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首页> 外文期刊>Biochemistry >Mechanism of recruitment and activation of the endosome-associated deubiquitinase AMSH
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Mechanism of recruitment and activation of the endosome-associated deubiquitinase AMSH

机译：内体相关的去泛素酶AMSH的募集和激活机制

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摘要

AMSH, a deubiquitinating enzyme (DUB) with exquisite specificity for Lys63-linked polyubiquitin chains, is an endosome-associated DUB that regulates sorting of activated cell-surface signaling receptors to the lysosome, a process mediated by the members of the endosomal sorting complexes required for transport (ESCRT) machinery. Whole-exome sequencing of DNA samples from children with microcephaly capillary malformation (MIC-CAP) syndrome identified recessive mutations encoded in the AMSH gene causatively linked to the disease. Herein, we report a number of important observations that significantly advance our understanding of AMSH within the context of the ESCRT machinery. First, we performed mutational and kinetic analysis of the putative residues involved in diubiquitin recognition and catalysis with a view of better understanding the catalytic mechanism of AMSH. Our mutational and kinetic analysis reveals that recognition of the proximal ubiquitin is imperative for the linkage specificity and catalytic efficiency of the enzyme. The MIC-CAP disease mutation, Thr313Ile, yields a substantial loss of catalytic activity without any significant change in the thermodynamic stability of the protein, indicating that its perturbed catalytic activity is the basis of the disease. The catalytic activity of AMSH is stimulated upon binding to the ESCRT-0 member STAM; however, the precise mechanism and its significance are not known. On the basis of a number of biochemical and biophysical analyses, we are able to propose a model for activation according to which activation of AMSH is allowed by facile, simultaneous binding to two ubiquitin groups in a polyubiquitin substrate, one by the catalytic domain of the DUB (binding to the distal ubiquitin) and the other (the proximal ubiquitin) by the ubiquitin interacting motif (UIM) from STAM. Such a mode of binding would stabilize the ubiquitin chain in a productive orientation, resulting in an enhancement of the activity of the enzyme. These data together provide a mechanism for understanding the recruitment and activation of AMSH at ESCRT-0, providing biochemical and biophysical evidence that supports a role for AMSH when it is recruited to the initial ESCRT complex: it functions to facilitate the transfer of ubiquitinated receptors (cargo) from one ESCRT member to the next by disassembling the polyubiquitin chain while leaving some ubiquitin groups still attached to the cargo.
机译：AMSH是一种对Lys63连接的多聚泛素链具有出色特异性的去泛素化酶（DUB），是一种与内体相关的DUB，它调节溶酶体的活化细胞表面信号受体的分选，该过程由所需的内体分选复合物的成员介导用于运输（ESCRT）机械。对患有小头畸形毛细血管畸形（MIC-CAP）综合征儿童的DNA样本进行全外显子测序，确定了AMSH基因编码的隐性突变与该病有因果关系。在此，我们报告了许多重要的观察结果，这些观察结果在ESCRT机制的背景下大大提高了我们对AMSH的理解。首先，我们对参与泛素识别和催化的推定残基进行了突变和动力学分析，以期更好地了解AMSH的催化机理。我们的突变和动力学分析表明，近端泛素的识别对于酶的连接特异性和催化效率至关重要。 MIC-CAP疾病突变Thr313Ile导致催化活性大大降低，而蛋白质的热力学稳定性没有任何显着变化，表明其扰动的催化活性是该疾病的基础。当结合到ESCRT-0成员STAM上时，AMSH的催化活性被激发。但是，确切的机制及其意义尚不清楚。根据许多生化和生物物理分析，我们能够提出一种激活模型，根据该模型，可以通过轻松，同时与多泛素底物中的两个泛素基团同时结合来激活AMSH。 DUB（与远端泛素结合）和另一个（近端泛素）通过来自STAM的泛素相互作用基序（UIM）产生。这种结合方式将使遍在蛋白链稳定在生产方向上，从而导致酶活性的增强。这些数据共同为理解AMSH在ESCRT-0处的募集和激活提供了一种机制，提供了将AMSH募集到初始ESCRT复合物中时支持其发挥作用的生化和生物物理证据：其作用是促进泛素化受体的转移（货物）从一个ESCRT成员转移到下一个成员，方法是拆解多聚泛素链，同时使一些泛素基团仍然附着在该货物上。

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