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首页> 外文学位 >Regulation of cell surface receptor trafficking through the ESCRT-0 complex by the deubiquitinating enzyme ESP8.
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Regulation of cell surface receptor trafficking through the ESCRT-0 complex by the deubiquitinating enzyme ESP8.

机译：去泛素化酶ESP8通过ESCRT-0复合物调节细胞表面受体运输。

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摘要

Regulation of cell surface receptor abundance and the spatial and temporal control of ligand-induced signaling is essential for proper cellular growth and homeostasis. Malfunctions along such regulatory pathways can lead to a wide variety of pathologies, including various cancers. Ubiquitination, the process of covalent modification of a target protein with one or more ubiquitin moieties, plays a key role in ligand-mediated downregulation of receptor tyrosine kinases and G protein-coupled receptors. Orchestrated by a cascade of ubiquitin activating, conjugating and ligating enzymes, ubiquitination serves as a signal for endosomal trafficking of cell surface receptors for lysosomal degradation. Moreover, ubiquitination of endosomal sorting machinery is critical for their function. The reversible nature of ubiquitination, imparted by deubiquitinating enzymes specific for cleavage of ubiquitin from modified substrate proteins enables coordinated and dynamic regulation of ubiquitin-dependent events in endocytosis.;The work presented herein investigates the role of the deubiquitinating enzyme Ubiquitin-Specific Protease 8 (USP8) in endocytosis and ligand-mediated downregulation of two receptors: Epidermal Growth Factor Receptor (EGFR) and Chemokine Receptor 4 (CXCR4). The central hypothesis of this study is based on the premise that USP8-mediated deubiquitination exhibits two distinct consequences with respect to receptor endocytosis---protection of direct receptor substrates from degradation and modulation of endosomal sorting machinery in support of general cargo trafficking. With particular emphasis on the functional relationship between USP8 and the early endosomal sorting complex required for transport (ESCRT-0), EGFR and CXCR4 illustrate the different modes of receptor regulation by USP8. To study USP8 function in this context, the following experimental aims were proposed: (i) to compare and contrast the implications of USP8 depletion on cellular abundance, signaling and ligand-mediated degradation of EGFR and CXCR4, (ii) to probe the effects of USP8 catalytic activity on ubiquitination and stability of endosomal adaptor proteins HRS and STAM, and (iii) to characterize the direct interactions of the three Arg-X-X-Lys (RXXK) motifs of USP8 with the SH3 domain of STAM and examine the contribution of these interactions to cellular activity of USP8. Collectively, the present body of work explores the role of USP8 as a global regulator of ubiquitin-mediated events governing receptor trafficking and degradation and seeks to contribute mechanistic insights into the biology of deubiquitination in endocytosis.;The investigation described herein reveals opposing consequences of USP8 activity for the regulation of EGFR and CXCR4 cellular abundance. USP8 loss-of-function studies, employing siRNA interference and over-expression of catalytically inactive mutants of USP8, demonstrate that USP8 opposes the early phase of EGF-induced EGFR ubiquitination and protects EGFR from ligand-mediated degradation. Importantly, the rate of EGFR turnover is most susceptible to USP8 activity in the presence of low, physiological concentrations of EGF. In