Inhibition of human mu-calpain by conformationally constrained calpastatin peptides.

The 27-mer peptide CP1B-[1-27] derived from exon 1B of calpastatin stands out among the known inhibitors for mu- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4-23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1-27]. Starting with this peptide, the turn-like region Glu(10)(i)-Leu(11)(i+1)-Gly(12)(i+2)-Lys(13)(i+3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1B-[4-23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu(10)-Lys(13) upon binding to mu-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.

A new cell-permeable calpain inhibitor.

The ubiquitous calpains, mu- and m-calpain, are implicated in a variety of vital (patho)physiological processes and therefore cell-permeable specific inhibitors represent important tools for defining the role of calpains in cells and animal models. A synthetic N-acetylated 27-mer peptide derived from exon B of the human calpastatin inhibitory domain 1 is known to be the most potent and selective reversible inhibitor of calpains. To improve the membrane permeability of this peptidic inhibitor, it was N-terminally extended with or disulfide-linked to the C-terminal 7-mer fragment of penetratin, a well-established vector for cell membrane translocation of bioactive compounds. Despite the shorter penetratin sequence, both constructs showed increased cell permeability and retained their full calpain inhibitory potency.

RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell-adhesive properties.

Secreted lysosomal cysteine proteases (cathepsins) are involved in degradation and remodeling of the extracellular matrix, thus contributing to cell adhesion and migration. Among the eleven human lysosomal cysteine proteases, only procathepsin X contains an RGD motif located in a highly exposed region of the propeptide, which may allow binding of the proenzyme to RGD-recognizing integrins. Here, we have tested procathepsin X for cell-adhesive properties and found that it supports integrin alpha(v)beta(3)-dependent attachment and spreading of human umbilical vein endothelial cells. Using site-directed mutants of procathepsin X, we proved that this effect is mediated by the RGD sequence within the proregion of the protease. Endogenous procathepsin X is transported to the plasma membrane, accumulates in vesicles at lamellipodia of the human umbilical vein endothelial cell, and is partly associated with the cell surface, as shown by immunofluorescence. In addition, procathepsin X is partly co-localized with integrin beta(3), as detected by immunogold electron microscopy. A direct interaction between endogenous procathepsin X and alpha(v)beta(3) was demonstrated by co-immunoprecipitation. Moreover, surface plasmon resonance analysis revealed significant and RGD-dependent binding of procathepsin X to integrin alpha(v)beta(3). Our results provide for the first time evidence that the extracellular function of cathepsin X may include binding to integrins thereby modulating the attachment of migrating cells to ECM components.

Mu-calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation.

Mu- and m-calpain are cysteine proteases requiring micro- and millimolar Ca2+ concentrations for their activation in vitro. Among other mechanisms, interaction of calpains with membrane phospholipids has been proposed to facilitate their activation by nanomolar [Ca2+] in living cells. Here the interaction of non-autolysing, C115A active-site mutated heterodimeric human mu-calpain with phospholipid bilayers was studied in vitro using protein-to-lipid fluorescence resonance energy transfer and surface plasmon resonance. Binding to liposomes was Ca2+-dependent, but not selective for specific phospholipid head groups. [Ca2+]0.5 for association with lipid bilayers was not lower than that required for the exposure of hydrophobic surface (detected by TNS fluorescence) or for enzyme activity in the absence of lipids. Deletion of domain V reduced the lipid affinity of the isolated small subunit (600-fold) and of the heterodimer (10- to 15-fold), thus confirming the proposed role of domain V for membrane binding. Unexpectedly, mutations in the acidic loop of the 'C2-like' domain III, a putative Ca2+ and phospholipid-binding site, did not affect lipid affinity. Taken together, these results support the hypothesis that in vitro membrane binding of mu-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca2+ requirement for activation.

Identification of calpain cleavage sites in the G1 cyclin-dependent kinase inhibitor p19(INK4d).

Calpains are a large family of Ca2+-dependent cysteine proteases that are ubiquitously distributed across most cell types and vertebrate species. Calpains play a role in cell differentiation, apoptosis, cytoskeletal remodeling, signal transduction and the cell cycle. The cell cycle proteins cyclin D1 and p21(KIP1), for example, have been shown to be affected by calpains. However, the rules that govern calpain cleavage specificity are poorly understood. We report here studies on the pattern of mu-calpain proteolysis of the p19(INK4d) protein, a cyclin-dependent kinase 4/6 inhibitor that negatively regulates the mammalian cell cycle. Our data show new characteristics of calpain action: mu-calpain cleaves p19(INK4d) immediately after the first and second ankyrin repeats that are structurally less stable compared to the other repeats. This is in contrast to features observed so far in the specificity of calpains for their substrates. These results imply that calpain may be involved in the cell cycle by regulating the cell cycle regulatory protein turnover through CDK inhibitors and cyclins.

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) by calpain.

