National Institute for Medical Research, Medical Research Council, London, United Kingdom.
PLoS One. 2010 Aug 26;5(8):e12430. doi: 10.1371/journal.pone.0012430.
Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology.
Ataxin-3 是神经退行性疾病脊髓小脑共济失调 3 型或 Machado-Joseph 病的致病蛋白，是泛素蛋白酶体途径中的一种半胱氨酸蛋白酶。它包含多个泛素结合位点，通过这些位点锚定不同连接的多泛素链，然后由 N 端催化（Josephin）结构域切割。Ataxin-3 羧基末端的泛素相互作用基序（UIMs）的特性已得到很好的确定。然而，关于 Josephin 结构域中最近确定的两个泛素结合位点如何有助于泛素链结合和切割，人们知之甚少。在本研究中，我们试图确定 Josephin 结构域对 ataxin-3 催化特性的具体贡献，并评估这些结合位点的拓扑结构和亲和力如何调节 ataxin-3 的活性。我们使用 NMR 对二泛素/Josephin 复合物的结构进行建模，并表明连接偏好是由两个结合位点的拓扑结构决定的。酶学研究进一步帮助我们确定了这些位点之间的精确层次关系。我们确定 Josephin 的结构决定了对 K48 连接链的特异性。靠近活性位点的位点 1 对于切割是必不可少的。我们的研究为更好地了解 ataxin-3 的细胞功能及其与病理学的联系铺平了道路。
