Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.     

Characterization of a large subunit catalase truncated by proteolytic cleavage

The large subunit catalase HPII from Escherichia coli can be truncated by proteolysis to a structure similar to small subunit catalases. Mass spectrometry analysis indicates that there is some heterogeneity in the precise cleavage sites, but approximately 74 N-terminal residues, 189 C-terminal residues, and a 9-11-residue internal fragment, including residues 298-308, are removed. Crystal structure refinement at 2.8 A reveals that the tertiary and quaternary structure of the native enzyme is retained with only very subtle changes despite the loss of 36% of the sequence. The truncated variant exhibits a 1.8 times faster turnover rate and enhanced sensitivity to high concentrations of H(2)O(2), consistent with easier access of the substrate to the active site. In addition, the truncated variant is more sensitive to inhibition, particularly by reagents such as aminotriazole and azide which are larger than substrate H(2)O(2). The main channel leading to the heme cavity is largely unaffected by the truncation, but the lateral channel is shortened and its entrance widened by removal of the C-terminal domain, providing an explanation for easier access to the active site. Opening of the entrance to the lateral channel also opens the putative NADPH binding site, but NADPH binding could not be demonstrated. Despite the lack of bound NADPH, the compound I species of both native and truncated HPII are reduced back to the resting state with compound II being evident in the absorbance spectrum only of the heme b-containing H392A variant.     

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.     

Nuclear translocation of intracellular domain of Protogenin by proteolytic cleavage

Protogenin (PRTG) is a transmembrane protein of immunoglobulin superfamily, which has multiple roles in embryogenesis as a receptor or an adhesion molecule. In this study, we present sequential proteolytic cleavage of PRTG. The cleavage first occurs at the extracellular domain, then at the interface of the transmembrane and the intracellular domain by γ-secretase, which results in the release of the intracellular domain of PRTG (PRTG-ICD). PRTG-ICD contains putative nuclear localization signal (NLS) at its N-terminal, and translocates to the nucleus in cultured cells and in the neuroepithelial cells of chick embryos. We propose that the PRTG-ICD is cleaved by γ-secretase and translocates to the nucleus, which is potentially implicated in signaling for neural differentiation and in cell adhesion mediated by PRTG.     

A pathway sensor for genome-wide screens of intracellular proteolytic cleavage

Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to β-actin. By including protease cleavage sites between GLUC and β-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and β-actin cleavage.     

The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells

The E-cadherin protein mediates Ca(2+)-dependent interepithelial adhesion. Association of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. We have identified a novel truncated E-cadherin species of 100-kDa (E-cad(100)) in prostate and mammary epithelial cells. E-cad(100) was generated by treatment of cells with ionomycin or TPA. Cell-permeable calpain inhibitors prevented E-cad(100) induction by ionomycin. Immunoblotting for spectrin and mu-calpain confirmed calpain activation in response to ionomycin treatment. Both the mu- and m-isoforms of calpain efficiently generated E-cad(100) in vitro. The E-cad(100) fragment was unable to bind to beta-catenin, gamma-catenin, and p120, suggesting that this cleavage event would disrupt the E-cadherin adhesion complex. Mutational analysis localized the calpain cleavage site to the cytosolic domain upstream of the beta- and gamma-catenin binding motifs of E-cadherin. Because E-cadherin is inactivated in many adenocarcinomas we hypothesized that calpain may play a role in prostate tumorigenesis. A prostate cDNA microarray data base was analyzed for calpain expression in which it was found that m-calpain was up-regulated in localized prostate cancer, and to an even higher degree in metastatic prostate cancer compared with normal prostate tissue. Furthermore, we examined the cleavage of E-cadherin in prostate cancer specimens and found that E-cad(100) accumulated in both localized and metastatic prostate tumors, supporting the cDNA microarray data. These findings demonstrate a novel mechanism by which E-cadherin is functionally inactivated through calpain-mediated proteolysis and suggests that E-cadherin is targeted by calpain during the tumorigenic progression of prostate cancer.     

