Comprehensive survey of p94/calpain 3 substrates by comparative proteomics – Possible regulation of protein synthesis by p94

Abstract

Calpain represents a family of Ca2+-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™ 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 Ca2+-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.

The effects of gender and muscle type on the mRNA levels of the calpain proteolytic system and beef tenderness during post-mortem aging

“…On the other hand, Ono et al (2007) claimed that calpain-3 have common substrates with the ubiquitous calpains. These substrates include filamin-C, annexin, translation factors (Ono et al, 2007), cardiac ankyrin repeat protein (Laure et al, 2010) as well as calpastatin (Ono et al, 2004).…”

“…These substrates include filamin-C, annexin, translation factors (Ono et al, 2007), cardiac ankyrin repeat protein (Laure et al, 2010) as well as calpastatin (Ono et al, 2004). Therefore, if a cleaved calpastatin have lower inhibitory potency than the intact one, then the involvement of calpain 3 in proteolysis would be supportive of μ-calpain activities and would lead to increased tenderness.…”

Dynamic distribution of muscle-specific calpain in mice has a key role in physical-stress adaptation and is impaired in muscular dystrophy

“…Each sample (45 μg of protein) was a mixture of equal amounts of protein from 3 individual mice and labeled with iTRAQ-114 (WT), -115 (p94KI), -116 (WT-ex), or -117 (p94KI-ex) reagents according to the manufacturer's instructions (ABI). The samples were subjected to fractionation (1,440 spots) using a DiNa LC system with HiQ-Sil-SCX, HiQSil-C18-3, and a MALDI-plate spotter (KYA Tech) as described previously (44). Spotted fractions were analyzed by MALDI-TOF mass spectrometry (ABI 4800), and proteins were identified using ProteinPilot 2.0 (ABI) and Swiss-Prot Database.…”

CaMPDB: A RESOURCE FOR CALPAIN AND MODULATORY PROTEOLYSIS

“…All samples from various time points were, then mixed (Fig. Al (3)), separated by 2D-nLC (2-Dimensional nano-scale Liquid-Chromatography; 1st step: strong cation ion-exchange chromatography; 2nd step: reverse-phase chromatography) into 684 fractions [10] (Fig. Al (4)).…”