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, 2 (5), 565-76

Comprehensive Survey of p94/calpain 3 Substrates by Comparative Proteomics--Possible Regulation of Protein Synthesis by p94

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Comprehensive Survey of p94/calpain 3 Substrates by Comparative Proteomics--Possible Regulation of Protein Synthesis by p94

Yasuko Ono et al. Biotechnol J.

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.

Figures

Figure 1
Figure 1
Proteomic analysis of COS7 cells expressing p94. (A) Overview of experimental design. (B) Number of peptides/proteins identified in the 2-D LC-MALDI analysis. More than half the identified proteins were represented by a single peptide. Of the proteins identified, 11% had more than six peptides; these included vimentin, several different heat shock proteins, and cytoplasmic actins, which are known to be abundant.
Figure 2
Figure 2
Statistical profiles of LC-MALDI analysis. (A, B) Error distribution of masses in Da (A) or ppm (B) between observed and calculated/theoretical molecular masses of 2801 identified MS/MS spectra out of 51 97 spectra. Dotted lines indicate means and ranges for one and two SDs. (C, D) Modified ratios of iTRAQT™ signals of 115 (CS-1) to 114 (WT-1) (C) or 116 (WT-2) to 114 (WT-1) (D). Values of ratios were modified as follows: Modified ratio = R − 1 (if R ≥ 1), 1/R − 1 (if 1 > R > 0), where R = iTRAQT™ signal ratio.
Figure 3
Figure 3
Scatter plots of iTRAQT™ signals (absolute values) between 114 (WT-1) and 115 (CS-1) (A), 114 (WT-1) and 116 (WT-2) (B), 115 (CS-1) and 117 (CS-2) (C), and 114 (WT-1) and 117 (CS-2) (D). Ranges of twofold and fourfold, one-half, and one-quarter are indicated by dotted lines. R2, correlation coefficient.
Figure 4
Figure 4
Examples of MS/MS spectra that exhibited significant differences in iTRAQT™ signal ratios. (A) MS/MS spectrum of precursor ion of 3410.5859, which was identified as elongation factor 2 with ion score 72 and I.C. 99.9% [calculated mass is 3410.5439, error = 0.0420 (1 2 ppm)]. Representative peaks are shown and calculated masses are indicated in parentheses. The predicted amino acid sequence is shown in the box, which corresponds to residue 801–829 of human EF2 with N-terminal iTRAQT™ and Cys-MMTS modification (indicated by *). The small square around m/z= 100 is the area magnified in (B). (B) Magnification of iTRAQ™ signals shown in (A). (C–G) Magnified iTRAQT™ signals of MS/MS spectrum (data not shown) identified as filamin A (C), annexin A1 (D), annexin A2 (E), vimentin (F), and p94 (G). The predicted peptide corresponds to residues 309–330 of human filamin A, 29–52 of human annexin A1, 1 79–195 of human annexin A2, 36–49 of human vimentin, and 733—748 of human p94.
Figure 5
Figure 5
Comparison of identified proteins by iTRAQ™ analysis. (A) Western blot analysis of selected proteins using specific antibodies. Equal amounts of protein extract from COS7 cells expressing either WT or CS mutant of p94 were loaded for each blot. Closed and open arrowheads indicate the full-length and the autolyzed fragments, respectively, detected by anti-pIS2 antibody. The epitope region is indicated by a vertical bar in (C). AnxA1, annexin A1; AnxA2, annexin A2. (B) 150K fragment of α-fodrin proteolyzed at the calpain-specific site detected by anti-GMMPR in a protein sample from COS7 cells expressing WT but not CS (open arrowhead). The anti-fodrin mAb detected several fragments of different sizes in addition to full-length unproteolyzed fodrin (closed arrowhead) in both the WT and CS lanes. *, Proteolyzed 150K fragment specific to the WT sample was also detected by the anti-fodrin mAb, but the signal overlaps with an unidentified band with an almost identical mobility, which was also present in the CS sample. (C) Positional relationships between previously determined proteolytic sites and peptides identified in the present analysis. The closed arrowheads indicate autolytic sites for p94 and proteolytic sites of conventional calpains for vimentin and α-fodrin. Closed, open, and gray circles indicate the positions in proteins of peptides whose relative abundance were decreased, unchanged, or increased, respectively, in COS7 cells expressing WT p94 relative to cells expressing p94:CS. Cys, His, and Asn indicate catalytic amino acid residues in p94. Vertical bars indicate the positions of epitopes for anti-pIS2 (for p94) and anti-GMMPR (for the calpain-specific 150K proteolytic fragment of fodrin). Bidirectional arrows indicate the autolytic fragment of p94 and the calpain specific proteolytic fragment of fodrin, which correspond to the open arrowheads in (A) and (B), respectively. NS, IS1, and IS2 are p94-specific insertion sequences. IIa and IIb comprise the calpain protease domain. III and IV are C2-like and 5EF-hand motif Ca2+-binding domains, respectively. 1A, 1 B, and 2, coiled coil domains. Sr1-19, spectrin repeat. (D) Relative quantities of p94 protein/peptide by iTRAQT™ method and Western blot analysis (WB). WT p94 amounts detected by both methods are represented as relative to those of CS (CS = 100). Difference between CS and WT by iTRAQ ([a]) corresponds to portions in WT degraded completely (to amino acid) or at least smaller than the length of trypsin peptides. Difference between iTRAQ and WB in WT ([b]) corresponds to portions in WT degraded partially, larger than the length oftrypsin peptides but small enough not to be detected by WB. Region [c] mainly corresponds to the 55-kDa band in WB (A, “p94”, open arrowhead).
Figure 6
Figure 6
Time course of in vitro translation of p94 and the μ-calpain large subunit (μCL). In vitro transcribed RNAfor rat p94 and human μCL were translated with rabbit reticulocyte lysate and [35S]Met in the presence (+inh.) or absence (−inh.) of 380 μg/mL leupeptin and E64, and sampled at the indicated time (min). Samples are separated by SDS-PAGE, and exposed to X-ray film (A) and an imaging plate. The radioactivities of 94-kDa (open triangle) and 80-kDa (closed triangle) bands were quantified using a Fujix Bioimage Analyzer, and plotted against the time of sampling (B). Note that the scales for the Y-axes differ by a factor of 10.

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