Calpains are non-lysosomal, Ca 2+ -dependent cysteine proteases, which are ubiquitously distributed across cell types and vertebrate species. The rules that govern calpain specificity have not yet been determined. To elucidate the cleavage pattern of calpains, we carried out calpain-induced proteolytic studies on the insulin-like growth factor binding proteins IGFBP-4 and -5. Proteolysis of IGFBPs is well characterized in numerous reports. Our results show that calpain cleavage sites are in the non-conserved unstructured regions of the IGFBPs. Compilation of the calpain-induced proteolytic cleavage sites in several proteins reported in the literature, together with our present study, has not revealed clear preferences for amino acid sequences. We therefore conclude that calpains seem not to recognize amino acid sequences, but instead cleave with low sequence specificity at unstructured or solvent-exposed fragments that connect folded, stable domains of target proteins.

Electrostatic interactions of domain III stabilize the inactive conformation of mu-calpain.

The ubiquitous mu- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human mu-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca2+ concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca2+ requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive micro-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca2+ sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain.

HaCaT keratinocytes secrete lysosomal cysteine proteinases during migration.

Cathepsin B, a lysosomal cysteine proteinase, was detected within vesicles of cellular protrusions forming cell-cell contact sites between keratinocytes of the stratum spinosum of human skin. This observation suggested the possibility that secretion of the protease into the pericellular spaces could be involved in the dissociation of cell-cell contacts to enable intraepidermal keratinocyte migration. To determine whether cathepsin B is indeed secreted from migrating keratinocytes, we first used subconfluent HaCaT cells as a culture model to study spontaneous keratinocyte migration. A cathepsin B-specific fluorescent affinity label proved the association of mature cathepsin B with the surfaces of HaCaT cells at the leading edges of growing cells. Second, we used scratch-wounds of confluent HaCaT monolayers as a model of induced keratinocyte migration. Cathepsin B was detected within lysosomes, i.e. vesicles within the perinuclear region of non-wounded cells. Expression of cathepsin B was up-regulated and cathepsin B-positive vesicles showed a redistribution from perinuclear to peripheral regions of keratinocytes at the wound margins within 4 h after wounding. Enzyme cytochemistry further showed that cell surface-associated cathepsin B was proteolytically active at the leading fronts of migrating keratinocytes. In addition, increased amounts of mature forms of cathepsin B were detected within the conditioned media of HaCaT cells during the first 4 h after scratch-wounding. In contrast, and as a control, the activity of the cytosolic enzyme lactate dehydrogenase was not significantly higher in media of wounded cells as compared with non-wounded controls, arguing for a specific induction of cathepsin B secretion upon wounding and migration of the cells. This was further substantiated by applying various cathepsin B-specific inhibitors after wounding. These experiments showed that the migration ability of keratinocytes was reduced due to the blockage of functional cathepsin B. Thus, our results strongly suggest that cell surface-associated cathepsin B is a protease that contributes to the remodelling of the extracellular matrix and thereby promotes keratinocyte migration during wound healing.

TMC-95A analogues with endocyclic biphenyl ether group as proteasome inhibitors.

TMC-95A, a cyclic tripeptide metabolite of Apiospora montagnei, is a potent competitive inhibitor of proteasome. Based on the X-ray structure of its complex with yeast proteasome, the synthetically challenging structure of this natural product was simplified in a first generation of analogues by replacing the highly oxidized side-chain biaryl system with a phenyl-oxindole group. In the present study, the TMC-95 biaryl group was substituted with a biphenyl ether with retainment of significant proteasome inhibition. Because of the facile synthetic access of tripeptides containing in i, i+2 positions residues of the isodityrosine type, this new generation of TMC-95 analogues may represent promising lead structures for further optimization of affinity and selectivity of proteasome inhibitors.

Rapid enzymatic test for phenotypic HIV protease drug resistance.

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (Ki). The Michaelis constant (Km) and the inhibition constant (Ki) of recombinant wild-type protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIV-positive patients scored from 'sensitive' to 'highly resistant' according to mutations detected by genotypic analysis. The measured Ki values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.

Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: enhancing cell permeability by conjugation with penetratin.

The ubiquitous calpains, mu- and m-calpain, have been implicated in essential physiological processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addition of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain versus cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concentration than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor 1) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains.

Subcellular localization and in vivo subunit interactions of ubiquitous mu-calpain.

Ubiquitously expressed calpains are Ca(2+)-dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca(2+)-induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins. All of the overexpressed variants of the catalytic subunit (DI-IV, DI-III, and DI-IIb) were active and Ca(2+)-dependent. The intact large subunit, but not its truncated variants, associates with the small subunit under resting and ionomycin-activated conditions. All of the variants were localized in cytoplasm and nuclei, except DI-IIb, which accumulates in the nucleus and in nucleoli as shown by microscopy and cell fractionation. Localization studies with mutated and chimeric variants indicate that nuclear targeting of the DI-IIb variant is conferred by the two N-terminal helices of DI. Only those variants that contain DIII migrated to membranes upon the addition of ionomycin, suggesting that DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain, but not dissociation of its intact subunits, contribute to regulate its proteolytic activity in vivo.

Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B-specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.

Epoxysuccinyl peptide-derived cathepsin B inhibitors: modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin.

Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cell-permeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cell-permeant epoxysuccinyl-based cathepsin B inhibitor [R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH; R=OMe] with the C-terminal heptapeptide segment of penetratin (R=epsilonAhx-Arg-Arg-Nle-Lys-Trp-Lys-Lys-NH2). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodrug-like epoxysuccinyl peptides E64d and CA074Me.

Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members.

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.