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B46 The HD-like type 4/SCA17 disease protein TBP is cleaved by calpains in vitro and in vivo
  1. Stefanie Anger1,2,
  2. Jonasz J Weber1,2,
  3. Huu P Nguyen1,2
  1. 1Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany
  2. 2Centre for Rare Diseases, University of Tuebingen, Tuebingen, Germany

Abstract

Background The Spinocerebellar ataxia type 17 (SCA17) also referred to as HD-like type 4 is a neurodegenerative disease caused by an elongation of the CAG repeat in the gene of the TATA box binding protein (TBP), an ubiquitary transcription factor. Although the underlying pathomechanism is not fully understood, fragmentation of polyglutamine-expanded TBP by yet unidentified proteases might contribute to the toxicity of the mutant protein in affected cerebellar neurons.

Aims The role of proteolysis of mutant proteins is a hallmark of many neurodegenerative diseases such as Parkinson disease, Huntington’s disease or Spinocerebellar ataxia type 3 and calpains, a family of calcium-dependent proteases, were found to be one of the key players. Thus we sought to investigate the contribution of calpains-dependent TBP cleavage to the pathogenesis in SCA 17.

Methods TBP fragmentation was investigated in vitro in cell-free and cell-based calpain activation assays. The activation state of the calpain system and the presence of calpain-dependent TBP fragments in vivo was analysed in our SCA17 rat model by western blotting.

Results Our calpain activation assays showed the occurrence of specific calpain-dependent fragments of wildtype and mutant TBP after addition of exogenous calpains or after triggering activity of endogenous calpains. Furthermore, pharmacologic inhibition of calpains reduced base-line cleavage of the disease protein and we identified corresponding TBP fragments in cerebellar lysates of SCA17 animals.

Conclusions We could identify TBP as a novel target of calpain-dependent cleavage in vitro and in vivo. Further analyses of cleavage sites, the contribution of specific TBP fragments to the molecular pathomechanism of SCA17 and ways to reduce fragmentation of TBP in vivo stand in the focus of our further investigations and could help to develop a therapeutic approach for this dreadful disease. As Huntingtin is also cleaved by calpains, effective therapeutic approaches in SCA17 could also be transferred to Huntington’s disease.

  • Calpain
  • TBP proteolysis
  • Spinocerebellar ataxia type 17
  • SCA17 rat
  • polyglutamine diseases

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