Human lysozyme inhibits the fibrillation of serum amyloid a protein from systemic AA amyloidosis.

BACKGROUND: Systemic AA amyloidosis is a world-wide occurring protein misfolding disease in humans and animals that arises from the formation of amyloid fibrils from serum amyloid A (SAA) protein and their deposition in multiple organs. OBJECTIVE: To identify new agents that prevent fibril formation from SAA protein and to determine their mode of action. MATERIALS AND METHODS: We used a cell model for the formation of amyloid deposits from SAA protein to screen a library of peptides and small proteins, which were purified from human hemofiltrate. To clarify the inhibitory mechanism the obtained inhibitors were characterised in cell-free fibril formation assays and other biochemical methods. RESULTS: We identified lysozyme as an inhibitor of SAA fibril formation. Lysozyme antagonised fibril formation both in the cell model as well as in cell-free fibril formation assays. The protein binds SAA with a dissociation constant of 16.5 ± 0.6 µM, while the binding site on SAA is formed by segments of positively charged amino acids. CONCLUSION: Our data imply that lysozyme acts in a chaperone-like fashion and prevents the aggregation of SAA protein through direct, physical interactions.

Cyclophilin A inhibition as potential treatment of human aortic valve calcification.

Aortic valve stenosis (AS) is a pathological condition that affects about 3% of the population, representing the most common valve disease. The main clinical feature of AS is represented by the impaired leaflet motility, due to calcification, which leads to the left ventricular outflow tract obstruction during systole. The formation and accumulation of calcium nodules are driven by valve interstitial cells (VICs). Unfortunately, to date, the in vitro and in vivo studies were not sufficient to fully recapitulate all the pathological pathways involved in AS development, as well as to define a specific and effective pharmacological treatment for AS patients. Cyclophilin A (CyPA), the most important immunophilin and endogenous ligand of cyclosporine A (CsA), is strongly involved in several detrimental cardiovascular processes, such as calcification. To date, there are no data on the CyPA role in VIC-mediated calcification process of AS. Here, we aimed to identify the role of CyPA in AS by studying VIC calcification, in vitro. In this study, we found that (i) CyPA is up-regulated in stenotic valves of AS patients, (ii) pro-calcifying medium promotes CyPA secretion by VICs, (iii) in vitro treatment of VICs with exogenous CyPA strongly stimulates calcium deposition, and (iv) exogenous CyPA inhibition mediated by CsA analogue MM284 abolished in vitro calcium potential. Thus, CyPA represents a biological target that may act as a novel candidate in the detrimental AS development and its inhibition may provide a novel pharmacological approach for AS treatment.

Influences of cyclosporin A and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229E replication.

The well-known immunosuppressive drug cyclosporin A inhibits replication of various viruses including coronaviruses by binding to cellular cyclophilins thus inactivating their cis-trans peptidyl-prolyl isomerase function. Viral nucleocapsid proteins are inevitable for genome encapsidation and replication. Here we demonstrate the interaction between the N protein of HCoV-229E and cyclophilin A, not cyclophilin B. Cyclophilin inhibitors abolish this interaction. Upon infection, cyclophilin A stays evenly distributed throughout the cell, whereas cyclophilin B concentrates at ER-bleb-like structures. We further show the inhibitory potential of non-immunosuppressive CsA derivatives Alisporivir, NIM811, compound 3 on HCoV-229E-GFP and -Luciferase replication in human Huh-7.5 hepatoma cells at 18 and 48 h time points post infection with EC50 s at low micromolar ranges. Thus, non-immunosuppressive CsA derivatives effectively inhibit HCoV-229E replication suggesting them as possible candidates for the treatment of HCoV infection. The interruption of interaction between CypA and N protein by CsA and its derivatives suggest a mechanism how CypA inhibitors suppress viral replication.

A fluorescence-based array screen for transglutaminase substrates.

Transglutaminases (EC 2.3.2.13) form an enzyme family that catalyzes the formation of isopeptide bonds between the γ-carboxamide group of glutamine and the ε-amine group of lysine residues of peptides and proteins. Other primary amines can be accepted in place of lysine. Because of their important physiological and pathophysiological functions, transglutaminases have been studied for 60 years. However, the substrate preferences of this enzyme class remain largely elusive. In this study, we used focused combinatorial libraries of 400 peptides to investigate the influence of the amino acids adjacent to the glutamine and lysine residues on the catalysis of isopeptide bond formation by microbial transglutaminase. Using the peptide microarray technology we found a strong positive influence of hydrophobic and basic amino acids, especially arginine, tyrosine, and leucine. Several tripeptide substrates were synthesized, and enzymatic kinetic parameters were determined both by microarray analysis and in solution.

