Role of tissue transglutaminase type 2 in calbindin-D28k interaction with ataxin-1.
Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeats within the disease protein, ataxin-1. The mutant ataxin-1 precipitates as large intranuclear aggregates in the affected neurons. These aggregates may protect neurons from mutant protein and/or trigger neuronal degeneration by encouraging recruitment of other essential proteins. Our previous studies have shown that calcium binding protein calbindin-D28k (CaB) associated with SCAl pathogenesis is recruited to ataxin-l aggregates in Purkinje cells of SCAl mice. Since our recent findings suggest that tissue transglutaminase 2 (TG2) may be involved in crosslinking and aggregation of ataxin-l, the present study was initiated to determine if TG2 has any role in CaB-ataxin-l interaction. The guinea pig TG2 covalently crosslinked purified rat brain CaB. Time dependent progressive increase in aggregation produced large multimers, which stayed on top of the gel. CaB interaction with ataxin-l was studied using HeLa cell lysates expressing GFP and GFP tagged ataxin-l with normal and expanded polyglutamine repeats (Q2, Q30 and Q82). The reaction products were analyzed by Western blots using anti-polyglutamine, CaB or GFP antibodies. CaB interacted with ataxin-1 independent of TG2 as the protein-protein crosslinker DSS stabilized CaB-ataxin-l complex. TG2 crosslinked CaB preferentially with Q82 ataxin-1. The crosslinking was inhibited with EGTA or TG2 inhibitor cystamine. The present data indicate that CaB may be a TG2 substrate. In addition, aggregates of mutant ataxin-l may recruit CaB via TG2 mediated covalent crosslinking, further supporting the argument that ataxin-l aggregates may be toxic to neurons.
Vig
P J S
PJ
Department of Neurology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA. pvig@neurology.umsmed.edu
Wei
J
J
Shao
Q
Q
Hebert
M D
MD
Subramony
S H
SH
Sutton
L T
LT
D000818:Animals; D000067696:Ataxin-1; D000067528:Ataxins; D015153:Blotting, Western; D064092:Calbindin 1; D064026:Calbindins; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011689:Purkinje Cells / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry*; D020754:Spinocerebellar Ataxias / Q000378:metabolism*; D011503:Transglutaminases / Q000737:chemistry*
C000593749:ATXN1 protein, human; D000067696:Ataxin-1; D000067528:Ataxins; C000593750:Atxn1 protein, mouse; C000593751:Atxn1 protein, rat; C576498:CALB1 protein, human; C576499:Calb1 protein, mouse; C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D003432:Cross-Linking Reagents; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D064030:S100 Calcium Binding Protein G; C000717608:TGM2 protein, human; C000717609:Tgm2 protein, rat; D049452:Green Fluorescent Proteins; C097188:polyglutamine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins