本文選題：OLA1(Obg　切入點(diǎn)：like　出處：《浙江大學(xué)》2017年博士論文

【摘要】：研究目的:Obg-likeATPase 1(OLA1)是一種GTP酶,屬于TRAFAC(翻譯因子相關(guān))類,Obg-HfIX超家族中的YchF亞群,具有水解GTP和ATP的活性,結(jié)合核糖體調(diào)節(jié)蛋白質(zhì)翻譯,在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、胞內(nèi)運(yùn)輸、細(xì)胞應(yīng)激及胚胎發(fā)育中起著重要作用。本課題組前期研究表明OLA1缺失可以引起小鼠不全性圍生期死亡,僅約20%Ola1-/-小鼠得以存活,而這些小鼠成年后表現(xiàn)為"個(gè)頭小"。本研究擬進(jìn)一步分析并尋找成年小鼠"個(gè)頭小"的原因,并通過(guò)建立組織特異性敲除小鼠的模型以便研究OLA1在特定組織器官中的功能。研究?jī)?nèi)容和方法:采用基因捕獲及Cre-loxP技術(shù)分別建立Ola1基因全身及組織特異性敲除的小鼠(Ola1-/-、AKO),以野生型小鼠(Ola1+/+)為對(duì)照,觀察基礎(chǔ)狀態(tài)及飲食誘導(dǎo)下小鼠表型的變化。采用原代培養(yǎng)方法建立小鼠胚胎成纖維細(xì)胞系(MEF),并用反相蛋白芯片技術(shù)(Reverse Phase Protein Assay,RPPA)從中篩選與OLA1相互作用的蛋白。結(jié)合Ola1-/-小鼠表型以及RPPA結(jié)果,篩選出相關(guān)分子并于細(xì)胞、組織水平驗(yàn)證。隨后,我們將對(duì)OLA1調(diào)節(jié)相關(guān)靶蛋白功能的分子機(jī)制進(jìn)行初步的探索。研究結(jié)果:前期我們課題組已經(jīng)報(bào)道了Ola1全身敲除小鼠表現(xiàn)為胚胎發(fā)育遲緩及圍生期高致死率,表明OLA1在生長(zhǎng)發(fā)育中的重要作用。通過(guò)對(duì)成年小鼠的進(jìn)一步分析發(fā)現(xiàn):相比于野生型小鼠,Ola1-/-小鼠仍表現(xiàn)為體型減小,各器官組織成比例減小,但脂肪組織減少更明顯,尤其是白色脂肪組織,呈現(xiàn)明顯"瘦"的表型。那么引起Ola1-/-小鼠白色脂肪組織減少的原因是OLA1影響了脂肪組織代謝,抑或是其他?于是我們從兩個(gè)方面進(jìn)行了驗(yàn)證。首先,我們采用Cre-loxp技術(shù)構(gòu)建了Ola1基因脂肪組織特異性敲除(Adipose Tissue Specific Knockout,AKO)小鼠模型。與其對(duì)應(yīng)野生型小鼠相比,在普通飲食及高脂飲食誘導(dǎo)下,AKO小鼠脂肪組織含量及相關(guān)代謝指標(biāo)與野生型小鼠相比均無(wú)明顯差異。這些結(jié)果表明OLA1不直接影響脂肪組織的代謝相關(guān)功能如脂肪組織新生,脂肪組織OLA1缺失不影響小鼠脂肪含量。另一方面,通過(guò)間接能量消耗測(cè)定、進(jìn)食監(jiān)測(cè)及小鼠認(rèn)知行為相關(guān)實(shí)驗(yàn),我們發(fā)現(xiàn)Ola1-/-小鼠能量消耗增加,進(jìn)食減少,自主活動(dòng)明顯增加,認(rèn)知記憶能力下降。據(jù)此,我們認(rèn)為Ola1-/-小鼠脂肪組織含量減少的原因在于自主活動(dòng)增加、進(jìn)食減少所致的全身能量消耗增加。接著,我們利用RPPA技術(shù)比較了 Ola1+/+與Olal-/-MEF細(xì)胞中蛋白的差異性表達(dá),發(fā)現(xiàn)GSK-3β的表達(dá)及活性在Ola1-/-MEF中明顯增高。隨后應(yīng)用western blotting我們驗(yàn)證了在OLA1缺失或下調(diào)的細(xì)胞中及胚胎組織中GSK-3β蛋白水平及活性增高。同時(shí)IHC結(jié)果表明Ola1-/-小鼠的胚胎及成年腦組織中GSK-3β亦增高。