LEKTI fragments specifically inhibit KLK5, KLK7, and KLK14 and control desquamation through a pH-dependent interaction

LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8-D11, and D9-D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8-D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum.     

Identification of novel prostate-specific antigen-binding peptides modulating its enzyme activity.

Prostate-specific antigen (PSA) is a serine protease with highly prostate-specific expression. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. PSA dissolves the seminal gel forming after ejaculation. It has been suggested to mediate invasion and metastasis of prostate cancer but also to exert antiangiogenic activity. We have identified peptides specific for PSA by screening cyclic phage display peptide libraries. PSA-binding peptides were isolated from four different libraries and produced as a fusion protein with glutathione S-transferase (GST). The phage and fusion proteins were shown to bind to PSA specifically as indicated by lack of binding to other serine proteinases. A peptide with four cysteines showed the highest affinity for PSA. Zn2+, an inhibitor of PSA activity, increased the affinity of the peptides to PSA. The binding specificity was characterized by cross-inhibition using monoclonal anti-PSA antibodies of known epitope specificities. The peptides bound to the same region as mAbs specific for free PSA indicating that they bind close to the active site of the enzyme. The peptides enhanced the enzyme activity of PSA against a chromogenic substrate. These results show that peptides binding to PSA and modulating its enzyme activity can be developed by phage display technique. The peptides have the potential to be used for identification of PSA variants and for imaging and targeting of prostatic tumors.     

Hdac2 regulates the cardiac hypertrophic response by modulating Gsk3 beta activity

In the adult heart, a variety of stresses induce re-expression of a fetal gene program in association with myocyte hypertrophy and heart failure. Here we show that histone deacetylase-2 (Hdac2) regulates expression of many fetal cardiac isoforms. Hdac2 deficiency or chemical histone deacetylase (HDAC) inhibition prevented the re-expression of fetal genes and attenuated cardiac hypertrophy in hearts exposed to hypertrophic stimuli. Resistance to hypertrophy was associated with increased expression of the gene encoding inositol polyphosphate-5-phosphatase f (Inpp5f) resulting in constitutive activation of glycogen synthase kinase 3beta (Gsk3beta) via inactivation of thymoma viral proto-oncogene (Akt) and 3-phosphoinositide-dependent protein kinase-1 (Pdk1). In contrast, Hdac2 transgenic mice had augmented hypertrophy associated with inactivated Gsk3beta. Chemical inhibition of activated Gsk3beta allowed Hdac2-deficient adults to become sensitive to hypertrophic stimulation. These results suggest that Hdac2 is an important molecular target of HDAC inhibitors in the heart and that Hdac2 and Gsk3beta are components of a regulatory pathway providing an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure.     

Mimetics of the disulfide bridge between the N- and C-terminal cysteines of the KLK3-stimulating peptide B-2

Human prostate produces kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen), which is widely used as a prostate cancer marker. Proteolytically active KLK3 has been shown to inhibit angiogenesis and its expression decreases in poorly differentiated tumors. Thus, it may be possible to control prostate cancer growth with agents that stimulate the proteolytic activity of KLK3. We have earlier developed synthetic peptides, which bind specifically to KLK3 and promote its proteolytic activity. These peptides are cyclic, all containing a disulfide bridge between the N- and C-terminal cysteines. To increase the in vivo stability of the KLK3-stimulating peptide B-2, we made differently cyclized analogues by replacing both terminal cysteines and the disulfide bridge between them. A replacement consisting of γ-amino butyric acid and aspartic acid, where the amino group from the former was linked to the main chain carboxyl group of the latter, was found to be, at high concentrations, more active than the B-2 peptide. Furthermore, as compared to the parent peptide, this analog had an improved stability in plasma and against the enzymatic degradation by KLK3. In addition, the series of analogues also provided valuable information of the structure–activity relationships of the B-2 peptide.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

Peptides and antitumor activity. Development and investigation of some peptides with antitumor activity

