Usp7/HAUSP通过去泛素化Ci/Gli调控Hh信号通路

发布时间：2022-01-09 23:26

　　Hedgehog （Hh）基因最早是在果蝇中通过EMS诱导的基因突变筛选出来的。Hh信号通路在昆虫到人类的各物种间具有保守性,其主要功能是调节体节形成、胚胎发育以及成体组织稳态。Hh信号通路的功能异常会导致许多人类疾病,包括出生缺陷和多种肿瘤。Hh信号通路的转导是通过转录因子Ci/Gli实现的。在果蝇内,Ci受到两条泛素化途径的调控。在没有Hh的情况下,Ci被Slimb-Cull E3连接酶泛素化,最终导致Ci部分降解。在Hh情况下,Ci被Hib-Cul3 E3连接酶泛素化,导致Ci完全降解。泛素化是一个由酶催化的过程,通过该过程将泛素链加到相应的靶蛋白上。泛素化的主要目的是促进靶蛋白的降解,该降解过程可以通过蛋白酶体或溶酶体。和其他的蛋白修饰一样,泛素化修饰也是一个可逆过程,体内的去泛素化酶可以将连在蛋白上的泛素链去除。虽然泛素化介导的Ci降解在Hh信号通路中起着至关重要的作用,但是去泛素化酶是否参与该过程,目前还未知。在本研究中,通过RNAi介导的筛选,我们找到了一个去泛素化酶Usp7能够正调控Hh信号通路。Usp7能够抑制Slimb-Cull和Hib-Cul3介导的Ci泛素化,最... 

【文章来源】：南京大学江苏省 211工程院校 985工程院校 教育部直属院校

【文章页数】：120 页

【学位级别】：博士

【文章目录】：
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



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