Calpain Research Articles

High incidence of 550delA mutation of CAPN3 in LGMD2 patients from Russia.

Autosomal recessive limb gird muscular dystrophy (LGMD2) is a clinically and genetically heterogeneous group of diseases that are characterized by progressive atrophy and weakness of the proximal limb muscles. At least eight genetic loci leading to LGMD2 are recognized. The proportion of particular gene involved in producing different forms of LGMD2 shows a marked geographical variation. We studied 19 LGMD2 patients from Russia (15 families) and found calpain 3 (CAPN3) gene mutations in most of the patients studied. Sequence analysis of the fourth exons revealed two sibs - heterozygous compound for a 15-bp deletion (nt598-612) and 550 adenine deletion, and two sibs homozygous for a 550delA. We developed assay based on allele specific amplification (ASA) for rapid screening of the 550delA. The ASA assay of the LGMD2 patients under study showed that 7 patients from 6 families were homozygous for 550delA and 7 patients from 4 families were heterozygous for 550delA. A linkage analysis employing four microsatellites flanking the LGMD2A locus was performed. We found complete haplotype identity in most cases what favors the possibility of a common founder. Heterozygous carriers of 550delA were found in general population. The crude estimate of the mutation frequency is 1/150. Hum Mutat 15:295, 2000.

Interactions of presenilin and calpain

Interactions of presenilin and calpain

Restriction of ?M-calcium-requiring calpain activation to the plasma membrane in human neuroblastoma cells: Evidence for regionalized influence of a calpain activator protein

Regulation of the microM-calcium-requiring form of calpain (mu calpain) was studied in SH-SY-5Y human neuroblastoma cells. Immunoblot analysis demonstrated that the vast majority of mu calpain is localized within cytosolic pools. Calpain activation was monitored as a function of autolysis within intact cells following calcium influx from the culture medium by calcium ionophores A23187 or ionomycin, or following release of calcium from intracellular stores by thapsigargin. Within intact neuronal cells, following an influx of calcium into the cytosolic from either extracellular or intracellular sources, mu calpain is preferentially activated at the plasma membrane as evidenced by autolytic generation of faster-migrating isoforms. By contrast, similar autolytic profiles for mu calpain in membrane or cytosolic fractions following addition of calcium were observed under cell-free conditions and within cells following death due to extended ionophore-mediated calcium influx. These differential activation profiles for cytosolic mu calpain within living cells and following cellular fractionation/cell death indicate the presence of a regulatory system within neuronal cells. As in previous studies in other systems, we demonstrate the presence of a calpain activator protein. Cycloheximide treatment depleted the autolytic capacity of membrane-associated mu calpain within 4-6 hr without a corresponding decline in total mu calpain protein levels, indicating that the activator protein undergoes rapid turnover in comparison to calpain; pulse-chase radiolabeling confirmed the half-life of mu calpain to exceed 24 hr. Our data suggest that this labile protein represents a major rate-limiting step for in situ calpain activation within neuronal cells, and that, given the tremendous latent mu calpain activity within the cytosol, the interplay of the activator protein and the endogenous inhibitor calpastatin are crucial for maintaining neuronal homeostasis.

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: II. Effect of withdrawal of growth factors.

