An endogenous activator of the Ca2+-dependent proteinase of human neutrophils that increases its affinity for Ca2+.

Pontremoli, S.; Melloni, Edon; Michetti, M.; Salamino, Franca; Sparatore, Bianca; Horecker, B.L.

doi:10.1073/pnas.85.6.1740

Cited by 47 publications

(29 citation statements)

References 24 publications

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“…The original hypothesis was initially proposed on two bases (see the Introduction) : (i) an attempt has been made to find a molecular function for sequences selected through a statistical analysis of primary structures of stable and unstable proteins ; and (ii) taking into account that the requirement of calcium for activating calpains in itro is one to three orders of magnitude (depending on the type of calpain) higher than physiological intracellular calcium concentrations, it has been suggested that PEST motifs might chelate calcium and thus activate calpains at the substrate level. However, it has more recently been reported that both lipid [40,41] and protein [42][43][44][45] activators increase dramatically (up to 100-fold) the affinity of calpains for calcium. This thus makes unlikely the hypothesis that the concentration of calcium at the level of substrates, through chelation by PEST motifs, is a prerequisite for cleavage by calpains.…”

Section: Discussionmentioning

confidence: 99%

PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein

Carillo

Pariat

Steff

et al. 1996

Biochemical Journal

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Cytoplasmic degradation of c-fos protein is extremely rapid. Under certain conditions, it is a multi-step process initiated by calcium-dependent and ATP-independent proteases called calpains. PEST motifs are peptide regions rich in proline, glutamic acid/aspartic acid and serine/threonine residues, commonly assumed to constitute built-in signals for rapid recognition by intracellular proteases and particularly by calpains. Using a cell-free degradation assay and site-directed mutagenesis, we report here that the three PEST motifs of c-fos are not required for rapid cleavage by calpains. Testing the susceptibility of PEST motif-bearing and non-bearing transcription factors including GATA1, GATA3, Myo D, c-erbA, Tal-1 and Sry, demonstrates that PEST sequences are neither necessary nor sufficient for specifying degradation of other proteins by calpains. This conclusion is strengthened by the observation that certain proteins, reportedly known to be cleavable by calpains, are devoid of PEST motifs

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Section: Discussionmentioning

confidence: 99%

PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein

Carillo

Pariat

Steff

et al. 1996

Biochemical Journal

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“…It was reported that isovalerylcarnitine reduced the Ca 2ϩ concentration required for maximal proteolytic activity of m-calpain to Ͻ10 M and increased its specific activity by 1.5-fold, but did not affect either the Ca 2ϩ requirement or the specific activity of -calpain (354,356). Other studies described a heat-stable polypeptide of ϳ40 -45 kDa isolated from human neutrophils (353) or rat skeletal muscle (357) that reduced the Ca 2ϩ concentration required for maximal activity of m-calpain to 20 M or less without affecting its specific activity. The 40-to 45-kDa "activator" also had no effect on the catalytic properties of -calpain.…”

Section: The Ca 2ϩ Requirement Problemmentioning

confidence: 99%

The Calpain System

Goll

Thompson

et al. 2003

Physiological Reviews

2,549

2,801

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Goll, Darrel E., Valery F. Thompson, Hongqi Li, Wei Wei, and Jinyang Cong. The Calpain System. Physiol Rev 83: 731–801, 2003; 10.1152/physrev.00029.2002.—The calpain system originally comprised three molecules: two Ca2+-dependent proteases, μ-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both μ- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55–65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six “domains” in the 80-kDa subunit: 1) a 19-amino acid NH2-terminal sequence; 2) and 3) two domains that constitute the active site, IIa and IIb; 4) domain III; 5) an 18-amino acid extended sequence linking domain III to domain IV; and 6) domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of μ- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.

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“…Caldonopain assay was conducted after 12 h incuba- [23] or there is a conformational change of the molecule to overcome the effect of this built-in inhibitor is yet to be established. We also propose that due to prolong incubation an appreciable amount of the endogenous inhibitor is possibly digested by the proteolytic enzyme and as a result, loses its inhibitory effect to a great extent.…”

Section: Discussionmentioning

confidence: 99%

Calcium dependent thiol protease caldonopain and its specific endogenous inhibitor inLeishmania donovani

1993

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A calcium dependent proteolytic enzyme was detected in the lysed promastigotes of Leishmania donovani, the causative agent of kala-azar. The protease was able to hydrolyse an added substrate, azocasein and showed high affinity for calcium. Rate of azocasein digestion was primarily slow but boosted up after eight hours. It was not inactivated when heated at 55 degrees C for 15 min at pH 7.4. Sulfhydryl reagents significantly reduced the enzymic activity but trypsin-like protease inhibitors hardly had any effect. The enzyme was not sensitive to calmodulin from a heterologous source but registered low activity when treated with chlorpromazine. The caseinolytic activity was stimulated when leishmanial cells were preincubated with ionophore A23187 in presence of 1 mM Ca2+. The enzyme is named caldonopain due to its similarity with a general class of calcium dependent protease calpain present in different tissues and cells. Caldonopain was found to be localized in cytosol along with its specific endogenous inhibitor caldonostatin. The ratio of caldonopain-caldonostatin unit was higher in the infected macrophage compared to the parasitic protozoa and Balb/c macrophage alone. It may be postulated that the amount of both calcium and its protein inhibitor may have a direct impact on the caldonopain-induced biological process to regulate cellular action of this pathogen.

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An endogenous activator of the Ca2+-dependent proteinase of human neutrophils that increases its affinity for Ca2+.

Cited by 47 publications

References 24 publications

PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein

PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein

The Calpain System

Calcium dependent thiol protease caldonopain and its specific endogenous inhibitor inLeishmania donovani

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