title: Comprehensive survey of p94/calpain 3 substrates by comparative proteomics--possible regulation of protein synthesis by p94.
abstract: Calpain represents a family of Ca(2+)-dependent cytosolic cysteine proteases found in almost all eukaryotes and some bacteria, and is involved in a variety of biological phenomena, including brain function. Several substrates of calpain are aggressively proteolyzed under pathological conditions, e.g., in neurodegenerating processes, fodrin is proteolyzed by calpain. Because very small amounts of substrate are proteolyzed by calpain under normal biological conditions, the molecular identities of calpain substrates are largely unknown. In this study, an extensive survey of the substrates of p94/calpain 3 in COS7 cells was executed using iTRAQ(TM) labeling and 2-D LC-MALDI analysis. p94 was used because: (i) several p94 splicing variants are expressed in brain tissue even though p94 itself is a skeletal-muscle-specific calpain, and (ii) it exhibits Ca(2+)-independent activity in COS cells, which makes it useful for evaluating the effects of p94 protease activity on proteins without perturbing the cells. Our approach revealed several novel protein substrates for p94, including the substrates of conventional calpains, components of the protein synthesis system, and enzymes of the glycolytic pathway. The results demonstrate the usefulness and sensitivity of this approach for mining calpain substrates. A combination of this method with other analytical methods would contribute to elucidation of the biological relevance of the calpain family.
authors: Department of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan Institute of Medical Science, Rinshoken, Tokyo, Japan.
authors: Yasuko
authors: Y
authors: Ono
authors: Chikako
authors: C
authors: Hayashi
authors: Naoko
authors: N
authors: Doi
authors: Fujiko
authors: F
authors: Kitamura
authors: Mayumi
authors: M
authors: Shindo
authors: Kenichi
authors: K
authors: Kudo
authors: Takuichi
authors: T
authors: Tsubata
authors: Mitsuaki
authors: M
authors: Yanagida
authors: Hiroyuki
authors: H
authors: Sorimachi
mesh_terms: D000818:Animals; D019556:COS Cells; D002154:Calpain / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology; D023281:Genomics / Q000379:methods*; D014176:Protein Biosynthesis / Q000502:physiology*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013379:Substrate Specificity
chemical_list: D020543:Proteome; D002154:Calpain; C078134:calpain p94