OLA1抑制GSK-3β的活性维持小鼠脂肪含量稳定

发布时间：2018-03-30 17:21

本文选题：OLA1(Obg　切入点：like　出处：《浙江大学》2017年博士论文

【摘要】：研究目的:Obg-likeATPase 1(OLA1)是一种GTP酶,属于TRAFAC(翻译因子相关)类,Obg-HfIX超家族中的YchF亚群,具有水解GTP和ATP的活性,结合核糖体调节蛋白质翻译,在细胞信号转导、胞内运输、细胞应激及胚胎发育中起着重要作用。本课题组前期研究表明OLA1缺失可以引起小鼠不全性围生期死亡,仅约20%Ola1-/-小鼠得以存活,而这些小鼠成年后表现为"个头小"。本研究拟进一步分析并寻找成年小鼠"个头小"的原因,并通过建立组织特异性敲除小鼠的模型以便研究OLA1在特定组织器官中的功能。研究内容和方法:采用基因捕获及Cre-loxP技术分别建立Ola1基因全身及组织特异性敲除的小鼠(Ola1-/-、AKO),以野生型小鼠(Ola1+/+)为对照,观察基础状态及饮食诱导下小鼠表型的变化。采用原代培养方法建立小鼠胚胎成纤维细胞系(MEF),并用反相蛋白芯片技术(Reverse Phase Protein Assay,RPPA)从中筛选与OLA1相互作用的蛋白。结合Ola1-/-小鼠表型以及RPPA结果,筛选出相关分子并于细胞、组织水平验证。随后,我们将对OLA1调节相关靶蛋白功能的分子机制进行初步的探索。研究结果:前期我们课题组已经报道了Ola1全身敲除小鼠表现为胚胎发育迟缓及围生期高致死率,表明OLA1在生长发育中的重要作用。通过对成年小鼠的进一步分析发现:相比于野生型小鼠,Ola1-/-小鼠仍表现为体型减小,各器官组织成比例减小,但脂肪组织减少更明显,尤其是白色脂肪组织,呈现明显"瘦"的表型。那么引起Ola1-/-小鼠白色脂肪组织减少的原因是OLA1影响了脂肪组织代谢,抑或是其他?于是我们从两个方面进行了验证。首先,我们采用Cre-loxp技术构建了Ola1基因脂肪组织特异性敲除(Adipose Tissue Specific Knockout,AKO)小鼠模型。与其对应野生型小鼠相比,在普通饮食及高脂饮食诱导下,AKO小鼠脂肪组织含量及相关代谢指标与野生型小鼠相比均无明显差异。这些结果表明OLA1不直接影响脂肪组织的代谢相关功能如脂肪组织新生,脂肪组织OLA1缺失不影响小鼠脂肪含量。另一方面,通过间接能量消耗测定、进食监测及小鼠认知行为相关实验,我们发现Ola1-/-小鼠能量消耗增加,进食减少,自主活动明显增加,认知记忆能力下降。据此,我们认为Ola1-/-小鼠脂肪组织含量减少的原因在于自主活动增加、进食减少所致的全身能量消耗增加。接着,我们利用RPPA技术比较了 Ola1+/+与Olal-/-MEF细胞中蛋白的差异性表达,发现GSK-3β的表达及活性在Ola1-/-MEF中明显增高。随后应用western blotting我们验证了在OLA1缺失或下调的细胞中及胚胎组织中GSK-3β蛋白水平及活性增高。同时IHC结果表明Ola1-/-小鼠的胚胎及成年脑组织中GSK-3β亦增高。Ola1-/-小鼠脑中GSK-3β表达及活性增高可以导致其自主活动增加,进食减少,使全身能量代谢失衡,作为能量储存载体的脂肪组织含量减少。随后我们以MEF细胞为基本工具,应用qRT-PCR、polysome profiling分别从转录水平、翻译水平研究了 OLA1对GSK-3β表达的调控,并结合放线菌酮追踪试验(CHX Chase Assay)评估OLA1对GSK-3β蛋白稳定性的影响,结果提示OLA1缺失后GSK-3β蛋白质稳定性增加进而引起蛋白水平上调。进一步,体外激酶抑制试验显示OLA1可以直接抑制GSK-3β的活性;免疫共沉淀结果表明OLA1和GSK-3β之间有直接的结合作用。综上所述,我们的研究首次阐明OLA1作为一种GTP酶,可以通过直接的相互作用促进GSK-3β的降解并抑制其活性。结论与创新1.在前期本课题组所报道的全身性Olaal基因敲除小鼠表型的基础上,本研究进一步发现了成年Ola1-/-小鼠的表型特征:作为能量存储载体的脂肪组织含量减少,呈现出明显"瘦"的体型。2.本研究成功构建了 floxed Ola1 E2小鼠品系(Ola1f/-),为研究Olal基因在特定组织器官背景下的功能提供了基础,并且构建了脂肪组织特异性敲除小鼠品系——AKO小鼠。AKO小鼠表现为正常的围生期存活率,没有生长发育迟缓。AKO小鼠在普通饮食和高脂饮食诱导后,均未表现出脂肪组织含量的变化。这些结果表明OLA1在脂肪组织相关代谢过程中没有直接的作用。3.本研究发现GSK-3β是OLA1作用的靶蛋白之一。OLA1可通过以下两种机制调节GSK-3β的活性:调节GSK-3β的稳定性影响其总蛋白水平;直接和GSK-3β相互作用抑制其激酶活性。通过阐明Ola1-/-小鼠脑中GSK-3β活性过度升高而导致其自发活动增加,进食减少,全身能量消耗增加,从而发现了 OLA1在机体能量代谢平衡中的间接作用。4.本研究进一步为OLA1在肿瘤中的研究提供了基础。GSK-3β参与许多信号通路如Wnt信号通路从而,而Wnt信号通路与多种肿瘤相关如结直肠癌等。因此,GSK-3β可作为桥梁为研究OLA1与其他相关肿瘤发生发展的关系奠定基础。
[Abstract]:Purpose: Obg-likeATPase 1 (OLA1) is a GTP enzyme, belonging to TRAFAC (translation factor), Obg-HfIX superfamily of YchF subgroups, with hydrolysis of GTP and the activity of ATP, binds to the ribosome to regulate protein translation in cell signal transduction, intracellular transport, plays an important role in cell stress and embryo development in the previous study showed that OLA1 deficiency can cause mice of perinatal death, only about 20%Ola1-/- mice survived, and these adult mice is shown as "small". This study intends to further analyze and find a "small mouse", and through the establishment of tissue specific knock in the mouse model for studying