addition to EGFR, USP8 catalytic activity impacts the ubiquitination and stability of early endocytic sorting adaptors, hepatocyte growth factor-regulated substrate (HRS) and signal transducing adaptor molecule (STAM). Three RXXK motifs within USP8 support a direct low-affinity polyvalent interaction with the SH3 domain(s) of STAM1/2 proteins, specifically localizing the USP8/STAM complex to the ESCRT-0 machinery. Importantly, the ability of USP8 to form a complex with STAM is required for USP8-mediated deubiquitination of activated EGFR. These findings suggest that USP8 cooperates with the ESCRT-0 sorting machinery to support recycling of signaling growth factor receptors. While USP8 also regulates the ubiquitination status of ESCRT-0 in a manner dependent upon the RXXK/SH3 interaction, stability of HRS and STAM is only marginally affected by its ability to directly interact with the SH3 domain of STAM, indicate that USP8 must possess additional means of localizing to the ESCRT-0 complex.;In contrast to EGFR, CXCR4 stability is enhanced in cells compromised for USP8 function. CXCR4 accumulates on enlarged early endosomes and co-localizes with abnormally distributed ESCRT-0 complex as a result of USP8 inactivation. However, regulation of CXCR4 trafficking and downregulation by USP8 is not susceptible to mutations in the RXXK motifs. Such evidence demonstrates that USP8 is not functionally beholden to a single mode of recruitment to the endosomal compartment and implicates USP8 as a multifaceted regulator of cell surface receptor endocytosis. Collectively, the findings described herein portray a complex cooperation between USP8 and the ESCRT-0 proteins that shapes ubiquitin-mediated events at the early-to-late endosomal transition.
机译：细胞表面受体丰度的调节以及配体诱导的信号传导的时空控制对于适当的细胞生长和体内平衡至关重要。这种调节途径的功能障碍会导致多种病理，包括各种癌症。泛素化是目标蛋白与一个或多个泛素部分共价修饰的过程，在配体介导的受体酪氨酸激酶和G蛋白偶联受体的下调中起关键作用。泛素化由一连串的遍在蛋白活化，缀合和连接酶精心策划，可作为内体运输溶酶体降解的细胞表面受体的信号。此外，内体分选机制的泛素化对其功能至关重要。泛素化的可逆性质是通过特异性地修饰修饰的底物蛋白的泛素化酶进行的泛素化酶赋予的，从而可以协调和动态调节内吞作用中泛素依赖性事件的发生;本文所述的工作研究了泛素化酶泛素特异性蛋白酶8（内吞作用和配体介导的两种受体下调：表皮生长因子受体（EGFR）和趋化因子受体4（CXCR4）。这项研究的中心假设是基于这样一个前提，即USP8介导的去泛素化在受体胞吞作用方面表现出两个截然不同的结果-保护直接受体底物免受降解和调节内体分拣机制的影响，以支持一般货物运输。 EGFR和CXCR4特别强调USP8与转运所需的早期内体分选复合物（ESCRT-0）之间的功能关系，阐明了USP8调节受体的不同模式。为了在这种情况下研究USP8的功能，提出了以下实验目标：（i）比较和对比USP8耗竭对EGFR和CXCR4的细胞丰度，信号传导和配体介导的降解的影响，（ii）探究USP8对内体衔接蛋白HRS和STAM的泛素化和稳定性的催化活性，以及​​（iii）表征USP8的三个Arg-XX-Lys（RXXK）基序与STAM的SH3结构域的直接相互作用，并研究这些作用与USP8细胞活性的相互作用。总的来说，本研究工作探讨了USP8作为泛素介导的事件的全球调节者的调控作用，该事件控制着受体的运输和降解，并寻求对内吞作用中去泛素化生物学机制的深入了解。本文所述的研究揭示了USP8的相反后果。调节EGFR和CXCR4细胞丰度的活性。 USP8功能丧失的研究采用siRNA干扰和USP8催化失活突变体的过表达，证明USP8反对EGF诱导的EGFR泛素化的早期阶段，并保护EGFR免受配体介导的降解。重要的是，在低的生理浓度的EGF存在下，EGFR周转率最容易受到USP8活性的影响。除EGFR外，USP8的催化活性还会影响早期内吞分选衔接子，肝细胞生长因子调节的底物（HRS）和信号转导衔接子分子（STAM）的泛素化和稳定性。 USP8中的三个RXXK基序支持与STAM1 / 2蛋白的SH3结构域的直接低亲和性多价相互作用，尤其是将USP8 / STAM复合体定位于ESCRT-0机制。重要的是，USP8介导的活化EGFR去泛素化要求USP8与STAM形成复合物。这些发现表明，USP8与ESCRT-0分选机制合作，以支持信号生长因子受体的回收。尽管USP8还以依赖于RXXK / SH3相互作用的方式调节ESCRT-0的泛素化状态，但HRS和STAM的稳定性仅受其与STAM的SH3域直接相互作用的能力的轻微影响，表明USP8必须拥有其他与EGFR相反，在USP8功能受损的细胞中，CXCR4的稳定性得到增强。由于USP8失活，CXCR4积聚在扩大的早期内体中，并与异常分布的ESCRT-0复合体共定位。但是，USP8对CXCR4转运的调节和下调对RXXK基序的突变不敏感。这些证据表明，USP8在功能上不依赖于向内体区室募集的单一模式，并且暗示USP8是细胞表面受体内吞作用的多方面调节剂。总体而言，本文所述发现描述了USP8与ESCRT-0蛋白之间的复杂合作，该蛋白在早期至晚期的内体转化过程中塑造了泛素介导的事件。

著录项

  • 作者

    Berlin, Ilana.;

  • 作者单位

    The University of Chicago.;

  • 授予单位 The University of Chicago.;
  • 学科 Biology Molecular.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 214 p.
  • 总页数 214
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 宗教;
  • 关键词

  • 入库时间 2022-08-17 11:38:24

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