Research on screening and identification of proteins interacting with ataxin-3.
OBJECTIVE: This study sought to isolate and identify the proteins that interact with ataxin-3, to confirm the interacted domain, and to provide new clues for exploring the function of ataxin-3 and the pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD). METHODS: Yeast two-hybrid screen (MATCHMAKER GAL4 Two-Hybrid System ... 3) and regular molecular biologic techniques were undertaken to screen human brain cDNA library with mutant ataxin-3 bait. Two baits from both normal and mutant C-terminus of ataxin-3 were created by subcloned methods to determine which domain of ataxin-3 interacts with the putative associated proteins and to find out optimal candidate proteins that interact with C-terminus of ataxin-3. Confocal microscope was used to observe whether ataxin-3 co-localized with the obtained interacting proteins in mammalian cells. RESULTS: Five novel ataxin-3 interacting proteins were obtained, among which were three known proteins, namely human rhodopsin guanosine diphosphate dissociation inhibitor alpha, small ubiquitin-like modifier 1, and human neuronal amiloride-sensitive cation channel 2; the other two were unknown. Interacting domain analysis revealed that an unknown protein interacted with the C-terminus near the polyglutamine tract of ataxin-3, the other four all interacted with the N-terminus. In the nucleus of SH-SY5Y cell, small ubiquitin-like modifier 1 co-localized with the wild-type ataxin-3 and with the intranuclear aggregates formed by the mutant ataxin-3. CONCLUSION: An unknown protein probably interacting with C-terminus of ataxin-3 is firstly discovered, and the initiative findings suggest first that the interaction of small ubiquitin-like modifier 1 with N-terminus of ataxin-3 and the relevant sumoylation probably participate in the post-translation modifying of ataxin-3 and in the pathogenesis of SCA3/MJD.
Mesh Terms:
Cell Line, Tumor, Green Fluorescent Proteins, Humans, Microscopy, Confocal, Mutation, Nerve Tissue Proteins, Nuclear Proteins, Plasmids, Protein Binding, Recombinant Fusion Proteins, Repressor Proteins, SUMO-1 Protein, Sodium Channels, Transfection, Two-Hybrid System Techniques
Cell Line, Tumor, Green Fluorescent Proteins, Humans, Microscopy, Confocal, Mutation, Nerve Tissue Proteins, Nuclear Proteins, Plasmids, Protein Binding, Recombinant Fusion Proteins, Repressor Proteins, SUMO-1 Protein, Sodium Channels, Transfection, Two-Hybrid System Techniques
Zhonghua Yi Xue Yi Chuan Xue Za Zhi
Date: Jun. 01, 2005
PubMed ID: 15952105
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