Hedgehog （Hh）基因最早是在果蠅中通過EMS誘導(dǎo)的基因突變篩選出來的。Hh信號通路在昆蟲到人類的各物種間具有保守性,其主要功能是調(diào)節(jié)體節(jié)形成、胚胎發(fā)育以及成體組織穩(wěn)態(tài)。Hh信號通路的功能異常會導(dǎo)致許多人類疾病,包括出生缺陷和多種腫瘤。Hh信號通路的轉(zhuǎn)導(dǎo)是通過轉(zhuǎn)錄因子Ci/Gli實現(xiàn)的。在果蠅內(nèi),Ci受到兩條泛素化途徑的調(diào)控。在沒有Hh的情況下,Ci被Slimb-Cull E3連接酶泛素化,最終導(dǎo)致Ci部分降解。在Hh情況下,Ci被Hib-Cul3 E3連接酶泛素化,導(dǎo)致Ci完全降解。泛素化是一個由酶催化的過程,通過該過程將泛素鏈加到相應(yīng)的靶蛋白上。泛素化的主要目的是促進靶蛋白的降解,該降解過程可以通過蛋白酶體或溶酶體。和其他的蛋白修飾一樣,泛素化修飾也是一個可逆過程,體內(nèi)的去泛素化酶可以將連在蛋白上的泛素鏈去除。雖然泛素化介導(dǎo)的Ci降解在Hh信號通路中起著至關(guān)重要的作用,但是去泛素化酶是否參與該過程,目前還未知。在本研究中,通過RNAi介導(dǎo)的篩選,我們找到了一個去泛素化酶Usp7能夠正調(diào)控Hh信號通路。Usp7能夠抑制Slimb-Cull和Hib-Cul3介導(dǎo)的Ci泛素化,最... 

【文章來源】：南京大學(xué)江蘇省 211工程院校 985工程院校 教育部直屬院校

【文章頁數(shù)】：120 頁

【學(xué)位級別】：博士

【文章目錄】：
Abstract
中文摘要
Abbreviations
Chapter Ⅰ:Review
    1. Hedgehog signal network in Drosophila
        1.1 Core components of Hedgehog pathway in Drosophila
            1.1.1 Hedgehog
            1.1.2 Patched
            1.1.3 Smoothened
            1.1.4 Cubitus interruptus
            1.1.5 Fused
            1.1.6 Suppressor of Fused
            1.1.7 Costal2
        1.2 The Hedgehog pathway in vertebrate
        1.3 Regulation of Hedgehog pathway
            1.3.1 Phosphorylation
            1.3.2 Ubiquitination
            1.3.3 Additional regulations
        1.4 Hedgehog-related diseases
    2. Ubiquitin-proteasome system
        2.1 The key components of ubiquitin-proteasome system
            2.1.1 The ubiquitin-activating enzyme,E1
            2.1.2 The ubiquitin-conjugating enzyme,E2
            2.1.3 The ubiquitin-protein ligase,E3
            2.1.4 The proteasome
        2.2 Multifaceted modes and roles of ubiquitination
    References
Chapter Ⅱ:Deubiquitination of Ci/Gli by Usp7/HAUSP regulates Hedgehogsignaling
    1. Abstract
    2. Introduction
    3. Materials and Methods
        3.1 Constructs, Mutants, and Transgenes
        3.2 Immunostaining and in situ hybridization of wing discs
        3.3 Cell culture, Transfection, Immunoprecipitation, Western blot, Cell immunostaining, Luciferase reporter assay
        3.4 Generating usp7 mutation clones
        3.5 GST fusion protein pull-down assay
        3.6 RNA interference
        3.7 Ci protein stability assays and ubiquitination assay
        3.8 In situ hybridization of zebrafish embryos
        3.9 MO knockdown
        3.10 RNA isolation, Reverse transcription, and Real-time PCR
        3.11 MTT cell proliferation assay
        3.12 BrdU incorporation assay
    4. Results
        4.1 Loss of usp7 specifically compromises Hh signaling through promoting Ci protein degradation
            4.1.1 Loss of usp7 downregulates Hh signaling in Drosophila
            4.1.2 Loss of usp7 downregulates Ci through promoting Ci degradation
            4.1.3 Loss of usp7 does not affect other signaling pathways
        4.2 Usp7 interacts with Ci
            4.2.1 Usp7 binds Ci through N-terminal MATH domain
            4.2.2 Ci binds Usp7 through its multiple P/AxxS motifs
            4.2.3 The interaction of Ci and Usp7 is not regulated by Ci ubiquitination
        4.3 Hh promotes Ci interaction with Usp7
        4.4 Usp7 antagonizes Ci degradation caused by both Slimb-Cull and Hib-Cul3 E3 ligases
        4.5 Usp7 deubiquitinase activity is essential for Ci stabilization
        4.6 Usp7 counteracts both Slimb-Cull and Hib-Cul3-dependent ubiquitination of Ci
        4.7 Usp7 regulates Ci through Usp7-GMPS complex
        4.8 HAUSP deubiquitinates Gli and promotes Hh signaling activity in mammalian systems
            4.8.1 HAUSP could functionally replace Usp7 in the regulation of Ci
            4.8.2 HAUSP binds Gli proteins in a manner promoted by Hh treatment
            4.8.3 HAUSP stabilizes Gli proteins
            4.8.4 HAUSP inhibits the ubiquitination of Gli proteins
            4.8.5 HAUSP positively regulates Hh pathway activity in mammalian cells
        4.9 zUsp7 plays a conserved positive role in the regulation of Hh signaling in zebrafish
        4.10 HAUSP promotes the proliferation of Hh-related cancer cells
    5. Discussion
    References
Acknowledgements
Publications



本文編號：3579634