Calpain 1–titin interactions concentrate calpain 1 in the Z‐band edges and in the N2‐line region within the skeletal myofibril

Abstract

Calpain 1 (microcalpain) and calpain 2 (millicalpain), the best characterized calpains, are known as intracellular calcium-dependent endoproteases and are expressed in different tissues of vertebrates. These ubiquitous cysteine proteases [1] play important roles in a large set of intracellular events [2][3][4][5], particularly in the selective proteolysis of factors involved in the cell cycle [6], during apoptosis in association with caspases [7], or in the cleavage of membrane-cytoskeleton complexes during cell motility phases [8]. Their activities are blocked by calpastatins (a specific inhibitor family largely expressed in the cell) and are regulated at the membrane level by phospholipids, which decrease the calcium requirements of calpains [1]. Calpain 1 (active in vitro at 50 lm Ca 2+ ions) and calpain 2 (active in vitro at 500 lm Ca 2+ ions) are composed of an 80 kDa and a 30 kDa subunit. The spatial structure of calpain 2 has recently been determined [9] and the organization of the six domains (dI-dIV in the 80 kDa subunit, and dV-dVI in the 30 kDa subunit) has been defined as well as the calcium-binding regions. In particular, it was found that dIV and dVI (calmodulin-like [2097][2098][2099][2100][2101][2102][2103][2104][2105][2106][2107]. Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK ⁄ N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl 2 and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.Abbreviations CP1 Ig, anti-(calpain 1) Ig; FITC, fluorescein isothiocyanate.

A Rising Titan:TTNReview and Mutation Update

“…Finally, the Ig motifs located at the Z/I junction bind specifically to the giant myofibrillar protein obscurin (ZIg8-ZIg9) [Young et al, 2001], to the ubiquitously expressed Ca 2+ -dependent protease calpain-1 (ZIg8-IIg5) [Raynaud et al, 2005], to sarcomeric actin (Zig-9-IIg-1) and to tropomyosin Raynaud et al, 2004] (Fig. 1).…”

“…S100A1 inhibits actin binding to the PEVK segment in a Ca 2+ -dependent manner and has been shown to interact with several sites along TTN's extensible region in situ [Yamasaki et al, 2001]. Tropomyosin, in a complex conformation with actin [Raynaud et al, 2004], nebulin (specifically the proline-rich sequences [ Ma and Wang, 2002]), and calpain-1 [Raynaud et al, 2005], also bind to the PEVK region in addition to their binding sites in the TTN Z-disk or Z/I junction regions (Fig. 1).…”

PKB and megalin determine the survival or death of renal proximal tubule cells

“…3C), indicating that high concentrations of albumin decrease the transcription of the megalin gene. Furthermore, the possibility that the effect of albumin could be due to lysosomal degradation was ruled out, because 0.5 M afilomycin A1 and 100 M U64, inhibitors of lysosomal degradation (25,26), did not change the albumin-induced decrease in megalin protein expression (data not shown).…”

El sistema proteolítico calpaina en la tenderización de la carne: Un enfoque molecular.

“…Inmunolocalization studies have shown that both, CAPN1 and CAPN2, are located intracellularly along the Z disk/I band regions in the form of intracellular stores (9). Raynaud et al (10) found that CAPN1 is concentrated on the N1 and N2 line region of titin and suggested that this might constitute a reservoir for the cell. Many calpain substrates, including titin, nebulin, filamin, troponin-T and desmin, which attaches the sarcolemma to the Z-disc, are co-localized and proteolyzed during meat tenderization (9).…”