Characterization of the bovine neurofilament NF-M protein and cDNA sequence, and identification of in vitro and in vivo calpain cleavage sites

Neurofilaments are the major components of the neuronal cytoskeleton, and accumulations of these proteins are associated with several important human diseases. Here we report the cloning and sequencing of bovine NF-M, the first NF-M cloned from a large domestic mammal. The bovine sequence proves to be generally more similar to that of human NF-M than the previously described mammalian sequences, suggesting that bovine neurofilaments are a useful model for biochemical studies of application to humans. However, we noted some unusual features within the 16 lys-ser-pro (KSP2) type phosphopeptide repeats and also note that the number of these repeats correlates well with the size of animal. We also characterized two in vitro calpain cleavage sites by direct peptide sequencing, finding that both are located in the glutamic acid rich E segment. Finally, we show biochemically that the more abundant and stable of these calpain fragments can also be detected in vivo.     

Myocilin mutations causing glaucoma inhibit the intracellular endoproteolytic cleavage of myocilin between amino acids Arg226 and Ile227

Myocilin is a secreted glycoprotein of unknown function that is ubiquitously expressed in many human organs, including the eye. Mutations in this protein produce glaucoma, a leading cause of blindness worldwide. To explore the biological role of myocilin and the pathogenesis of glaucoma, we have analyzed the expression of recombinant wild type and four representative pathogenic myocilin mutations (E323K, Q368X, P370L, and D380A) in transiently transfected cell lines derived from ocular and nonocular tissues. We found that wild type myocilin undergoes an intracellular endoproteolytic processing at the C terminus of Arg226. This cleavage predicts the production of two fragments, one of 35 kDa containing the C-terminal olfactomedin-like domain, and another of 20 kDa containing the N-terminal leucine zipper-like domain. Here we have analyzed the 35-kDa processed fragment, and we have found that it is co-secreted with the nonprocessed protein. Western immunoblot analyses showed that human aqueous humor and some ocular tissues also contain the processed 35-kDa myocilin, indicating that the endoproteolytic cleavage occurs in vivo. Mutant myocilins accumulated in the endoplasmic reticulum of transfected cells as insoluble aggregates. Interestingly, the four pathogenic myocilins inhibited the endoproteolytic processing with varying efficiency. Furthermore, the mutation P370L, which produces the most severe glaucoma phenotype, also elicited the most potent endoproteolytic cleavage inhibition. We propose that the endoproteolytic processing might regulate the activity of myocilin and that the inhibition of the processing by pathogenic mutations impairs the normal role of myocilin.     

Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein define a requirement for dibasic residues for intracellular cleavage.

We investigated the amino acid sequence requirements for intracellular cleavage of the Rous sarcoma virus glycoprotein precursor by introducing mutations into the region encoding the cleavage recognition site (Arg-Arg-Lys-Arg). In addition to mutants G1 (Arg-Arg-Glu-Arg) and Dr1 (deletion of all four codons) that we have reported on previously (L. G. Perez and E. Hunter, J. Virol. 61:1609-1614, 1987), we constructed two additional mutants, AR1 (Arg-Arg-Arg-Arg), in which the highly conserved lysine is replaced by an arginine, and S19 (Ser-Arg-Glu-Arg), in which no dibasic pairs remain. The results of these studies demonstrate that when the cleavage sequence is deleted (Dr1) or modified to contain unpaired basic residues (S19), intracellular cleavage of the glycoprotein precursor is completely blocked. This demonstrates that the cellular endopeptidase responsible for cleavage has a stringent requirement for the presence of a pair of basic residues (Arg-Arg or Lys-Arg). Furthermore, it implies that the cleavage enzyme is not trypsinlike, since it is unable to recognize arginine residues that are sensitive to trypsin action. Substitution of the mutated genes into a replication-competent avian retrovirus genome showed that cleavage of the glycoprotein precursor was not required for incorporation into virions but was necessary for infectivity. Treatment of BH-RCAN-S19-transfected turkey cells with low levels of trypsin resulted in the release of infectious virus, demonstrating that exogenous cleavage could generate a biologically active glycoprotein molecule.     

