A023 Calpain-derived TBP fragments contribute to the molecular pathogenesis in spinocerebellar ataxia type 17, also known as Huntington’s disease-like 4

<h3>Background</h3> Spinocerebellar ataxia type 17 (SCA17), also known as Huntington’s disease-like 4 (HDL4), is a neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) tract in the transcription factor TATA box-binding protein (TBP). Previous research unveiled calcium-dependent calpain proteases as important modulators of the SCA17 molecular pathology. Similar to other polyQ diseases such as Huntington’s disease (HD), calpains were reported to be overactivated in SCA17 resulting in elevated cleavage of TBP. <h3>Aim</h3> We aimed at determining the exact calpain-dependent cleavage sites in TBP to understand the relevance of emerging fragments in SCA17. <h3>Methods</h3> Calpain-dependent TBP fragments were characterized using in silico and in vitro approaches, followed by cleavage sites identification via mass spectrometry (MS). By overexpressing corresponding TBP fragment constructs in cell models, we investigated their aggregation, degradation and localization as well as their impact on calpain system activation using western blotting, filter retardation assays and microscopy. Global cellular perturbations caused by the presence of TBP fragments were analyzed through omics-based approaches. <h3>Results</h3> We identified a major calpain-mediated cleavage site after threonine 106, located C-terminal of TBP’s polyQ tract. While polyQ-containing N-terminal fragments were highly aggregation prone, the nuclear localized C-terminal counterpart caused an overactivation of calpains. Importantly, quantitative MS analysis demonstrated alterations in the cellular proteome induced by TBP fragment constructs, underlying the observed calpain overactivation. <h3>Conclusions</h3> We emphasize the relevance of calpain-mediated TBP fragmentation in SCA17. Translating these findings to other polyQ diseases such as HD may help understand the role of arising fragments in molecular pathomechanisms and develop therapeutic strategies across diseases.