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.

Dimerization of beta-site beta-amyloid precursor protein-cleaving enzyme.

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

Binding mode of TMC-95A analogues to eukaryotic 20S proteasome.

The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities. X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.

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.

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.

Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems.

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.

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.

Synthesis of a TMC-95A ketomethylene analogue by cyclization via intramolecular Suzuki coupling.

[structure: see text] A TMC-95A analogue extended at the C-terminus with NlePsi[COCH(2)]Gly-Ala-Ala-NH(2) was synthesized via side-chain cyclization of the linear precursor by a Suzuki cross-coupling reaction in solution to analyze the effect of additional P' residues on the inhibitory potency against yeast proteasome.

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.

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.