Research on screening and identification of proteins interacting with ataxin-3 / 中华医学遗传学杂志
Chinese Journal of Medical Genetics
;
(6): 242-247, 2005.
Article
in English
| WPRIM
| ID: wpr-321116
ABSTRACT
<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Protein Binding
/
Repressor Proteins
/
Recombinant Fusion Proteins
/
Nuclear Proteins
/
Transfection
/
Sodium Channels
/
Microscopy, Confocal
/
Two-Hybrid System Techniques
/
SUMO-1 Protein
Type of study:
Diagnostic study
/
Prognostic study
/
Screening study
Limits:
Humans
Language:
English
Journal:
Chinese Journal of Medical Genetics
Year:
2005
Type:
Article
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