Dual intracellular signaling by proteolytic cleavage of membrane-anchored heparin-binding EGF-like growth factor

Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a membrane-anchored precursor (proHB-EGF) that is cleaved to release a soluble HB-EGF by specific metalloproteases. Proteolytic cleavage of proHB-EGF yields amino- and carboxy-terminal fragments (HB-EGF and HB-EGF-C). Recent studies indicate that the processing of proHB-EGF is strictly regulated and involved in a variety of biological processes and that not only HB-EGF but also HB-EGF-C functions as a signaling molecule. ProHB-EGF generates dual intracellular signaling molecules by its proteolytic cleavage.     

Characterization of Bacillus subtilis mutants with a temperature-sensitive intracellular protease.

A colony screening procedure was devised to detect Bacillus subtilis mutants containing temperature-sensitive trypsin-like intracellular protease activity. The enzyme was characterized as a non-sulfhydryl serine protease on the basis of inhibitor studies. It was also inhibited by D- or L-histidine but not by any other amino acid tested. The long-term survival at 45 degrees C of these mutants in a minimal salts medium was decreased, with rapid lysis occurring within 24 h. A D-histidine function in long-term survival and inhibition accounted for the presence of additional protease mutants among survivors of histidine auxotrophs selected for their ability to utilize D-histidine. In addition to being lysed when incubated at 45 degrees C under nongrowth conditions, all of the protease mutants had a decreased rate of protein turnover and produced spores deficient in a major low-molecular-weight spore coat polypeptide. The morphology of the undercoat layers was altered, but there was no effect on spore heat resistance or on germination. The missing spore coat polypeptide appeared to be processed from a larger precursor by cleavage to produce N-terminal histidine. A defect in this protease could account for the lack of processing and thus the absence of this polypeptide in spore coats.     

Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1

Modification of proteins by small ubiquitin-like modifier (SUMO) plays an important role in the function, compartmentalization, and stability of target proteins, contributing to the regulation of diverse processes. SUMO-1 modification can be regulated not only at the level of conjugation; it may also be reversed by a class of proteases known as the SUMO-specific proteases. However, current understanding of the regulation, specificity, and function of these proteases remains limited. In this study, we characterize aspects of the compartmentalization and proteolytic activity of the mammalian SUMO-specific protease, SENP1, providing insight into its function and regulation. We demonstrate the presence of a single nonconsensus nuclear localization signal within the N terminus of the protein, the mutation of which results in pronounced cytoplasmic accumulation in contrast to the nuclear accumulation of the parental protein. In addition, we observe that the N terminus of the protein may be essential for the correct regulation of the protease, since expression of the core domain alone results in limited expression and loss of SUMO-1, indicative of constitutive catalytic activity. Consistent with the prediction that the protease is a member of the cysteine family of proteases, we mutated a key cysteine residue and observed that expression of this catalytic mutant had a dominant negative phenotype, resulting in the accumulation of high molecular weight SUMO-1 conjugates. Furthermore, we demonstrate that SENP1 may itself be a target for SUMO-1 modification occurring at a nonconsensus site. Finally, we demonstrate that SENP1 localization is influenced by expression and localization of SUMO-1-conjugated target proteins within the cell.     

Proteomic identification of protease cleavage sites: cell-biological and biomedical applications

Proteolysis shapes proteomes by protein degradation or restricted proteolysis, which generates stable cleavage products. Proteolytic (in-)activation of enzymes and cytokines is an essential aspect of the functional proteome status. Proteome-wide identification and quantification of proteolytic processing is accessible by complementary techniques for the focused analysis of protein termini. These innovative strategies are now widely applied and have transformed protease research. Pioneering studies portrayed apoptotic and caspase-dependent cleavage events. Protease-centric investigations focused predominantly on matrix metalloproteinases (MMPs), granzymes and aspartyl and cysteine cathepsins. The first in vivo degradomic studies were performed with mice lacking either cysteine cathepsins or matrix metalloproteinases. Process-centric degradomic analyses investigated infectious processes and mitochondrial import. Peptidomic analyses yielded disease biomarkers representing cleavage fragments from bodily fluids. The diversity of degradomic endeavors illustrates the importance of portraying proteolytic processing in health and disease. The present review provides an overview of the current status of degradomic studies.     

