锌通过SREBP旁路调节线虫中SCDs和脂肪积累的研究

	锌通过SREBP旁路调节线虫中SCDs和脂肪积累的研究
其他题名	Zinc bypasses SREBP to directly regulate SCD activity and fat accumulation in C. elegans
	张静静
学位类型	博士
导师	梁斌
	2016-07
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	Srebps 遗传抑制子图位克隆锌离子 Scds 脂肪代谢
其他摘要	固醇调节元件结合蛋白（Sterol regulatory element binding proteins，SREBPs）是属于bHLH-Zip转录因子家族的成员，是调控脂肪酸、甘油三酯和胆固醇等脂类代谢的重要转录因子。早期研究表明SREBPs与代谢性疾病，如肥胖、2-型糖尿病、动脉粥样硬化、高血脂症和脂肪肝等密切相关。近年来，关于SREBPs的调控网络越来越受到人们关注，对于其靶基因、调节机制和抑制剂等均有研究，但是对于其遗传抑制子至今尚无报道。根据遗传抑制子的作用方式可以将其归为两大类，基因内抑制和基因外抑制。基因内抑制子主要通过回复突变、第二位点代偿性突变和顺式作用下的显性突变等发挥功能；基因外抑制的作用途径主要是无义介导的衰变途径紊乱、旁路抑制、非特异性的生理效应、基因剂量的改变和调制器的活性等。由于遗传抑制子的各种各样的作用方式，对某一关键基因的遗传抑制子的研究成为揭示该基因的表达、功能及其相互作用因子的强而有力的手段。因此，寻找SREBPs的遗传抑制子，将加深和拓展我们对SREBPs调控脂代谢机理、以及肥胖相关代谢性疾病发病机理的认识，同时可能发掘出新的脂代谢调控基因，并为这些疾病的预防和治疗提供新的作用靶点。模式生物秀丽线虫由于其与哺乳动物小鼠和人中的能量代谢通路高度保守，且具有大规模遗传筛选的优势，逐渐成为研究脂肪代谢和进行遗传筛选的模式生物。而且秀丽线虫中有SREBPs的同源基因sbp-1，其功能和哺乳动物中的SREBPs类似。因此，我们利用秀丽线虫sbp-1基因突变体，通过甲基磺酸乙酯（Ethylmethylsulfone，EMS）随机诱变筛选到14个SREBPs的遗传抑制子，并根据线虫的单核苷酸多态性，采用分离分群分析法和全基因重测序的方法，对其中三个抑制子（kun82、kun83和 kun84）进行图位克隆，寻找抑制子的候选基因，结果表明kun82是在smg-2基因第七个外显子上发生错义突变（G805D），使该基因所编码的Upf1同源蛋白质功能缺失，进而影响包含有过早翻译终止密码子的RNA降解途径；kun83是在dhc-1第八个外显子发生变异（A2443T），dhc-1编码一种胞质动力蛋白的重链；kun84在基因sur-7第四个外显子上发生突变（P191L），sur-7编码蛋白属于阳离子转运蛋白家族成员，与重金属锌离子的跨膜运输相关。之后我们重点针对遗传抑制子sur-7通过遗传学和分子生物学等方法阐明sur-7与sbp-1调控脂代谢的相互作用机制进行深入研究。结果如下：1，我们通过遗传杂交方法构建sur-7基因不同位点突变体与sbp-1(ep79) 的双突变株sur-79(tm6523);sbp-1(ep79) 和sur-79(ku119);sbp-1(ep79)，经尼罗红固定染色、气相色谱和薄层层析等方法表明sur-7能够恢复sbp-1功能缺失导致的表型，如脂滴大小和脂肪含量恢复至野生型水平、SCDs活性及其产物C18:1-n9含量也恢复至野生型水平、生长发育也部分恢复。同时我们还发现sur-7所属的阳离子扩散家族中另一成员cdf-1也能够抑制突变体sbp-1(ep79) 的表型。 2，通过构建sur-7(tm6523);KQ377(Psbp-1::sbp-1::gfp) 进行荧光观察和qPCR检测sur-7突变背景下sbp-1的转录水平的结果表明sur-7既不影响sbp-1在转录水平的表达，也不影响sbp-1的蛋白水平和成熟加工。3，综上，我们推测sur-7通过旁路抑制sbp-1的作用影响线虫的脂肪堆积，并通过实验验证了这一推测的正确性，验证方法及结果如下：3.1）我们通过外源添加硫酸锌和螯合剂TPEN的方法，通过尼罗红固定染色及薄层层析分析的方法，发现sur-7和cdf-1对sbp-1的抑制作用依赖于锌离子的正常水平，且锌离子水平在一定条件下影响线虫的脂肪代谢，如锌过载致使单突变体sur-7和cdf-1的脂肪含量降低，相反地，锌离子的缺乏能够增大线虫中的脂滴、增加线虫中脂肪的积累；3.2）采用RNA干扰的方法筛选sbp-1的下游靶基因并利用气相色谱分析法检测脂肪酸含量的变化，结果表明锌离子水平通过影响线虫中SCDs的活性，改变单不饱和脂肪酸（C16:1-n7和C18:1-n9）与饱和脂肪酸（C16:0和C18:0）的比例，进而影响线虫中甘油三酯的堆积；3.3）通过RNA干扰降低脂肪酸内源合成途径相关基因acs-1的表达，线虫中SCDs的活性降低，进而使线虫中脂滴减小、甘油三酯的含量降低，恢复sur-7对sbp-1的遗传抑制作用，抵抗锌离子缺乏导致的脂肪积累；3.4）此外，我们还发现sur-7对sbp-1的遗传抑制作用依赖于脂肪酸合成相关基因let-767和lpin-1的正常表达。而锌离子缺乏导致的脂滴增大和脂肪积累部分依赖于let-767。结论：线虫中锌离子绕过SREBP通过旁路途径直接调控SCDs的活性，影响脂肪酸的内源合成，进而调节脂肪的代谢; Sterol regulatory element binding proteins (SREBPs) are members of membrane-bound, basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors family. SREBPs play a key role in regulating synthesis of fatty acid, cholesterol, triglycerides, and phospholipids. Early studies demonstrated that SREBPs controlled lipogenesis founction and closely associated with metabolic diseases like obesity, type 2 diabetes, atherosclerosis, hyperlipidemia, fatty liver and so on. In recent years, scientists paid more attention to the regulation and control network of SREBPs, which has been studied for its target genes, regulation mechanism and inhibitors, but