Altered expression of HSPA5, HSPA8 and PARK7 in spinocerebellar ataxia type 17 identified by 2-dimensional fluorescence difference in gel electrophoresis.
BACKGROUND
Expansion of the CAG repeat of the TATA-box binding protein (TBP) gene has been identified as the causative mutations in spinocerebellar ataxia 17 (SCA17). TBP is ubiquitously expressed in both central nervous system and peripheral tissues. The underlying molecular changes of SCA17 are rarely explored.


METHODS
To study the molecular mechanisms underlying SCA17, we generated stably induced isogenic 293 cells expressing normal TBP-Q(36) and expanded TBP-Q(61) and analyzed the expressed proteins using two-dimensional difference in gel electrophoresis (2D-DIGE), followed by mass spectrometry and immunoblotting.


RESULTS
Upon induction with doxycycline, the expanded TBP-Q(61) formed aggregates with significant increase in the cell population at subG1 phase and cleaved caspase-3. Proteomics study identified a total of 16 proteins with expression changes greater than 1.5 fold. Among the 16 proteins, PARK7, GLRX3, HNRNPA1, GINS1, ENO1, HNRPK and NPM1 are increased, and SERPINA5, HSPA5, VCL, KHSRP, HSPA8, HNRPH1, IMMT, VCP and HNRNPL are decreased in cells expressing TBP-Q(61) compared with those expressing TBP-Q(36). The altered expression of HSPA5, HSPA8 and PARK7 were further validated by 2D and Western immunoblot analyses.


CONCLUSIONS
The results illustrate the utility of proteomics to identify alterations of proteins which underlie pathogenesis of SCA17, and may serve as potential therapeutic targets.
Lee
Li-Ching
LC
Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.
Chen
Chiung-Mei
CM
Chen
Fen-Lin
FL
Lin
Pei-Ying
PY
Hsiao
Ya-Chin
YC
Wang
Pin-Rong
PR
Su
Ming-Tsan
MT
Hsieh-Li
Hsiu-Mei
HM
Hwang
Ji-Chuu
JC
Wu
Chung-Hsin
CH
Lee
Guan-Chiun
GC
Singh
Sher
S
Lin
Yenshou
Y
Hsieh
Sen-Yung
SY
Lee-Chen
Guey-Jen
GJ
Lin
Jung-Yaw
JY
D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D005453:Fluorescence; D005786:Gene Expression Regulation; D050883:HSC70 Heat-Shock Proteins / Q000032:analysis* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis* / Q000235:genetics; D018832:Molecular Chaperones / Q000032:analysis* / Q000235:genetics; D000090243:Nucleophosmin; D015513:Oncogene Proteins / Q000032:analysis* / Q000235:genetics; D010641:Phenotype; D000071617:Protein Deglycase DJ-1; D020754:Spinocerebellar Ataxias / Q000235:genetics* / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000235:genetics
D000091342:Endoplasmic Reticulum Chaperone BiP; D050883:HSC70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; C494371:HSPA8 protein, human; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; C000716967:NPM1 protein, human; D015513:Oncogene Proteins; D035181:TATA-Box Binding Protein; D000090243:Nucleophosmin; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1