Ola1-/-小鼠腦中GSK-3β表達(dá)及活性增高可以導(dǎo)致其自主活動(dòng)增加,進(jìn)食減少,使全身能量代謝失衡,作為能量?jī)?chǔ)存載體的脂肪組織含量減少。隨后我們以MEF細(xì)胞為基本工具,應(yīng)用qRT-PCR、polysome profiling分別從轉(zhuǎn)錄水平、翻譯水平研究了 OLA1對(duì)GSK-3β表達(dá)的調(diào)控,并結(jié)合放線菌酮追蹤試驗(yàn)(CHX Chase Assay)評(píng)估OLA1對(duì)GSK-3β蛋白穩(wěn)定性的影響,結(jié)果提示OLA1缺失后GSK-3β蛋白質(zhì)穩(wěn)定性增加進(jìn)而引起蛋白水平上調(diào)。進(jìn)一步,體外激酶抑制試驗(yàn)顯示OLA1可以直接抑制GSK-3β的活性;免疫共沉淀結(jié)果表明OLA1和GSK-3β之間有直接的結(jié)合作用。綜上所述,我們的研究首次闡明OLA1作為一種GTP酶,可以通過(guò)直接的相互作用促進(jìn)GSK-3β的降解并抑制其活性。結(jié)論與創(chuàng)新1.在前期本課題組所報(bào)道的全身性O(shè)laal基因敲除小鼠表型的基礎(chǔ)上,本研究進(jìn)一步發(fā)現(xiàn)了成年Ola1-/-小鼠的表型特征:作為能量存儲(chǔ)載體的脂肪組織含量減少,呈現(xiàn)出明顯"瘦"的體型。2.本研究成功構(gòu)建了 floxed Ola1 E2小鼠品系(Ola1f/-),為研究Olal基因在特定組織器官背景下的功能提供了基礎(chǔ),并且構(gòu)建了脂肪組織特異性敲除小鼠品系——AKO小鼠。AKO小鼠表現(xiàn)為正常的圍生期存活率,沒有生長(zhǎng)發(fā)育遲緩。AKO小鼠在普通飲食和高脂飲食誘導(dǎo)后,均未表現(xiàn)出脂肪組織含量的變化。這些結(jié)果表明OLA1在脂肪組織相關(guān)代謝過(guò)程中沒有直接的作用。3.本研究發(fā)現(xiàn)GSK-3β是OLA1作用的靶蛋白之一。OLA1可通過(guò)以下兩種機(jī)制調(diào)節(jié)GSK-3β的活性:調(diào)節(jié)GSK-3β的穩(wěn)定性影響其總蛋白水平;直接和GSK-3β相互作用抑制其激酶活性。通過(guò)闡明Ola1-/-小鼠腦中GSK-3β活性過(guò)度升高而導(dǎo)致其自發(fā)活動(dòng)增加,進(jìn)食減少,全身能量消耗增加,從而發(fā)現(xiàn)了 OLA1在機(jī)體能量代謝平衡中的間接作用。4.本研究進(jìn)一步為OLA1在腫瘤中的研究提供了基礎(chǔ)。GSK-3β參與許多信號(hào)通路如Wnt信號(hào)通路從而,而Wnt信號(hào)通路與多種腫瘤相關(guān)如結(jié)直腸癌等。因此,GSK-3β可作為橋梁為研究OLA1與其他相關(guān)腫瘤發(fā)生發(fā)展的關(guān)系奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Valentina RUBIO;;Knockdown of OLA1,a regulator of oxidative stress response,inhibits motility and invasion of breast cancer cells[J];Journal of Zhejiang University(Science B:An International Biomedicine，Biochemistry & Biotechnology Journal);2009年11期

2 ;Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells[J];Cell Research;2006年07期

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本文編號(hào)：1686873