Cells offer different types of cytoskeletal anchorages: transitory structures such as focal contacts and perennial ones such as the sarcomeric cytoskeleton of muscle cells. The turnover of these structures is controlled with different timing by a family of cysteine proteases activated by calcium, the calpains. The large number of potential substrates present in each of these structures imposes fine tuning of the activity of the proteases to avoid excessive action. This phenomenon is thus guaranteed by various types of regulation, ranging from a relatively high calcium concentration necessary for activation, phosphorylation of substrates or the proteases themselves with either a favorable or inhibitory effect, possible intervention of phospholipids, and the presence of a specific inhibitor and its possible degradation before activation. Finally, formation of multiprotein complexes containing calpains offers a new method of regulation.
Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-length protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various preformed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102K (EEDQ); these were fluorescent only when fluorescent S-1 was used. They were identified by immunostaining with specific antibodies directed against selected parts of he NH2-terminal actin segment of residues 1-113.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
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