Identification of CLP-1 atypical calpain substrates in Caenorhabditis elegans

  • Laurence Newman

Student thesis: Master's ThesisMaster of Science (MSc)

Abstract

Calpains are regulatory calcium ion activated cysteine proteases present in nearly all eukaryotes (1). Dysregulation of Ca2+ homeostasis, which results in aberrant calpain activation is implicated in contributing to age-related pathologies, such as Alzheimer’s disease and sarcopenia (2,3). However, it has been difficult to identify substrates targeted by calpain activation, because cleavage specificity is not determined by primary sequence alone (4).
Calpains are categorised into typical and atypical based on their domain architecture.
Current understanding of human calpain activity is based primarily on studies of ‘typical’ calpains, which contain EF hand domains; less is understood about the six ‘atypical’ calpains that lack this domain. The genome of the model organism Caenorhabditis elegans encodes only atypical calpain proteins. Earlier studies have shown that these proteins are differentially expressed in a variety of tissues (5). Overexpression of CLP-1 in body wall muscle was shown to cause a low penetrance paralysis accompanied by muscle degeneration in adults (5). The severity of paralysis increased when CLP-1 was overexpressed in egl-19(gf) mutants that produce elevated levels of intracellular calcium. The substrates targeted by CLP-1 are unknown.
To identify potential CLP-1 substrates, a proteomic screen was performed. A CLP- 1::mRFP fusion protein was expressed in muscle and protein complexes co-purifying with CLP-1 after immunoprecipitation were identified by mass spectrometry. A bioinformatics tool WormGRAB was created to collect information on proteins identified by mass spectrometry. A subset of proteins associated with muscle integrity and normal locomotion
were identified from the proteomic screen as well as other potential substrates that had not been previously associated with maintenance of muscle.
Further analysis of the intracellular localisation of CLP-1 in body wall muscle was also pursued to understand how it might be targeted to substructures of the sarcomere. The involvement of calpains in heat stress was also investigated.
Date of Award23 Jan 2019
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorPatricia E Kuwabara (Supervisor) & Nigel J Savery (Supervisor)

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