背景應(yīng)激顆粒（Stress granules,SG）是細胞在應(yīng)激條件下形成的細胞質(zhì)聚集體,伴有整體翻譯抑制作用。SG包含RNA結(jié)合蛋白,凋亡/生存相關(guān)的mRNA和翻譯起始因子。SG的形成促進細胞存活,并參與許多人類病理機制。在哺乳動物中,有一個重要的信號通路稱為“Hedgehog（Hh）信號通路”。它在胚胎發(fā)育過程中很重要,它的損傷或過度活化可能導(dǎo)致多種發(fā)育異常和腫瘤的發(fā)生發(fā)展。Hh配體結(jié)合到Ptch1進而激活Hh信號通路,最終將GliFL轉(zhuǎn)換為GliA,并觸發(fā)Hh靶基因的表達。Gli轉(zhuǎn)錄因子家族受SPOP和Sufu調(diào)控。SPOP與Gli2/3相互作用并促進Gli2/3經(jīng)蛋白酶體途徑降解,而Sufu保護Gli2/3免受降解進而負調(diào)節(jié)Hh途徑。研究報道表明,SG通過招募特定的蛋白,例如Raptor,RACK1和TRAF2等來調(diào)節(jié)某些信號通路。這些蛋白被招募到SG中以調(diào)控應(yīng)激反應(yīng)和抑制細胞發(fā)生凋亡。以上信號通路和SG之間的相互作用驅(qū)使我們探究Hh信號通路和SG之間可能存在的功能互作。目的首次鑒定Hh信號通路蛋白SPOP和Sufu與應(yīng)激顆粒在功能上存在關(guān)聯(lián),這將有助于我們了解Hh信號通路和S... 

【文章來源】：浙江師范大學(xué)浙江省

【文章頁數(shù)】：77 頁

【學(xué)位級別】：碩士

【文章目錄】：
摘要
ABSTRACT
Literature Review
    1 Stress granules(SG)
        1.1 Stress granules assembly and significance
        1.2 Stress granules regulation and dynamics
        1.3 Stress granules and signaling pathways
    2 Hedgehog signaling pathway
        2.1 Characteristics of hedgehog pathway
        2.2 Hedgehog pathway signal transduction
        2.3 Significance of Hedgehog signaling
    3 SPOP Protein
        3.1 Structure of SPOP protein
        3.2 SPOP as a regulator of cellular function
        3.3 Liquid-liquid phase separation of SPOP with substrates
    4 Sufu Protein
        4.1 Structure of Sufu protein
        4.2 Regulation between Sufu and Gli
    5 Research purpose and significance
Chapter1 Unbiased Screen of Common Regulators of SG and Hh Signaling
    1 Experimental materials
        1.1 Cell strain
        1.2 Plasmids
        1.3 Main instruments
        1.4 Main Reagents
        1.5 Preparation of major reagents
    2 Experimental methods
        2.1 Thawing frozen cells
        2.2 Cell freezing
        2.3 Immunofluorescence experiment
        2.4 Experimental data analysis
    3 Experimental results
        3.1 SG and Hh shares6 common regulators
        3.2 SPOP and Sufu localize into stress granules
    4 Discussions
Chapter2 SPOP Regulate Stress Granules Dynamics
    1 Experimental materials
    2 Experimental methods
        2.1 Cells Passaging
        2.2 Passaging of adherent cells
        2.3 Plasmid transfection
        2.4 Stress treatments
        2.5 Triton Extraction
        2.6 Experimental data analysis
    3 Experimental results
        3.1 SPOP localize into stress granules
        3.2 Prostate cancer associated SPOP mutants are failed to localize in SG
        3.3 Nuclear SPOP,but not cytoplasmic SPOP inhibits SG
        3.4 SPOP regulates phase transition of Caprin1 protein
    4 Discussions
Chapter3 Sufu Regulate Stress Granules Dynamics
    1 Experimental materials
        1.1 Cell strain
        1.2 Plasmids
        1.3 Main instruments
        1.4 Main Reagents
        1.5 Preparation of major reagents
    2 Experimental methods
        2.1 Preparation of DH5αcompetent cells
        2.2 Plasmids transformation and expanding culture
        2.3 Plasmid extraction
        2.4 Western Blot experiment
        2.5 Co-immunoprecipitation (Co-IP) experiment
        2.6 Experimental data analysis
    3 Results of the experiment
        3.1 Sufu transiently localizes in SG or inhibits SG
        3.2 Sufu interacts with SG protein TTP and TIA
        3.3 Sufu N-terminal inhibits SG while C-terminal promote SG
    4 Discussions
Chapter4 SG inhibition mechanism of Sufu is IDR dependent
    1 Experimental materials
    2 Experimental methods
        2.1 Sufu point mutations and internal deletions
        2.2 Experimental data analysis
    3 Experimental results
        3.1 Sufu SG inhibition mechanism is dependent on Gli2-binding ability
        3.2 SG inhibition mechanism of Sufu is IDR dependent
    4 Discussions
Conclusion and Perspectives
References
Appendix A Abbreviations
Appendix B Primers
Research Achievements During Study
Acknowledgement



本文編號：3165475