Endogenous Calpain-3 Activation Is Primarily Governed by Small Increases in Resting Cytoplasmic [Ca2+] and Is Not Dependent on Stretch

Abstract

Proteolytically active calpain-3/p94 is clearly vital for normal muscle function, since its absence leads to limb girdle muscular dystrophy 2A, but its function and regulatory control are poorly understood. Here we use single muscle fibers, individually skinned by microdissection, to investigate the diffusibility and autolytic activation of calpain-3 in situ. Virtually all calpain-3 present in mature muscle fibers is tightly bound in the vicinity of the titin N2A line and triad junctions and remains so irrespective of fiber stretching or raised [Ca 2؉ ]. Most calpain-3 is evidently bound within the contractile filament lattice, because (i) its slow diffusional loss is slowed further by locking myosin and actin into rigor and (ii) detergent dispersion of membranes causes rapid washout of most ryanodine receptors and sarcoplasmic reticulum Ca 2؉ pumps with little accompanying washout of calpain-3. Calpain-3 autolyzes (becoming proteolytically active) in a tightly calcium-dependent manner. It remains in its nonactivated full-length form if [Ca 2؉ ] is maintained at <50 nM, the normal resting level, even with brief increases to 2-20 M during repeated tetanic contractions, but it becomes active (though still bound) if [Ca 2؉ ] is kept slightly elevated at 200 nM (ϳ20% autolysis in 1 h). Calpain-3 did not spontaneously autolyze even when free in solution with 200 nM Ca 2؉ for up to 60 min. These findings explain why calpain-3 remains quiescent with normal exercise but is activated following eccentric (stretching) contractions, when resting [Ca 2؉ ] is elevated, and how a protease such as calpain-3 can be very Ca 2؉ -sensitive yet highly specific in its actions.

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract

“…4). These are levels known to activate calpain (28). In the cortical areas, there was also an approximately fourfold difference in Ca 2+ concentrations between the K6W-Ub lens vs. WT-Ub lens (∼800 vs. 200 nM, respectively) (Fig.…”

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

“…3C) and even after scaling-up the expression, the amount of full length ADGB was insufficient for further biophysical characterization. This truncation is most probably a result of proteolysis by intracellular proteases or by auto-cleavage by the N-terminal calpain-like domain, a phenomenon commonly observed among calpains [53][54][55][56]. To further evaluate if ADGB autolytic activity would be the source of this potential auto-cleavage we added three different protease inhibitors to the growth medium; the cysteine protease inhibitor E64, the acid protease inhibitor Pepstatin A and the calpain specific Calpain II inhibitor.…”

Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle

“…Furthermore, we hypothesized that at rest the majority of the glycogen-related proteins would remain associated with glycogen and/or membranous compartments in muscle and that their diffusibility would be augmented following glycogen utilizing in vitro stimulation. To understand how tightly associated the various catabolic and anabolic enzymes are associated with glycogen, we used our technique of allowing diffusible proteins to move out of mechanically skinned quiescent muscle fibers in physiological buffer with or without either amylase, which degrades the glycogen granule at ␣-1,4 linkages or Triton X-100, which allows proteins associated with membranous compartments to be identified (15,17). These findings are of particular physiological relevance because the entire pool of a given protein can be assessed.…”