Role of tissue transglutaminase type 2 in calbindin–D28k interaction with ataxin-1

Abstract

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 SCA1 pathogenesis is recruited to ataxin-1 aggregates in Purkinje cells of SCA1 mice. Since our recent findings suggest that tissue transglutaminase 2 (TG2) may be involved in cross-linking and aggregation of ataxin-1, the present study was initiated to determine if TG2 has any role in CaB-ataxin-1 interaction. The guinea pig TG2 covalently cross-linked purified rat brain CaB. Time dependent progressive increase in aggregation produced large multimers, which stayed on top of the gel. CaB interaction with ataxin-1 was studied using HeLa cell lysates expressing GFP and GFP tagged ataxin-1 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 cross-linker DSS stabilized CaB-ataxin-1 complex. TG2 cross-linked CaB preferentially with Q82 ataxin-1. The cross-linking 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-1 may recruit CaB via TG2 mediated covalent cross-linking, further supporting the argument that ataxin-1 aggregates may be toxic to neurons.

Bergmann Glial S100B Activates Myo-inositol Monophosphatase 1 and Co-localizes to Purkinje Cell Vacuoles in SCA1 Transgenic Mice

“…S100B and IMPA1 cross-linking was performed as described earlier [34]. The reaction was performed at 37°C for 30 min in 100 μl PBS containing appropriate amounts of purified bovine brain S100B (Sigma Chemical Co.) and IMPA1 (Calbiochem Nova Biochem, San Diego, CA, USA) followed by an additional 20-min incubation at room temperature in the presence of 0.5 mM disuccinimidyl suberate (DSS).…”

“…For Western blot analysis, the above samples or cerebellar cytosolic/membrane fractions were subjected to SDS-PAGE (4–20% acrylamide gels; Bio-Rad Laboratories, Hercules, CA, USA) as described earlier [34]. Equal amounts of proteins were loaded.…”

Roles of Post-translational Modifications in Spinocerebellar Ataxias

“…ATXN1 has been demonstrated to be a substrate of TG type 2 (TG2), and SCA1 transgenic mice have showed elevated nuclear TG2 in the Purkinje cells of the cerebellum ( D’Souza et al, 2006 ). In addition, TG2 mediates the recruitment of the calcium binding protein calbindin-D28k (CaB) to ATXN1 ( Vig et al, 2007 ). The interaction between CaB and myoinositol monophosphatase (IMPase) is necessary for inositol-1, 4,5-trisphosphate (IP3)-mediated Ca 2+ signalling, which is significantly involved in synaptic integration and plasticity in the peripheral nervous system ( Schmidt et al, 2005 ).…”

Beyond the Glutamine Expansion: Influence of Posttranslational Modifications of Ataxin-1 in the Pathogenesis of Spinocerebellar Ataxia Type 1

“…Tissue transglutaminase 2 (TG2) catalyzes the transglutamination reaction in a calcium-dependent manner, and is highly expressed in PCs of the cerebellum [73]. Interestingly, ATXN1 was reported as a substrate of TG2, and elevated nuclear TG2 expression was found in the cerebellar PCs of SCA1 mice [74, 75]. Thus, transglutamination is likely to be involved in the process of mutant ATXN1 aggregate formation (Fig.…”