We developed a group of synthetic analogs of GnRH and Somatostatin to inhibit the tumor growth of different kind. The GnRH analogs decreasing the gonadotroph and steroid hormone levels act on the hormone dependent tumors and influence their growth. One of the most effective antitumor analog was patented under the name FOLLIGEN which inhibited the breast cancer caused by DMBA in rats without any side-effects. Other inhibitory analogs of GnRH with long-lasting effect were effective in the treatment of breast, ovary and prostate tumors. Another analog [alpha-Asp(DEA)]6,Gln8-hGnRH showed a very low endocrine but high antitumor effect in both in vitro and in vivo experiments. Its tritium labeled derivative exhibited specific binding sites on human tumor cell lines. We synthesized the analogs of GnRH-III with effective selective antitumor activity which does not alter the ovarian cycle of rats but inhibits the colony-formation of human breast cancer cell lines and has a significant antiproliferative effect. We also synthesized conjugates of potent GnRH analogs with a branched chain polylysine backbone which induce a 33-35% decrease of cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines and 45-50% inhibition of cell proliferation. Another conjugate decreased the tumor growth of MDA-MB-231 xenografts by 80% in a treatment of 9 weeks and even tumor free animals could be found among the ones treated. Using these radiolabeled peptide hormone analogs we found that human tumor cell lines and xenografts specifically bind the GnRH conjugates. We also synthesized a series of Somatostatin analogs which inhibit tyrosine kinases and the growth of several breast, prostate and colon tumor cell lines. One of our best analogs was a heptapeptide, TT-232, which strongly inhibited the tyrosine kinase activity and the cell-proliferation in different colon tumor cells. However, it did not inhibit the growth hormone release either in vitro or in vivo from rat pituitary cells. The TT-232 was found to be effective on 60 human tumor cell lines, it significantly inhibited the tumor growth on different animal tumor models, and induced apoptosis, as a result of which some animals became tumor free. The TT-232 inhibited the tumor growth of PC3 prostate xenografts with 60% and caused a 100% survival of mice 60 days after the transplantation. It is being preclinically tested at present. We have shown that the new GnRH analogs acting without any hormonal effect and the Somatostatin analogs with strong antitumor and tyrosine kinase inhibitory activity but no hormonal effect may represent a breakthrough in the research of the antitumor peptides, having direct effect on tumor cells.     

Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis

Natriuretic peptides of various forms are present in animals and plants, and display structural similarities to cyclic antibacterial peptides. Pretreatment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide (BNP) or C-type natriuretic peptide (CNP) increases bacterium-induced glial cell necrosis. In eukaryotes, natriuretic peptides act through receptors coupled to cyclases. We observed that stable analogs of cAMP (dibutyryl cAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natriuretic peptide and CNP on bacteria. Further evidence for the involvement of bacterial cyclases in the regulation of P. aeruginosa PAO1 cytotoxicity by natriuretic peptides is provided by the observed doubling of intrabacterial cAMP concentration after exposure to CNP. Lipopolysaccharide (LPS) extracted from P. aeruginosa PAO1 treated with both dibutyryl cAMP and 8-bromo-cGMP induces higher levels of necrosis than LPS extracted from untreated bacteria. Capillary electrophoresis and MALDI-TOF MS analysis have shown that differences in LPS toxicity are due to specific differences in the structure of the macromolecule. Using a strain deleted in the vfr gene, we showed that the Vfr protein is essential for the effect of natriuretic peptides on P. aeruginosa PAO1 virulence. These data support the hypothesis that P. aeruginosa has a cyclic nucleotide-dependent natriuretic peptide sensor system that may affect virulence by activating the expression of Vfr and LPS biosynthesis.     

3'Sulfogalactolipid binding specifically inhibits Hsp70 ATPase activity in vitro

A method for the generation of soluble glycosphingolipid derivatives that retain the receptor activity of the parent (BBRC 257:391-394, Carb Res 335:91-100) was used to investigate the consequence of 3'sulfogalactolipid (SGL) specific binding within the N-terminal ATPase-containing domain of Hsc70. Sulfogalactosyl ceramide (SGC) was deacylated, and the resulting sulfogalactosylsphingosine coupled to an alpha-adamantane or a norbornane rigid hydrophobic frame. The resulting conjugate preferentially partitioned into water, as opposed to organic solvent. In the range of 100-300 microM, these conjugates inhibited the specific binding of bovine brain Hsc70 to immobilized SGLs. A similar dose-related inhibition of bovine brain Hsc70 ATPase activity was seen between 100 and 300 microM adamantylSGC (adaSGC). Adamantyl conjugates of glycolipids not bound by Hsp70s had no effect. Kinetic analysis indicated that adaSGC was a noncompetitive inhibitor of Hsc70 ATPase activity, a special case of mixed inhibition since the K(m) values were not statistically different, 0.89 +/- 0.024 microM to 0.93 +/- 0.038 microM, but the V(max) decreased from 0.20 +/- 0.012 pmol min(-1) microg(-1) to 0.15 +/- 0.016 pmol min(-1) microg(-1). A reproducible 5 min lag was observed prior to ATPase inhibition that could be eliminated by preincubation of adaSGC with Hsc70 or by adding the cochaperone Hdj-1. The dependence of ATPase inhibition on the rate of hydrolysis indicates that adaSGC binding occurs at a specific stage of the ATPase cycle. These studies identify a new mechanism for the regulation of Hsp70 ATPase activity.     