We have examined the reversal of the regulatory effect of growth factors on calpain/calpastatin activity in transfected Schwann cells (tSc) after their subsequent withdrawal. Removal of nerve growth factor (NGF) or cyclic adenosine monophosphate (cAMP) from tSc resulted in a smaller loss of mu calpain (37%) and mcalpain (36.5 %) activity compared to treated cells from which the growth factors were not withdrawn. The mu calpain activity increased approximately 12% following withdrawal of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) at 24 hr, while the increased mcalpain activity was more than 30-40% compared with that of cells that were continuously treated. The activity of both isoforms returned to their normal levels (untreated) at 48-72 hr following withdrawal of various growth factors, including NGF, cAMP, aFGF, bFGF, platelet-derived growth factor aa (PDGFaa), and PDGFbb. The inhibitory activity of calpastatin was greater than control following withdrawal of NGF, cAMP, PDGFaa, or PDGFbb at 24 hr and this inhibitory activity was less with treatment by aFGF and bFGF. The control activity was restored at 48 hr following withdrawal of these factors. The intensity of the cytoplasmic calpain immunoreactivity was significantly decreased in the nuclear and non-nuclear regions of the cytoplasm, respectively, following withdrawal of cAMP at 144 hr. Removal of bFGF from the medium resulted in an increase of cytoplasmic calpain immunoreactivity in the nuclear regions and cytoplasm, while there was dramatic loss of myelin calpain immunoreactivity from both the nuclear region and cytoplasm. The changes in calpain activity and immunoreactivity in tSc following withdrawal of growth factors suggest that release of calpain from membrane to cytosol may be regulated by these factors.

Immunolocalization of cytoplasmic and myelin mCalpain in transfected Schwann cells: I. effect of treatment with growth factors

We have examined the effect of growth factors on the activity and localization of calpain in transfected Schwann cells (tSc). Axolemma-enriched fraction, cAMP, or NGF showed concentration-dependent inhibition of both mu calpain and mcalpain activity. In contrast, both acidic FGF and basic FGF stimulated mu calpain (37%) and mcalpain (58%) of tSC while PDGF-aa and PDGF-bb inhibited both calpain activities. The inhibitor (calpastatin) activity was approximately 90% following treatment with NGF, cAMP, PDGF-aa, and PDGF-bb compared to control while this activity was 40% with FGF-treated samples. Immunofluorescence studies indicated localization of cytoplasmic calpain in the nuclear region following growth factor treatment in the cytoplasm. Growth factor treatment caused a decrease in the intensity of calpain immunoreactivity. Treatment with cAMP or FGF resulted in strong immunoreactivity of mcalpain in the nuclear region and cytoplasm compared to untreated. The growth factors did not cause translocation of calpain to the outer surface of the cell membrane. The increased immunoreactivity seen with myelin calpain antibody was greater than cytosolic antibody. The changes seen in calpain activity and immunoreactivity following treatment with growth factors suggest that these factors may regulate calpain-calpastatin expression and translocation to the membrane for interaction with lipids for enzyme activation.

Calpain and phagocytosis in cytoskeleton disrupture

Calpain and phagocytosis in cytoskeleton disrupture

Calpains and cathepsin D of the injured myocardium: Role of iodothyronines

Calpains and cathepsin D of the injured myocardium: Role of iodothyronines

Limited proteolysis and differential down-regulation of protein kinase C subspecies in KM3 cells.

Protein kinase C (PKC) is known to play crucial roles in signal transduction. This enzyme was originally found as an undefined protein kinase, which could be activated by limited proteolysis by a Ca2(+)-dependent neutral protease, calpain. PKC was thought to be a single entity for many years, but recent, molecular cloning and biochemical analysis has shown that this enzyme consists of multiple subspecies with closely related structures. The present studies were undertaken to explore the susceptibility of PKC subspecies to proteolysis by calpain in KM3 cells, a pre-B, pre-T cell line. The PKC from KM3 cells was resolved into two subspecies, type II (mainly beta II) and type III (alpha), upon hydroxyapatite column chromatography. This cell line was also shown to contain calpain I and calpain II, which show different sensitivities to Ca2+. Both type II and type III PKC were hydrolysed by either calpain I or calpain II, but type II PKC was cleaved more effectively than type III. The simultaneous presence of phospholipid and diacylglycerol enhanced the cleavage of these PKC subspecies by calpain I. When these cells were treated with phorbol ester, the rates of translocation and down-regulation of these PKC subspecies were different; type II PKC was translocated and depleted from the cell more rapidly than type III enzyme. The results presented herein suggest different roles of the PKC subspecies in the cellular responses to external stimuli.