On the sequential determinants of calpain cleavage

The structural clues of substrate recognition by calpain are incompletely understood. In this study, 106 cleavage sites in substrate proteins compiled from the literature have been analyzed to dissect the signal for calpain cleavage and also to enable the design of an ideal calpain substrate and interfere with calpain action via site-directed mutagenesis. In general, our data underline the importance of the primary structure of the substrate around the scissile bond in the recognition process. Significant amino acid preferences were found to extend over 11 residues around the scissile bond, from P(4) to P(7)'. In compliance with earlier data, preferred residues in the P(2) position are Leu, Thr, and Val, and in P(1) Lys, Tyr, and Arg. In position P(1) ', small hydrophilic residues, Ser and to a lesser extent Thr and Ala, occur most often. Pro dominates the region flanking the P(2)-P(1)' segment, i.e. positions P(3) and P(2)'-P(4)'; most notable is its occurrence 5.59 times above chance in P(3)'. Intriguingly, the segment C-terminal to the cleavage site resembles the consensus inhibitory region of calpastatin, the specific inhibitor of the enzyme. Further, the position of the scissile bond correlates with certain sequential attributes, such as secondary structure and PEST score, which, along with the amino acid preferences, suggests that calpain cleaves within rather disordered segments of proteins. The amino acid preferences were confirmed by site-directed mutagenesis of the autolysis sites of Drosophila calpain B; when amino acids at key positions were changed to less preferred ones, autolytic cleavage shifted to other, adjacent sites. Based on these preferences, a new fluorogenic calpain substrate, DABCYLTPLKSPPPSPR-EDANS, was designed and synthesized. In the case of micro- and m-calpain, this substrate is kinetically superior to commercially available ones, and it can be used for the in vivo assessment of the activity of these ubiquitous mammalian calpains.     

Molecular determinants of survival motor neuron (SMN) protein cleavage by the calcium-activated protease, calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN.     

A role for the intracellular protease calpain in the activation of store-operated calcium entry in human platelets

Here, we report a novel role for the cysteine protease calpain in store-operated calcium entry. Several structurally and mechanistically unrelated inhibitors of calpain inhibited Ca2+ entry activated in human platelets by thapsigargin-evoked Ca2+ store depletion or the physiological agonist thrombin, whereas inhibitors of other cysteine proteases were without effect. The use of the cell-permeable fluorogenic calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-l-leucyl-l-methionine amide revealed rapid activation of calpain which was closely temporally correlated with Ca2+ store depletion even in the absence of a rise in cytosolic [Ca2+]. Calpain inhibition prevented the tyrosine phosphorylation of several proteins upon Ca2+ store depletion, suggesting that calpain may lie upstream of protein tyrosine phosphorylation that is known to be required for the activation of store-operated Ca2+ entry in human platelets. Earlier studies using calpain inhibitors may need reinterpretation in the light of this finding that calpain plays a role in the activation of physiological Ca2+ entry pathways.     

Characterization of the rubella virus nonstructural protease domain and its cleavage site.

The region of the rubella virus nonstructural open reading frame that contains the papain-like cysteine protease domain and its cleavage site was expressed with a Sindbis virus vector. Cys-1151 has previously been shown to be required for the activity of the protease (L. D. Marr, C.-Y. Wang, and T. K Frey, Virology 198:586-592, 1994). Here we show that His-1272 is also necessary for protease activity, consistent with the active site of the enzyme being composed of a catalytic dyad consisting of Cys-1151 and His-1272. By means of radiochemical amino acid sequencing, the site in the polyprotein cleaved by the nonstructural protease was found to follow Gly-1300 in the sequence Gly-1299-Gly-1300-Gly-1301. Mutagenesis studies demonstrated that change of Gly-1300 to alanine or valine abrogated cleavage. In contrast, Gly-1299 and Gly-1301 could be changed to alanine with retention of cleavage, but a change to valine abrogated cleavage. Coexpression of a construct that contains a cleavage site mutation (to serve as a protease) together with a construct that contains a protease mutation (to serve as a substrate) failed to reveal trans cleavage. Coexpression of wild-type constructs with protease-mutant constructs also failed to reveal trans cleavage, even after extended in vitro incubation following lysis. These results indicate that the protease functions only in cis, at least under the conditions tested.