Hh信号通路蛋白SPOP和Sufu调控应激颗粒动态

发布时间：2021-04-28 12:26

　　背景应激颗粒（Stress granules,SG）是细胞在应激条件下形成的细胞质聚集体,伴有整体翻译抑制作用。SG包含RNA结合蛋白,凋亡/生存相关的mRNA和翻译起始因子。SG的形成促进细胞存活,并参与许多人类病理机制。在哺乳动物中,有一个重要的信号通路称为“Hedgehog（Hh）信号通路”。它在胚胎发育过程中很重要,它的损伤或过度活化可能导致多种发育异常和肿瘤的发生发展。Hh配体结合到Ptch1进而激活Hh信号通路,最终将GliFL转换为GliA,并触发Hh靶基因的表达。Gli转录因子家族受SPOP和Sufu调控。SPOP与Gli2/3相互作用并促进Gli2/3经蛋白酶体途径降解,而Sufu保护Gli2/3免受降解进而负调节Hh途径。研究报道表明,SG通过招募特定的蛋白,例如Raptor,RACK1和TRAF2等来调节某些信号通路。这些蛋白被招募到SG中以调控应激反应和抑制细胞发生凋亡。以上信号通路和SG之间的相互作用驱使我们探究Hh信号通路和SG之间可能存在的功能互作。目的首次鉴定Hh信号通路蛋白SPOP和Sufu与应激颗粒在功能上存在关联,这将有助于我们了解Hh信号通路和S... 

【文章来源】：浙江师范大学浙江省

【文章页数】：77 页

【学位级别】：硕士

【文章目录】：
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



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