OLA1 in specific tissues and organs function. The research contents and methods: establish mice systemic and tissue specific knockout of the Ola1 gene by gene trapping and Cre-loxP Technology (Ola1-/-, AKO), in the wild Mice (Ola1+/+) as control, to observe the change of the basic condition and diet induced phenotype of mice. By using the method of primary culture of mouse embryonic fibroblast cell line (MEF), and protein chip technology (Reverse Phase Protein by Assay, RPPA) in the screening of proteins interacting with OLA1. Combined with the Ola1-/- phenotype of mice and the results of RPPA, screened and related molecules in the cell and tissue level verification. Then, it makes a preliminary exploration of molecular mechanism will be related to regulation of target protein function of OLA1. Results: our previous research group has reported Ola1 knockout mice exhibited systemic fetal growth retardation and perinatal mortality. OLA1 shows an important role in growth and development. Through further analysis of adult mice found: compared to wild-type mice, Ola1-/- mice showed decreased body organs, tissue decreased in proportion, but Adipose tissue decreased significantly, especially in white adipose tissue, showing obvious phenotype of "thin". Then the cause of white adipose tissue of Ola1-/- mice reduced the OLA1 of adipose tissue metabolism, or other? So we verified from two aspects. First, we use the Cre-loxp technology to construct Ola1 gene in adipose tissue specific knockout (Adipose Tissue Specific Knockout, AKO). The corresponding model mice compared to wild-type mice induced by normal diet and high-fat diet, AKO mouse adipose tissue content and related metabolic indexes compared with wild type mice showed no significant difference. These results suggest that OLA1 does not directly affect adipose tissue metabolism the function such as fat tissue of newborn, OLA1 deletion does not affect the content of fat in adipose tissue of mice. On the other hand, through the determination of indirect energy consumption, consumption monitoring and cognitive behavior in mice Related experiments, we found that Ola1-/- mice increased energy consumption, reduce consumption, autonomic activities increased significantly, decreased cognitive ability. Therefore, we believe that the reasons for the decrease of Ola1-/- content in adipose tissue of mice autonomic activities increased, eating whole body energy due to the reduction of consumption increase. Then, we use RPPA Technology to compare the differential expression of protein and Ola1+/+ in Olal-/-MEF cells, found that the expression and activity of GSK-3 beta increased significantly in Ola1-/-MEF. Then we verify the application of Western blotting GSK-3 protein level and activity increased in OLA1 deficient or down regulated cells and embryonic tissues. At the same time IHC results showed that Ola1-/- mouse embryonic and