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Studies on the Function of Suppressor of Cytokine Signaling-3in the Fatty Acid β-oxidation

Author: LuoBin
Tutor: WangYeFu
School: Wuhan University
Course: Biology , biomedical engineering
Keywords: SOCS3 VLCAD Leptin-resistance Fatty acid β-oxidation Obesity
CLC: R589.2
Type: PhD thesis
Year: 2013
Downloads: 27
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Abstract

Obesity is a major global public health issue that has drawn the attention of physicians and other health care professionals; meanwhile it also has an association with cardiovascular disease, metabolic disorders and other diseases. The high level of leptin is reversely existed in the blood of obese patients. Leptin resistance as a key factor results in obesity. The current studies demonstrated that leptin-resistance is mainly attributed to the surplus SOCS3in the cell.Suppressor of cytokine signaling(SOCS)proteins have been involved in cytokine signaling as a family of negative regulators. The SOCS family comprises eight proteins, including SOCS1-SOCS7and CIS. Based on their amino acid sequence alignment, the result indicates that all the members of family contain a central SH2domain and a conserved C-terminal SOCS box. Those domains contain very important function, which has involved in the regulation of cytokine signaling by different mechanisms.SOCS3is thought as an important negative regulator of IL-6, leptin, erythropoietin and insulin signaling pathways. SOCS3protein binds to the activation loop of Jak family tyrosine kinases and directly inhibits their kinase activity through its SH2domain and kinase inhibitory region. SOCS3also can draw the activated signaling proteins into the the ubiquitination pathway for proteasomal degradation to inhibit signal pathway in cell. Leptin as a polypeptide, a product of the Ob gene, is an endocrine signal released from adipocytes to regulate body energy balance and reduce food intake. It can cross the blood-brain barrier to bind with the leptin receptor (OB-R) in the hypothalamus. Leptin induces activation of STAT1and STAT3through binding with its receptor OB-R. The phosphorylation of STAT3activates the expression of SOCS3gene. The high level of leptin is reversely existed in the blood of obese patients. The OB-R is continuously stimulated by the high level of leptin in the obese body, leading to the overexpresion of SOCS3in the cell. SOCS3as the negative feedback regulator of leptin signaling pathway not only directly bind to the JAK and inhibit the activity of enzyme, but also draw JAK into the ubiquitination pathway for proteasomal degradation. The experiment demonstrated that the fat content of mice with SOCS3gene knock out is lower than the mice without the SOCS3gene knock out after feeding the high fat diet. SOCS3may play an important part in the fatty acid oxidation and energe metabolism. The regulation mechanism of of SOCS3has not studied thoroughtly.To further investigate potential treatment of obesity and the suppression cytokine signal transduction mechanism of SOCS3protein, we developed T7select phage display system with purified HIS-SOCS3as bait to screen a human liver cDNA library in order to search for interaction of short peptides. After screening and sequencing analysis, we found that phage-presenting peptide RGGVVTSNPLGF show significant binding to SOCS3. The peptide sequence was similar to the sequence of amino acids644-655of C-terminal extra-polypeptide of very-long-chain acyl-CoA dehydrogenase (VLCAD). The result imply that SOCS3may interact with VLCAD. To further to prove it, We also carried out yeast two hybrid experiments to testify the interaction between SOCS3protein and VLCAD. First of all, we construct two pGBKT7-SOCS3and pGADT7-VLCAD expression plasmid for yeast hybrid system. Then, the yeast strain AH109which was transformed with pGBKT7-SOCS3and pGADT7-VLCAD expression plasmid grows on the-Trp-Leu-His plate. The β-galactosidase activity was measured. The result of experiment is positive. It indicated that SOCS3protein can bind with VLCAD in the yeast system. We identified protein VLCAD as a potential SOCS3interacting protein at the first time.The acyl-CoA dehydrogenases are mitochondrial flavoenzymes that catalyze the initial step in fatty acid β-oxidation. This reaction involves the2,3-dehydrogenation of acyl-CoA thioesters with formation of the trans-2-enoyl-CoA product. Four distinct acyl-CoA dehydrogenases, SCAD, MCAD, LCAD and VLCAD have been identified in cell. The location of462GLU is considered as an important catalytic residue, meanwhile VLCAD shows the highest central activity of enzyme in four distinct acyl-CoA dehydrogenases.Based on their amino acid sequence alignment, the sequence of VLCAD has the high similarity to SCAD (60%), MACD (48%), and LCAD (35%). VLCAD is a unique dehydrogenase in that it contains about25kDa of extra polypeptide at the carboxyl-terminal side compared to the other dehydrogenases, has a10-fold higher specific activity than that of LCAD toward palmitoyl-CoA as substrate. At present, the function of the special25KDa of extra polypeptide is still unknown.To confirm the interaction between SOCS3and VLCAD, we performed in vitro GST pull-down experiment and in vivo immunoprecipitation. In order to further research on the interaction of region, we construct pCMA-VLCAD-C-terminal extra polypeptide eukaryotic expression plasmid, which transfects into Mouse macrophages RAW264.7cells. After36h of culture, the cells were stimulated with LPS. Then, whole-cell lysates were immunoblotted with antibodies to HA-tag at1,2,4,8h after LPS stimulation. Our studies find that LPS definitely stimulates the SOCS3expression, meanwhile the reversely reduced extra-polypeptide expression was detected by immunoblotting with antibodies to HA-tag as the increasing expression of SOCS3under LPS stimulation. The degradation mechanism of VLCAD C-terminal probably is that extra-polypeptides that SOCS3-targeted25KDa VLCAD C-terminal extra-polypeptide entered the ubiquitination pathway for proteasomal degradationCompetitive binding inhibition of plus SOCS3protein in cell may be a potential treatment of obesity. Then, the12aa peptide of VLCAD C-terminal was synthesized and injected in the Kun Ming mice. The PBS and a scrambled12aa peptide were used as control. The weight of mice was measured every three days. After one month, the gain in weight of mice injected with12aa peptide of VLCAD C-terminal increased slowest. We also measure the total triglycerides (TG) and total cholesterol (TC) in the mice blood as important physiological index. Finally, we identified that TG and TC content of mice injected with specific VLCAD C-terminal12peptides are lower than the control group. Certainly, VLCAD C-terminal12peptides has the effect on the anti-obesity. The mechanism of anti-obesity is that the binding site of SOCS3is possibly competitive occupied by the synthetic VLCAD C-terminal12peptides, to prevent the interaction of SOCS3and VLCAD. Releasing more VLCAD involvement in fatty acid β-oxidation led to increasing weight loss and strengthening the fat metabolism. VLCAD is a rate-limiting enzyme in the long-chain fatty acid P-oxidation system.In conclusion, we for the first time demonstrated that SOCS3protein can interact with VLCAD, meanwhile the sequence of amino acids644-655of C-terminal extra-polypeptide of VLCAD as the binding area has a specific binding to SOCS3, which mediates the ubiquitination pathway for proteasomal degradation of25KDa VLCAD C-terminal extra-polypeptide and make the VLCAD disfunctional. These results explain why the fatty acid oxidation is slower in the obese body in the mechanism. SOCS3is an important factor for lipid metabolism and a potential drug-target for treatment of widespread obesity. This small peptide may potentially be useful therapeutics for the treatment of obesity.

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