【作者】 罗斌；

【导师】 王业富； 齐义鹏；

【作者基本信息】 武汉大学 ， 生物学、生物医药工程， 2013， 博士

【摘要】 肥胖症问题越来越受到世界关注,同时它还伴随发生心血管疾病和脂质代谢失常等疾病。通常肥胖患者血液内都呈现高水平leptin, leptin的抵抗是造成肥胖症的关键诱因之一,而细胞内多余的SOCS3又是造成leptin抵抗的主要原因。细胞负反馈调控因子SOCS蛋白参与细胞内各种信号通路的调控,SOCS1-SOCS7以及CIS这八个蛋白组成了SOCS3家族。通过对它们氨基酸序列比对发现：它们均包含一个SH2结构域和一个C端较保守的SOCS-box功能区域,这两个功能性结构域对SOCS家族成员十分重要,它们是SOCS蛋白调控细胞内各种信号通路的主要功能区域。SOCS3是一个重要的负反馈调节蛋白,它参与IL-6、leptin、促红细胞生成素和胰岛素等信号途径的调控。SOCS3蛋白能够通过自身的SH2结构与JAK家族的酪氨酸激酶结合直接抑制其酶活性或者SOCS3结合信号分子将其带入蛋白酶降解途径,从而抑制细胞内的信号通路。leptin是一个脂肪组织分泌的细胞因子,其功能是维持体内能量平衡和降低食物摄取量,它通过血脑屏障与位于下丘脑的受(体瘦素受体,简称OB-R)结合,引起STAT1和STAT3活化。磷酸化的STAT3能够诱导SOCS3基因表达。通常肥胖症患者血液中的leptin含量高,能长时间持续的刺激OB-R使得细胞内SOCS3过表达。SOCS3作为leptin信号通路负反馈机制,过剩的SOCS3和JAK结合直接抑制酶的活性,并且将JAK信号分子带入蛋白酶降解途径。SOCS3的基因敲除小鼠用高脂饲料喂养后,其体重和脂肪含量减轻,因此,SOCS3可能在能量代谢和脂肪酸氧化中扮演重要角色。然而,SOCS3调控目标蛋白的机制还研究得不够透彻。为了进一步研究SOCS3蛋白抑制细胞因子信号转导的机制和其潜在的肥胖症治疗功能,我们以表达纯化的HIS-SOCS3为诱饵蛋白利用噬菌体展示技术从人肝的cDNA文库中筛选能与其相互作用的短肽。通过对筛选出来的一条短肽的序列RGGVVTSNPLGF分析,我们发现该短肽序列与编码VLCAD蛋白C末端644-655位的12个氨基酸序列相同,暗示SOCS3与VLCAD艮可能具有相互作用。为了证明这一点,我们用酵母双杂交系统构建了两个表达质粒pGBKT7-SOCS3和pGADT7-VLCAD,共转化酵母菌AH109,我们发现,重组菌落能够在-Trp-Leu-His的平板上生长,β半乳糖苷酶显色实验阳性。实验结果充分证明SOCS3蛋白能与VLCAD蛋白相互作用。我们首次发现了超长链乙酰辅酶A (VLCAD)是一个重要的SOCS3结合蛋白。乙酰辅酶A脱氢酶属于线粒体中的黄素酶,具有催化脂肪酸β氧化起始反应的活性。细胞中存在四种不同乙酰辅酶A脱氢酶：短链乙酰辅酶脱氢酶(SCAD),中链乙酰辅酶脱氢酶(MCAD),长链乙酰辅酶脱氢酶(LCAD)和超长链乙酰辅酶脱氢酶(VLCAD)位于462位的GLU被认为是酶的重要活性中心。其中以VLCAD活性最强。通过氨基酸序列比较发现,这四种乙酰辅酶A脱氢酶的C-端都含有加长的25KD尾巴。当棕榈酸乙酰A作为酶的底物时,VLCAD的活性比LCAD高10倍。目前对这条25KD的特殊序列的功能还不了解。进一步,我们还通过体外GST pull-down实验和体内免疫共沉淀实验,验证了SOCS3蛋白与VLCAD蛋白的相互作用。为了深入研究这种相互作用的功能区,我们又将VLCADC端含有220个氨基酸的extra-peptide构建至真核表达载体pCMV-HA上,转染至小鼠巨噬细胞RAW264.7,用LPS刺激细胞,刺激时间依次为1h,2h,4h和8h,细胞裂解后Western blot检测蛋白的表达情况。我们发现,由于LPS的刺激诱导了SOCS3蛋白的表达,同时,相应的减少了VLCAD的表达,这意味着VLCAD的C端部分能够被SOCS3蛋白降解。其降解机制估计是由于SOCS3蛋白能够结合VLCADC端25KD的尾巴,并且将其带入到蛋白酶体降解途径。为了证明SOCS3蛋白参与VLCAD的降解而带来的减肥功效,我们合成了VLCADC末端的12个氨基酸短肽,将其从腹腔注射至昆明小鼠体内,每三天加强注射一次并称量小鼠体重,24天后我们观察到注射PBS的空白对照小鼠体重增加最为显著,注射随机十肽的对照小鼠体重增加次之,而注射VLCAD特异性12短肽的小鼠体重增加最为缓慢。同时我们还检测小鼠血液中的总甘油三脂(TG)和总胆固醇(TC)含量作为减肥的重要生理指标,我们发现注射VLCAD特异性12短肽小鼠的TG和TC含量比对照小鼠显著降低,具有一定的减肥作用。减肥功能的机制可能是由于合成的特异性12短肽在小鼠体内竞争性结合了SOCS3蛋白,从而释放更多的VLCAD酶分子,VLCAD是脂肪酸β氧化反应第一步的限速酶,因此加快了体内更多脂肪酸的代谢速度。而降低了老鼠的体重。综合以上结果,我们的研究首次证明了SOCS3蛋白与超长链乙酰辅酶A相互结合,结合的功能区是VLCAD-C末端12氨基酸。随着SOCS3与VLCAD-C末端25KD尾巴的结合而将VLCAD带入泛素化降解途径,影响VLCAD的正常功能而降低机体脂肪酸代谢。这可能是解释为什么肥胖症患者脂肪酸代谢缓慢的重要原因之一。我们认为,SOCS3是脂肪β氧化反应代谢重要负调控因子和治疗肥胖症潜在的药物作用位点。合成的VLCAD-C末端12氨基酸短肽也有可能成为治疗肥胖症的重要靶标药物。更多还原

【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.更多还原