NALD) and other peroxisome-related metabolic diseases. In addition, studies have found that Phyh protein can interact with some proteins, which can lead to Rafer prime Farm disease. For example, PAHX-AP# 1 protein in brain tissue can interact with Phyh protein, which has certain relationship with symptoms of nervous system in Rafer prime Farm.SCP2, abundant in the liver, mainly exits in the mitochondria, peroxisome, endoplasmic reticulum and cytoplasm. On the one hand, as a regulatory factor in cholesterol metabolism, it involves in cholesterol biosynthesis and conversion of cholesterol into bile acids, cholesterol esters and steroid hormones; on the other hand as a cholesterol transporter, it involves in cholesterol transport in cells and among plasma membranes, and can directly transport newly synthesized cholesterol rapidly in liver from the endoplasmic reticulum to bile. All along, many studies show that high expression level of SCP2 is an important reason for cholesterol stone formation.To prove whether Phyh and SCP2 interact with each other, we verified the interaction in vitro and vivo. Firstly, we constructed prokaryotic and eukaryotic expression plasmids of Phyh, pGEX-4T-Phyh and pcDNA3-HA-Phyh respectively; prokaryotic and eukaryotic expression plasmids of SCP2, pET14b-SCP2 and pcDNA3-Flag-SCP2 respectively. Next, to study whether they can interact with each other in vitro, the vitro binding assay was conducted and we found that both can be specifically bind to each other. Then, we verified the interaction in vivo. At first we used immunofluorescence to observe the expression and co-localization of both in the mitochondria. The results showed that both can be located in the mitochondria, and co-localization exited. Then we used the co-immunoprecipitation technology to investigate whether they can bind in vivo, and we found that they can not bind to each other in NIH3T3 cells. Therefore, we assumed that the two can only interact with each other in certain stress stimuli. We added two experimental groups, LPS and NaAsO2 stimulation for 1h in NIH/3T3 cells respectively, the results are the same as before. After analysis on the results, we thought that as a result of complex cellular circumstances, Phyh and SCP2 can only interact with each other in special conditions or in the action of other cellular substances; or the interaction is so mild and transient that it can not be verified through current experiments. We should still do further research to verify the interaction between them.In summary, to verify whether there exists interaction between Phyh and SCP2 or not, we first build the prokaryotic and eukaryotic expression plasmid of Phyh and SCP2, followed by observing their binding in vitro through the vitro binding assay, and then we observed the co-localization situation of the two proteins in cells by immunofluorescence. Finally, we verify the existence of interaction between the two in vivo by the co-immunoprecipitation experiment.Though the study above, we drew conclusions as follows:1. The vitro binding assay showed that Phyh and SCP2 can specifically combine with each other in vitro.2. The immunofluorescence assay showed that Phyh and SCP2 were co-localized in the mitochondria.3. Co-immunoprecipitation experiments show that the Phyh and SCP2 can not specifically bind to each other in vivo, as a result, further study should be made to prove whether they have interactions in vivo.The findings in this issue enriched the information on protein-protein interactions and provided new ideas and methods for the study of new protein-protein interactions.