adult brain GSK-3 beta beta also increased GSK-3 the expression of.Ola1-/- in mouse brain and can lead to increased activity of the independent activity increase, reduce consumption, so that the body energy metabolism imbalance, as The energy stored in adipose tissue content of the carrier is reduced. Then we used MEF cells as the basic tool, the application of qRT-PCR and polysome profiling respectively from the transcription level and translation level on the regulation of OLA1 on the expression of GSK-3 beta, combined with cycloheximide (CHX Chase Assay) tracing test impact assessment OLA1 on GSK-3 protein stability results. These results suggest that OLA1 deficiency after GSK-3 beta protein stability increase and cause protein levels increase. Further, that OLA1 can directly inhibit GSK-3 beta activity in vitro kinase inhibition test; results show that the binding between OLA1 and direct GSK-3 beta co immunoprecipitation. In summary, our study is the first to clarify the OLA1 as a GTP enzyme, can be with each other a direct role in promoting degradation of GSK-3 beta and inhibit the activity of systemic Olaal gene. The conclusion and innovation 1. in our previous report table knockout mice Type on the basis of the further study found the phenotypic characteristics of adult Ola1-/- mice as adipose tissue content of energy storage carrier is reduced, figure.2. shows an obvious "thin" this study successfully constructed floxed Ola1 E2 mice (Ola1f/-), for the study of Olal gene in specific tissues and organs under the background of the function provides the basis, and build the adipose tissue specific knockout mice, AKO mice.AKO mice showed normal perinatal survival rate, no growth retardation in.AKO mice induced by normal diet and high fat diet, showed no changes in fat content. The results show that no direct role in.3. the OLA1 in adipose tissue metabolism in the process of this study found that GSK-3 beta OLA1 targeting protein of.OLA1 GSK-3 beta can be adjusted through the following two mechanisms: active stability regulation of GSK-3 beta. The total protein level; and direct interaction of GSK-3 beta inhibit its kinase activity. By explaining the excessive increase in the GSK-3 activity in mouse brain Ola1-/- due to the increased locomotor activity, reduce food intake and body energy consumption increased, leading to the discovery of the OLA1 indirect effects on energy metabolism balance in this study for further study of.4. in tumor the OLA1 provides the basis for.GSK-3 is involved in many signaling pathways such as Wnt pathway and Wnt pathway, which is associated with many kinds of tumors such as colorectal cancer. Therefore, GSK-3 can be used as a bridge between the beta of OLA1 and other related to the occurrence and development of tumor foundation.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.2

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