Gene expression changes associated with the anti-angiogenic activity of kallikrein-related peptidase 3 (KLK3) on human umbilical vein endothelial cells

Kallikrein-related peptidase 3 (KLK3, also known as prostate-specific antigen, PSA) is a chymotrypsin-like kallikrein that has anti-angiogenic properties. We have previously shown in a human umbilical vein endothelial cell (HUVEC) model that the anti-angiogenic effect of KLK3 is related to its enzyme activity. However, the mechanism of this effect remains to be clarified. To this end, we used a DNA microarray to study KLK3-induced changes in gene expression associated with reduction of HUVEC tube formation. Among the 41,000 genes studied, 311 were differentially expressed between control and KLK3-treated cells. These changes were enriched in several pathways, including those associated with proteasome, ubiquitin-mediated proteolysis, focal adhesion and regulation of the actin cytoskeleton. Furthermore, the changes were opposite to those previously described to occur during tubulogenesis. In conclusion, our results show that KLK3 induces gene expression changes in HUVECs. Although these changes might be relevant for the mechanism by which KLK3 exerts its anti-angiogenic activity, it cannot be judged from the present results whether they reflect the primary mechanism mediating the effect of KLK3 or are secondary to morphogenic differentiation.     

Prostate--a novel target for evaluation of FSH modulating peptides

Effects of prostatic inhibin peptide and its synthetic fragments on FSH biosynthesis by the human pituitary and prostate, were examined in vitro. The results showed that FSH biosynthesis by prostatic tissue is modulated by these peptides in a similar fashion to that observed at the pituitary level.     

Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells

Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins’ functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host-pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.     

Parameters modulating the maximum insertion pressure of proteins and peptides in lipid monolayers

The lipid monolayer model membrane is useful for studying the parameters responsible for protein and peptide membrane binding. Different approaches have been used to determine the extent of protein and peptide binding to lipid monolayers. This review focuses on the use of the “maximum insertion pressure” (MIP) to estimate the extent of protein and peptide penetration in lipid monolayers. The MIP data obtained with different proteins and peptides have been reviewed and discussed which allowed to draw conclusions on the parameters modulating the monolayer binding of proteins and peptides. In particular, secondary structure components such as amphipathic α-helices of proteins and peptides as well as electrostatic interactions play important roles in monolayer binding. The MIPs have been compared to the estimated lateral pressure of biomembranes which allowed to evaluate the possible association between proteins or peptides with natural membranes. For example, the MIP of a membrane-anchored protein with a glycosylphosphatidylinositol (GPI) was found to be far below the estimated lateral pressure of biomembranes. This allowed us to conclude that this protein is probably unable to penetrate the membrane and should thus be hanged at the membrane surface by use of its GPI lipid anchor. Moreover, the values of MIP obtained with myristoylated and non-myristoylated forms of calcineurin suggest that the myristoyl group does not contribute to monolayer binding. However, the acylation of a peptide resulted in a large increase of MIP. Finally, the physical state of lipid monolayers can have a strong effect on the values of MIP such that it is preferable to perform measurements with lipids showing a single physical state. Altogether the data show that the measurement of the maximum insertion pressure provides very useful information on the membrane binding properties of proteins and peptides although uncertainties must be provided to make sure the observed differences are significant.     

Antifungal activity of C3a and C3a-derived peptides against Candida

Antimicrobial peptides are generated during activation of the complement system [Nordahl et al. Proc. Natl. Acad. Sci. U. S. A. 2004, 101:16879-16884]. Here we show that the anaphylatoxin C3a exerts antimicrobial effects against the yeast Candida. Fluorescence microscopy and electron microscopy analysis demonstrated that C3a-derived peptides bound to the cell surface of Candida, and induced membrane perturbations and release of extracellular material. Various Candida isolates were found to induce complement degradation, leading to generation of C3a. Arginine residues were found to be critical for the antifungal and membrane breaking activity of a C3a-derived antimicrobial peptide, CNY21 (C3a; Cys57-Arg77). A CNY21 variant with increased positive net charge displayed enhanced antifungal activity. Thus, C3a-derived peptides can be utilized as templates in the development of peptide-based antifungal therapies.     

EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity

EHDs [EH (Eps15 homology)-domain-containing proteins] participate in different stages of endocytosis. EHD2 is a plasma-membrane-associated EHD which regulates trafficking from the plasma membrane and recycling. EHD2 has a role in nucleotide-dependent membrane remodelling and its ATP-binding domain is involved in dimerization, which creates a membrane-binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide-free mutant (EHD2 T72A) failed to associate. To elucidate the possible function of EHD2 during endocytic trafficking, we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3 [NIMA (never in mitosis in Aspergillus nidulans)-related kinase 3], a serine/threonine kinase. EHD2 was also found in association with Vav1, a Nek3-regulated GEF (guanine-nucleotide-exchange factor) for Rho GTPases. Since Vav1 regulates Rac1 activity and promotes actin polymerization, the impact of overexpression of EHD2 on Rac1 activity was tested. The results indicated that wt (wild-type) EHD2, but not its P-loop mutants, reduced Rac1 activity. The inhibitory effect of EHD2 overexpression was partially rescued by co-expression of Rac1 as measured using a cholera toxin trafficking assay. The results of the present study strongly indicate that EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity.     

Cationic amino acid transporter-2 regulates immunity by modulating arginase activity

Cationic amino acid transporters (CAT) are important regulators of NOS2 and ARG1 activity because they regulate L-arginine availability. However, their role in the development of Th1/Th2 effector functions following infection has not been investigated. Here we dissect the function of CAT2 by studying two infectious disease models characterized by the development of polarized Th1 or Th2-type responses. We show that CAT2(-/-) mice are significantly more susceptible to the Th1-inducing pathogen Toxoplasma gondii. Although T. gondii infected CAT2(-/-) mice developed stronger IFN-gamma responses, nitric oxide (NO) production was significantly impaired, which contributed to their enhanced susceptibility. In contrast, CAT2(-/-) mice infected with the Th2-inducing pathogen Schistosoma mansoni displayed no change in susceptibility to infection, although they succumbed to schistosomiasis at an accelerated rate. Granuloma formation and fibrosis, pathological features regulated by Th2 cytokines, were also exacerbated even though their Th2 response was reduced. Finally, while IL-13 blockade was highly efficacious in wild-type mice, the development of fibrosis in CAT2(-/-) mice was largely IL-13-independent. Instead, the exacerbated pathology was associated with increased arginase activity in fibroblasts and alternatively activated macrophages, both in vitro and in vivo. Thus, by controlling NOS2 and arginase activity, CAT2 functions as a potent regulator of immunity.     

Anti-innexin 2 aptamers specifically inhibit the heterologous interaction of the innexin 2 and innexin 3 carboxyl-termini in vitro

We recently demonstrated that heteromerization of innexins 2 and 3 from Drosophila melanogaster (Dm) is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins are thought to interact via their C-terminal cytoplasmic domains during the assembly of heteromeric gap junction channels. However, the mechanisms that control heteromeric versus homomeric channel formation are still largely unknown. Here we report the isolation of both non-modified and 2'-fluoro-2'-deoxy-modified RNA anti-innexin 2 aptamers by in vitro selection. The aptamers bind to a proximal epitope on the carboxyl-tail of Dm innexin 2 protein and specifically inhibit the heterologous interaction of innexin 2 and innexin 3 carboxyl-termini in vitro. These domain-specific inhibitors represent the first step towards functional studies focusing on the activity of these domains in vivo.     

Interactions between lung stretch and PaCO2 in modulating ventilatory activity in dogs

The effects of changes in airway pressure (Paw) and arterial PCO2 (PaCO2) on ventilatory activity were studied in anesthetized thoracotomized dogs in which both lungs were ventilated separately. Pulmonary artery occlusion on one side and contralateral vagotomy allowed the reflex effects on ventilation of changes in Paw and PaCO2 to be elicited independently of each other. Ventilatory activity was assessed from integrated efferent phrenic activity, analyzed with respect to burst amplitude (Phr), burst frequency (f), and inspiratory TI) and expiratory duration (TE). While Phr increased linearly with PaCO2, it was independent of Paw. Both PaCO2 and Paw affected f in a complex nonadditive way; this response was entirely mediated by effects on TE, TI being unaffected by either stimulus. The analog of ventilation, estimated as Phr x f, increased linearly with PaCO2 and decreased linearly with Paw, but the effects of both stimuli appeared to be additive. It is concluded that the apparently simple effect of Paw and PaCO2 on ventilation results from more complex effects these stimuli exert on its components.