it has no reported about the genetic suppressor of SREBPs so far. Many different kinds of suppression have been reported, according to the modes of suppressed action, the suppressors can be divided into intragenic and extragenic classes. Intragenic types include same-site replacement, compensatory second site mutation, alteration in splicing, and dominant mutations by loss-of-function in cis. Extragenic suppression can occur by a variety of informational mechanisms, such as loss of nonsense-mediated decay (NMD), bypass, non-physiological effects, dosage effects, modulators of activity and so on. Because of the various informations of the suppression, genetic suppressors provide some of the most powerful tools available for exploring gene expression, function and interaction. Therefore, the suppressors of SREBPs are extremely important to reveal the roles of SREBPs in the regulation of lipid metabolism, and to provide new insights into the pathogenesis of metabolic disease. As well as may bring new potential target for prevention and therapy of these diseases.sbp-1, a homologue of mammalian srebp-1 in C. elegans, has similar functions like SREBPs. In a highly conserved mechanism, sbp-1(ep79), which contained a genetic deletion of 2800bp in C-terminal, was decreased fat content, slow growth, egg less and changed fatty acids content. In addation to the highly conservative energy metabolism pathways with mammalian and the advantage of easier for large scale genetic screening. Thus, we took the advantages of genome screen in C. elegans to search for the genetic suppressors of sbp-1(ep79) mutation by Ethylmethylsulfone (EMS). Eventually, we got 14 mutants that significantly rescued the fat storage and fatty acid compositions of sbp-1(ep79) to wild type (WT). Then, we mapped and cloned three mutants (kun82, kun83 and kun84) by Bulk Segregation Analysis (BSA) and whole-genome sequencing (WGS). The results showed that smg-2(kun82) contented a missense mutation (G805D) on the seventh exon, which encodes a homologous of Upf1, a key member of complex about NMD pathway. dhc-1, encodes a cytoplasmic dynein heavy chain was considered as kun83. sur-7 that encodes a slightly divergent member of the cation diffusion facilator (CDF) family of transmembrane proteins, which regulate the transport of heavy metal ions like zinc, was the candidate gene of kun84, which occurred a missense mutation at P191L. Then we focused on the question of how did sur-7 regulate fat metabolism with sbp-1 in C.elegans by the methods of genetics and molecular biology. The results as follows. 