The Novel Extracellular Cyclophilin A (CyPA) - Inhibitor MM284 Reduces Myocardial Inflammation and Remodeling in a Mouse Model of Troponin I -Induced Myocarditis.

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.

Inhibition of extracellular cyclophilins with cyclosporine analog and development of atherosclerosis in apolipoprotein E-deficient mice.

Cyclophilins exert both intracellular and extracellular activities related to immune responses and inflammation, which have been implicated in pathogenesis of atherosclerosis. Pan-inhibition of cyclophilins has both pro- and antiatherosclerotic properties, but specific contributions of extracellular and intracellular cyclophilins to these effects have not been characterized. Here, using selective inhibitor of extracellular cyclophilins, we investigated the role of these molecules in atherosclerosis. Apolipoprotein E-null mice fed a high-fat diet received intraperitoneal injections every second day of either vehicle or two analogs of cyclosporine A (CsA): [Melle](4)-CsA (NIM811), a nonimmunosupressive cell-permeable inhibitor of both intracellular and extracellular cyclophilins; and [(4R)-4-[(6-carboxy-1H-benzo[d]imidazol-2-yl)-methyl]-4-methyl-l-threonine](1)-CsA (MM284), cell-impermeable analog only inhibiting extracellular cyclophilins. Development of atherosclerosis and composition of plaques in aorta and innominate artery were studied. Both analogs increased abundance and cross-sectional size of the atherosclerotic plaques in aorta but did not affect development of atherosclerosis in innominate artery. Neither compound affected abundance of macrophages and amount of vascular cell adhesion molecule-1 or nitrotyrosine in the plaques of both arteries. Both compounds reduced the amount of collagen in innominate artery without affecting abundance of collagen in aortic sinus. MM284, but not NIM811, significantly reduced plasma concentration of tumor necrosis factor-α (TNFα); neither compound affected plasma concentrations of interleukin (IL)-6, IL-10 or monocyte chemoattractant protein-1. Ratio between different populations of immune cells in blood or isolated from lymph nodes and spleen as well as plasma lipoprotein profile were unaffected by both compounds. In conclusion, selective inhibition of extracellular cyclophilins reduced TNFα levels in plasma but increased atherosclerosis.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

OBJECTIVE: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. APPROACH AND RESULTS: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. CONCLUSIONS: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

Cyclophilin-facilitated membrane translocation as pharmacological target to prevent intoxication of mammalian cells by binary clostridial actin ADP-ribosylated toxins.

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 µM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.

Design of cyclic peptides featuring proline predominantly in the cis conformation under physiological conditions.

Turns are secondary-structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four-residue ß-turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The ßVI-type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c-(-DXaa-Ser-Pro-DXaa-Lys-Pro-) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D-amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis-Pro/cis-Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space.

The FKBP38 catalytic domain binds to Bcl-2 via a charge-sensitive loop.

FKBP38 is a regulator of the prosurvival protein Bcl-2, but in the absence of detailed structural insights, the molecular mechanism of the underlying interaction has remained unknown. Here, we report the contact regions between Bcl-2 and the catalytic domain of FKBP38 derived by heteronuclear NMR spectroscopy. The data reveal that a previously identified charge-sensitive loop near the putative active site of FKBP38 is mainly responsible for Bcl-2 binding. The corresponding binding epitope of Bcl-2 could be identified via a peptide library-based membrane assay. Site-directed mutagenesis of the key residues verified the contact sites of this electrostatic protein/protein interaction. The derived structure model of the complex between Bcl-2 and the FKBP38 catalytic domain features both electrostatic and hydrophobic intermolecular contacts and provides a rationale for the regulation of the FKBP38/Bcl-2 interaction by Ca(2+).

Crystal structure and functional analysis of the protein disulfide isomerase-related protein ERp29.

The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 A, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties.

Secreted cyclophilin A, a peptidylprolyl cis-trans isomerase, mediates matrix assembly of hensin, a protein implicated in epithelial differentiation.

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

A novel calmodulin-Ca2+ target recognition activates the Bcl-2 regulator FKBP38.

The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290-Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.

Inhibition of human ether à go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-termini.

Human ether à go-go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array-based screen of the entire cytosolic protein of hEAG1 identified three putative CaM-binding domains, two in the C-terminus (BD-C1: 674-683, BD-C2: 711-721) and one in the N-terminus (BD-N: 151-165). Binding of GST-fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca2+, BD-N and BD-C2 provided dissociation constants in the nanomolar range, BD-C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM-EF-hand mutants showed that CaM binding to the N- and C-terminus are primarily dependent on EF-hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed.