Primary and secondary screens for thiol-disulfide oxidoreductases that change total soluble mutant huntingtin protein levels.

COS1 cells were transfected with plasmids encoding human thiol-disulfide oxidoreductase proteins and N171-40Q mutant huntingtin protein (mhtt). Twenty-four hours later cells were lysed and analyzed by reducing SDS-PAGE and Western blot analysis to quantify total soluble N171 mhtt. N171-40Q mutant huntingtin levels (MAB5492 – Millipore) were normalized to actin then to co-transfection control. TRX1 and TMX3 were chosen as targets that may decrease mhtt levels; glutaredoxin 1 (GLX1), PDIA6 and FLJ44606 were chosen as targets that may increase mhtt. See results for more details. Shown are mean ± SEM. n=3, B-F. Secondary screen. COS1 cells were co-transfected with plasmids encoding N171-40Q and wild-type or enzymatically non-functional (control) human thiol-disulfide oxidoreductases. Twenty-four hours later cells were lysed for reducing SDS-PAGE and western blot analysis. Candidate gene expression was confirmed by PCR. The letter/number codes above the right western blot lanes are the substitution of the mutant inactive protein. n=3-5, B. Glutaredoxin 1 (GLX1) increases soluble N171-40Q mhtt levels. n=5, C. Thioredoxin 1 (TRX1) decreases soluble N171-40Q mhtt levels. n=5, D. TMX3 decreases soluble N171-40Q mhtt levels. n=4, E. FLJ44606 has no effect on N171-40Q mhtt levels. n=4, F. Protein disulfide isomerase A6 (PDIA6) has no effect on N171-40Q mhtt levels. n=5. P-values: *<0.05 and **<0.01.