1, We respectively crossed sur-7(tm6523) and sur-7(ku119) with sbp-1(ep79), then using the methods of Nile red staining fixed, gas chromatography and thin-layer chromatography and others, we confirmed that sur-7 was a suppressor of sbp-1, it can rescue the phenotypes of sbp-1(ep79), such as the size of lipid droplets enlarged, the fat content increased to similar with the wild type, the activity of SCDs enhanced, the content of C18:1-n9 also increased, and the growth rate was rescued too. At the same time, we also found cdf-1, another member belongs to the family of CDF, like sur-7, can rescue the phenotypes of sbp-1(ep79).2, We crossed sur-7(tm6523) with KQ377(Psbp-1::sbp-1::GFP) then observed the fluorescence of sbp-1 in sur-7(tm6523);KQ377(Psbp-1::sbp-1::GFP). As well as the detected sbp-1 mRNA level when limited sur-7 by qPCR, the results showed that sur-7 didn’t affected the expression of sbp-1, beyond that, sur-7 neither regulated the protein level or the mature processing of SBP-1.3, According to above results, we hypothesized that sur-7 bypassed SBP-1 to regulated the fat accumulation in C.elegans, and the correctness of this theory was verified by experiment, and the results are as follows: 3.1) We tested whether superfluous or deficiency zinc effected lipid droplets and fat accumulation, by adding zinc sulfate or TPEN which is a Zn-chelate into NGM plates. The results suggested that as the gentic suppressors of sbp-1, sur-7 and cdf-1 rescued phenotypes of sbp-1(ep79) dependence on the normal or lower concentration of zinc. Then we found that zinc regulated the fat metabolism of nematodes under certain conditions. For instance, zinc overload caused fat content reduced in single mutant sur-7 and cdf-1, but not in wild type and sbp-1. On the contrary, zinc deficiency led to the lipid droplets enlarged and the fat accumulation increased.3.2) We screened the target genes of sbp-1 by RNA interference, and detected the changes of fatty acids content by gas chromatography. The results showed that zinc controlled the ratio of monounsaturated fatty acid (C16:1-n7 and C18:1-n9) of and saturated fatty acid (C16:0 and C18:0) by directly regulated the activity of SCDs. Thereby zinc regulated the accumulation of triglyceride in C.elegans. 3.3) When knockdown the expresssion of acs-1 by RNA interference, which played an important role in the fatty acid synthetic pathway, the activity of SCDs was weakened, the lipid droplets were diminidhed and decreased, the content of triglyceride was reduced. acs-1(RNAi) rescued the phenotypes of sur-7;sbp-1 to similar with sbp-1, and resisted fat accumulation caused by zinc deficiency.3.4) In addition, we also found that sur-7 suppressed sbp-1 in dependence on the fatty acid synthesis related genes, let-767 and lpin-1. While the lipid droplets and fat accumulation caused by zinc deficiency were partially dependent on let-767.Conclusion: sur-7 and cdf-1 were genetic suppressors of sbp-1, all the three regulated zinc level, and zinc bypassed SBP-1 to regulate the activity of SCDs and the endogenous synthesis of fatty acids, then regulated the lipid metabolism in C.elegans.
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/11970
专题	科研部门_脂类代谢与疾病（梁斌）
作者单位	中国科学院昆明动物研究所
推荐引用方式 GB/T 7714	张静静. 锌通过SREBP旁路调节线虫中SCDs和脂肪积累的研究[D]. 北京. 中国科学院研究生院,2016.

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