The kyphoscoliosis peptidase (KY) protein is a Z-disc–associated factor implicated in skeletal muscle maintenance, yet its molecular function has remained poorly defined. This thesis set out to characterise KY’s role in muscle integrity, proteostasis, and stress adaptation by integrating cellular imaging, in vivo expression by electroporation, biochemical assays, structural modelling, and proteomic profiling.
Initial investigations into KY’s subcellular behaviour under stress revealed a striking species-specific distinction: while human KY accumulated in cytoplasmic and nuclear puncta following heat shock, mouse KY did not exhibit this redistribution. Under normal conditions, KY displayed a diffuse sarcoplasmic distribution with clear Z-disc localisation, consistent with its role as a structural component of the sarcomere. These changes were further modulated by proteasomal and autophagic inhibition, pointing to KY’s dynamic response to proteotoxic stress.
To test KY’s functional role in muscle maintenance, in vivo rescue experiments were performed in ky/ky mice. Re-expression of full-length KY successfully restored muscle fibre cross-sectional area, whereas deletion of the TGN/PROT domain abolished rescue capacity. Interestingly, point mutations disrupting the catalytic triad did not impair function, suggesting that the domain serves a structural or scaffolding role rather than enzymatic activity. AlphaFold-based modelling predicted structural conservation of the catalytic fold, but the predicted active site geometry suggested catalytic inactivity, consistent with experimental evidence showing no enzymatic function.
Further analysis demonstrated that Z-disc localisation was preserved even when the TGN/PROT domain was removed, implicating other regions of the protein in sarcomeric anchoring. Proteomic screening using immunoprecipitation–mass spectrometry (IP–MS) identified several stress-related KY interactors, including PPM1B and HSP70 family members, linking KY to autophagy and chaperone-mediated proteostasis. Functional assays using GFP–LC3–RFP–LC3ΔG reporters confirmed impaired autophagic flux in KY-deficient cells, evidenced by disrupted LC3 processing and cargo accumulation.
ZAKβ, a stress-activated kinase that promotes muscle hypertrophy through MAPK signalling, was also examined in relation to KY. Expression of constitutively active ZAKβ induced hypertrophy in both wild-type and KY-deficient muscle, establishing that KY is unnecessary for ZAKβ-driven growth. Altogether, this work positions KY as a non-enzymatic scaffold that integrates proteostatic, structural, and stress-response pathways essential for maintaining skeletal muscle homeostasis.
