Structures and functions of the human Josephin domain-containing proteins

描述（由申请人提供）：含Josephin结构域的蛋白质是去泛素化酶（DUBs）的五个主要家族之一。该家族的创始成员是ataxin-3，一种聚谷氨酰胺蛋白，是最常见的遗传性共济失调，脊髓小脑性共济失调3型（SCA3，也称为Machado-Joseph病）的病原体。Ataxin-3的DUB活性位于分子的n端，即所谓的Josephin结构域。Ataxin-3与许多与维持蛋白质稳态相关的细胞过程有关，包括错误折叠蛋白质向聚合体的运输以及通过内质网相关降解途径（ERAD）的蛋白质通量。Ataxin-3也可以抑制扩展重复聚谷氨酰胺结构域的毒性。所有这些功能都需要DUB活性，从而确立了Josephin结构域的生物学意义，并暗示Josephin DUB活性与蛋白质错误折叠诱导的神经变性之间存在联系。ataxin-3在人类基因组中编码有三个同源物：ataxin-3样蛋白（AT3L）、Josephin-1和Josephin-2。所有三种都具有DUB活性，但在催化效率和特异性方面存在很大差异。含有Josephin结构域的同源物在大多数真核生物中是保守的，包括植物和原生动物，这意味着该结构域及其表达的DUB活性负责有价值的生物学功能。我们建议分析人类Josephin结构域蛋白的分子结构和功能，以了解它们如何识别和作用于底物和其他相互作用的蛋白。我们将使用x射线晶体学来确定不同Josephin结构域的结构，单独或与泛素底物复合；表征四种人类Josephin结构域蛋白的DUB活性，评估它们如何结合和切割小的和大的泛素底物；探讨它们的泛素结合活性；并探讨ataxin-3与其细胞结合伙伴的相互作用。

DESCRIPTION (provided by applicant): The Josephin domain-containing proteins are one of the five major families of deubiquitinating enzymes (DUBs). The founding member of the family is ataxin-3, a polyglutamine protein and the causative agent of the most common inherited ataxia, spinal cerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease). Ataxin-3's DUB activity resides in the molecule's N-terminal half, the so-called Josephin domain. Ataxin-3 is linked to many cellular processes that are related to maintaining protein homeostasis, including the transport of misfolded proteins to aggresomes and the flux of proteins through the endoplasmic reticulum-associated degradation pathway (ERAD). Ataxin-3 can also suppress the toxicity of expanded repeat polyglutamine domains. All of these functions require DUB activity, establishing the biological significance of the Josephin domain and hinting at a link between Josephin DUB activity and neurodegeneration induced by protein misfolding. Three homologs of ataxin-3 are encoded in the human genome: The ataxin-3-like protein (AT3L), Josephin-1, and Josephin-2. All three possess DUB activity, but differ substantially in catalytic efficiency and specificity. Josephin domain-containing homologs are conserved in most eukaryotes, including plants and protozoans, implying that this domain and the DUB activity it expresses are responsible for valuable biological functions. We propose to analyze the molecular structure and function of the human Josephin domain-containing proteins in order to understand how they recognize and act upon substrates and other interacting proteins. We will use X-ray crystallography to determine structures for different Josephin domains, alone and in complex with ubiquitin substrates; characterize the DUB activities of the four human Josephin domain-containing proteins, assessing how they bind and cleave both small and large ubiquitin substrates; explore their ubiquitin-binding activities; and probe the interactions of ataxin-3 with its cellular binding partners. PUBLIC HEALTH RELEVANCE: The Josephin domain proteins appear to function in quality control pathways in human cells. Understanding how the Josephin proteins function will help reveal how cells protect themselves from the potentially toxic effects of protein misfolding. The insights obtained from this work will inform efforts to design therapies for disorders in which the cell's quality control pathways are disrupted, such as Huntington's disease, Alzheimer's disease, and Parkinson's disease.