Department of Chemistry, Purdue University , West Lafayette, Indiana 47907, United States.
Biochemistry. 2013 Nov 5;52(44):7818-29. doi: 10.1021/bi401106b. Epub 2013 Oct 23.
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.
AMSH 是一种具有精氨酸特异性的去泛素化酶（DUB），能够特异性识别 Lys63 连接的多泛素链，它是一种内体相关的 DUB，可调节激活的细胞表面信号受体向溶酶体的分拣，这一过程由内体分选复合物必需的运输（ESCRT）机器的成员介导。对患有小头毛细血管畸形（MIC-CAP）综合征的儿童的 DNA 样本进行全外显子组测序，发现了编码 AMSH 基因的隐性突变，该突变与疾病有因果关系。在此，我们报告了一些重要的观察结果，这些结果极大地促进了我们对内体分选复合物中的 AMSH 的理解。首先，我们对参与二泛素识别和催化的假定残基进行了突变和动力学分析，以便更好地理解 AMSH 的催化机制。我们的突变和动力学分析表明，对近端泛素的识别对酶的连接特异性和催化效率至关重要。MIC-CAP 疾病突变 Thr313Ile 导致催化活性显著丧失，而蛋白质的热力学稳定性没有任何显著变化，表明其催化活性的紊乱是疾病的基础。当与 ESCRT-0 成员 STAM 结合时，AMSH 的催化活性会受到刺激；然而，确切的机制及其意义尚不清楚。根据一系列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，当多泛素底物中的两个泛素基团能够同时轻易结合时，AMSH 的激活是允许的，一个由 DUB 的催化结构域（结合到远端泛素）结合，另一个（近端泛素）由 STAM 的泛素相互作用基序（UIM）结合。这种结合方式将稳定泛素链的活性构象，从而增强酶的活性。这些数据共同提供了一种理解 AMSH 在 ESCRT-0 处募集和激活的机制，提供了生化和生物物理证据，支持 AMSH 在被募集到初始 ESCRT 复合物时的作用：它的功能是通过将多泛素化的受体（货物）从一个 ESCRT 成员转移到下一个成员来促进其转移，同时将一些泛素基团仍连接到货物上。
