'Oxidation Inhibits Substrate Proteolysis by Calpain I but Not Autolysis

Abstract

In this study, the effects of oxidation on calpain I autolysis and calpain-mediated proteolysis were examined. Calpain I was incubated with increasing concentrations of free calcium in the presence or absence of oxidant, and autolytic conversion of both the 80-and 30-kDa subunits was measured by immunoblotting utilizing monoclonal antibodies which recognize both autolyzed and non-autolyzed forms of each subunit, respectively. Autolytic conversion of the 80-kDa subunit of calpain I was not detected until free calcium concentration was greater than 40 M, whereas autolysis of the 30-kDa subunit did not occur until the free calcium concentration was greater than 100 M. In addition, autolytic conversion of either the 80-or 30-kDa subunit was not inhibited by the presence of oxidant. Calpain I activity was measured using the fluorescent peptide N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin or the microtubule-associated protein tau as substrate. Calpain I was found to have proteolytic activity at free calcium concentrations below that required for autolysis. Calpain I activity was strongly inhibited by oxidant at all calcium concentrations studied, suggesting that proteolytic activity of both the non-autolyzed 80-kDa and autolyzed 76-kDa forms was susceptible to oxidation. Interestingly, whereas oxidation did not inhibit autolytic conversion, the presence of high substrate concentrations did result in a significant reduction of autolysis without altering calpain proteolytic activity. Calpain I activity that had been inhibited by the presence of oxidant was recovered immediately by addition of the reducing agent dithiothreitol.Calpains are a family of calcium-dependent thiol proteases that require both calcium and a reduced environment for activity. Calpains are present in virtually all vertebrate cells and have been postulated to play a role in many physiological processes (1-4). Calpains I and II are ubiquitously expressed, whereas the remaining isoforms are tissue-specific and are found predominantly in muscle (5, 6). Although homologous, calpains I and II require different concentrations of calcium for activity in vitro. Calpain II requires 200-1000 M calcium (7), and calpain I requires 3-50 M calcium (7) for half-maximal activity, a level which has been shown to be reached in the presynaptic terminals of neurons (8) and under pathological conditions (9). The focus of this study was calpain I because it is present in neurons and has been postulated to play a role in neuronal death associated with ischemia (10, 11) and certain neurodegenerative disorders (12-15).Calpain I is a heterodimer composed of a unique 80-kDa catalytic subunit and a 30-kDa regulatory subunit which is identical to the 30-kDa subunit of calpain II (16). How these subunits interact and what regulatory mechanisms are involved in the process of calpain activation remain unresolved. Initially it was proposed that the 80-kDa form of calpain I was an inactive pro-enzyme that must undergo calcium-dependent conformational chan...

Temporal relationship of peroxynitrite-induced oxidative damage, calpain-mediated cytoskeletal degradation and neurodegeneration after traumatic brain injury

“…This repeatedly demonstrated biphasic time course of calpain-mediated proteolysis can potentially be explained by an initial partial ROS-mediated inhibition of calpain activity in the early posttraumatic hrs during which PN generation and evidence of oxidative damage are at their highest level. Consistent with this hypothesis, it has been shown that ROS can inhibit calpain activity via oxidation of the sulfhydryl groups of cysteine residues at the active site of the enzyme (Benuck et al, 1992) (Guttmann et al, 1997) (Guttmann and Johnson, 1998). Recent work by another group has shown that another cysteine protease caspase-3 in the traumatized brain is similarly inhibited by PN specifically, and that this is prevented by application of the sulfhydryl reducing agent dithiothreitol (Lau et al, 2006).…”

Rapid Calpain I Activation and Cytoskeletal Protein Degradation Following Traumatic Spinal Cord Injury: Attenuation with Riluzole Pretreatment

“…It should be noted, however, that calpain J autolysis is not a necessary condition for expression of proteolytic activity (Guttmann et al, 1997). Therefore, it is possible that calpain J proteolytic activity after spinal cord injury may be greater than that indicated by the presence of the autolyzed 76-kDa subunit.…”

Ceramide Selectively Decreases Tau Levels in Differentiated PC12 Cells Through Modulation of Calpain I

“…Suc-LLVY-AMC, as previously reported (Bronk and Gores, 1993;Guttmann et al, 1997). The nonfluorescent Sue-LLVY-AMC, after cleavage by calpains, emits strong fluorescence at a wavelength of 460-480 nm, which was monitored by using a BIOTEK FL500 microplate fluorescence reader.…”