The global rise in the proportion of aged individuals has led to an increased awareness of age-related degenerative diseases. C. elegans has become a popular model for studying the molecular mechanisms of ageing and age-related diseases.Calpains are regulatory Ca2+ activated cysteine proteases present in nearly all eukaryotes1. Misregulation of calpain activity resulting from dysregulation of Ca2+ homeostasis has been implicated to be a causative agent in age-related pathologies such as Alzheimer's disease and sarcopenia. However, it has been difficult to identify the substrates targeted by age-dependent calpain activation, as cleavage specificity is determined by substrate tertiary structure and less so by primary sequence2.Calpains consist of a core catalytic domain and other discrete domains. Our present understanding of calpain activity is based primarily on studies of 'typical' calpains that contain an EF hand domain; less is understood about the 6 human genes encoding 'atypical' calpains lacking this domain. The C. elegans genome lacks typical calpain genes, yet encodes 10 atypical calpain genes (
clp-1 to
clp-7, F44F1.3, T11A5.6, W05G11.4), which we have shown are differentially expressed in hypodermis, muscle and other tissues3. It was found that overexpression of the protein CLP-1 in body wall muscle caused muscle degeneration and paralysis in 1.5% of adults. The level of paralysis increased to 23.5% when CLP-1 was overexpressed in an
egl-19(gf) mutant, which has elevated intracellular Ca2+ levels. Muscle degeneration and paralysis was not observed when catalytically inactive CLP-1 was overexpressed. It was also found that the paralysis phenotype was suppressed when aspartyl protease activity was suppressed by RNAi, suggesting that overactivation of CLP-1 might upregulate the necrotic pathway.We speculate that CLP-1 may be proteolysing key structural proteins in the sarcomere. To identify potential CLP-1 substrates, we are taking a proteomic approach by screening for CLP-1 binding partners using co-immunoprecipitation followed by mass spectrometry. To facilitate this study, we have expressed an epitope-tagged catalytically inactive CLP-1 in body wall muscle. We hypothesised that potential CLP-1 substrates would retain the ability to be bound by inactive CLP-1 yet fail to be cleaved. Potential CLP-1 substrates identified from this screen will be validated by quantitative Western blotting.Refs: 1. Sorimachi et al. 2011, Proc Jpn Acad Ser B Phys Biol Sci. 87(6): 287-327. 2. Cuerrier et al. 2005, J Biol Chem. 9;280(49):40632-41. 3. Joyce et al. 2012, PLoS Genet. 8,
e1002602.