József Lázár

Structural insights into transient receptor potential vanilloid type 1 (TRPV1) from homology modeling, flexible docking, and mutational studies

The transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel composed of four monomers with six transmembrane helices (TM1–TM6). TRPV1 is found in the central and peripheral nervous system, and it is an important therapeutic target for pain relief. We describe here the construction of a tetrameric homology model of rat TRPV1 (rTRPV1). We experimentally evaluated by mutational analysis the contribution of residues of rTRPV1 contributing to ligand binding by the prototypical TRPV1 agonists, capsaicin and resiniferatoxin (RTX). We then performed docking analysis using our homology model. The docking results with capsaicin and RTX showed that our homology model was reliable, affording good agreement with our mutation data. Additionally, the binding mode of a simplified RTX (sRTX) ligand as predicted by the modeling agreed well with those of capsaicin and RTX, accounting for the high binding affinity of the sRTX ligand for TRPV1. Through the homology modeling, docking and mutational studies, we obtained important insights into the ligand-receptor interactions at the molecular level which should prove of value in the design of novel TRPV1 ligands.

Opposite roles of protein kinase C isoforms in proliferation, differentiation, apoptosis, and tumorigenicity of human HaCaT keratinocytes

We have previously shown that the protein kinase C (PKC) system plays a pivotal role in regulation of proliferation and differentiation of the human keratinocyte line HaCaT which is often used to assess processes of immortalization, transformation, and tumorigenesis in human skin. In this paper, using pharmacological and molecular biology approaches, we investigated the isoform-specific roles of certain PKC isoenzymes (conventional cPKCα and β; novel nPKCδ and ε) in the regulation of various keratinocyte functions. cPKCα and nPKCδ stimulated cellular differentiation and increased susceptibility of cells to actions of inducers of apoptosis, and they markedly inhibited cellular proliferation and tumor growth in immunodeficient mice. In marked contrast, cPKCβ and nPKCε increased both in vitro and in vivo growth of cells and inhibited differentiation and apoptosis. Our data present clear evidence for the specific, antagonistic roles of certain cPKC and nPKC isoforms in regulating the above processes in human HaCaT keratinocytes.

Protein kinase C isozymes regulate proliferation and high cell density‐mediated differentiation in HaCaT keratinocytes

Abstract:u2002 Protein kinase C (PKC) isoforms play pivotal roles in the regulation of differentiation of normal human epidermal keratinocytes (NHEK). In this study, we investigated the participation of the PKC system in the proliferation and high cell density‐induced differentiation of the human immortalized keratinocyte line HaCaT. HaCaT keratinocytes possessed a characteristic PKC isoform pattern (PKCα, β, γ,δ,ε,η,θ,ζ), which altered during proliferation and differentiation. The GF109203X compound, a selective PKC inhibitor, suppressed the expressions of the late (granular cell) differentiation markers involucrin (INV) and filaggrin (FIL), and the terminal marker keratinocytes‐specific transglutaminase‐1 (TG), but did not affect the level of the early (spinous cell) marker keratin 10 (K10) and cellular proliferation. Phorbol 12‐myristate 13‐acetate (PMA), an activator of PKC, inhibited proliferation, elevated intracellular calcium concentration, decreased the expression of K10, and increased the expressions of INV, FIL, and TG. These data indicate that the endogenous activation of PKC regulates the expressions of the late differentiation markers, and that the exogenous activation of PKC by PMA results in the induction of terminal differentiation. Because the cellular effects of PMA were accompanied by differential down‐regulations of the sensitive PKC isoforms in proliferating and differentiating cultures, our findings argue for the differential roles of the existing PKC isoforms in the regulation of cellular proliferation and high cell density‐induced differentiation of HaCaT cells.

Screening TRPV1 antagonists for the treatment of pain: lessons learned over a decade

Background: The capsaicin receptor TRPV1, a polymodal nociceptor whose expression is up-regulated in a number of painful inflammatory disorders, represents a promising therapeutic target for pain relief. Potent small molecule TRPV1 antagonists are now undergoing clinical trials in patients with inflammatory or neuropathic pain. This review focuses on the multiplicity of factors regulating this channel and on their contributions to the emerging complexity of responses to TRPV1 and partial antagonists. For example, it is now clear that antagonists of capsaicin response can also antagonize, have no effect, or stimulate response to heat or protons. The complexity of TRPV1 regulation affords the potential to optimize agents for a specific therapeutic indication. An encouraging advance is the dissection of therapeutic efficacy of antagonists from induction of hyperthermia, a side effect that initially had raised concerns about the suitability of systemically administered TRPV1 antagonists for therapy. Objectives and methods: To discuss the challenges facing the development of clinically useful TRPV1 antagonists based on our experience and a comprehensive review of the literature. Results/conclusions: TRPV1 is a polymodal receptor. Some antagonists block all modalities of TRPV1 stimulation whereas others are more selective in their pharmacological profile. A number of antagonists can, conversely, potentiate certain modes of TRPV1 activation (e.g., protons and heat). The selectivity of TRPV1 antagonists is species-dependent, posing a problem for extrapolation from animal models to patients. At present, this rich pharmacology of TRPV1 antagonists complicates drug development but for the future it promises great opportunities for drug design.

Characterization of the Interaction of Phorbol Esters with the C1 Domain of MRCK (Myotonic Dystrophy Kinase-related Cdc42 Binding Kinase) α/β

C1 domains mediate the recognition and subsequent signaling response to diacylglycerol and phorbol esters by protein kinase C (PKC) and by several other families of signal-transducing proteins such as the chimerins or RasGRP. MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), a member of the dystrophia myotonica protein kinase family that functions downstream of Cdc42, contains a C1 domain with substantial homology to that of the diacylglycerol/phorbol ester-responsive C1 domains and has been reported to bind phorbol ester. We have characterized here the interaction of the C1 domains of the two MRCK isoforms α and β with phorbol ester. The MRCK C1 domains bind [20-3H]phorbol 12,13-dibutyrate with Kd values of 10 and 17 nm, respectively, reflecting 60–90-fold weaker affinity compared with the protein kinase C δ C1b domain. In contrast to binding by the C1b domain of PKCδ, the binding by the C1 domains of MRCK α and β was fully dependent on the presence of phosphatidylserine. Comparison of ligand binding selectivity showed resemblance to that by the C1b domain of PKCα and marked contrast to that of the C1b domain of PKCδ. In intact cells, as in the binding assays, the MRCK C1 domains required 50–100-fold higher concentrations of phorbol ester for induction of membrane translocation. We conclude that additional structural elements within the MRCK structure are necessary if the C1 domains of MRCK are to respond to phorbol ester at concentrations comparable with those that modulate PKC.

Distinct features of recombinant rat vanilloid receptor-1 expressed in various expression systems

In this study, we expressed rat vanilloid receptor 1 (VR1) in various heterologous expression systems using different VR1-encoding vectors, and examined how the VR1 agonists capsaicin and resiniferatoxin affected intracellular calcium. Our results clearly show that the magnitude and kinetics of response as well as the extent of tachyphylaxis differ markedly between systems. Using green fluorescent protein-tagged VR1, we show that much of the VR1 is localized to intracellular membranes. Consistent with this localization, VR1 agonists are able to liberate calcium from intracellular stores in the absence of extracellular calcium. As with other parameters of response, the three expression systems differ in the degree to which, in the absence of extracellular calcium, capsaicin and resiniferatoxin can liberate calcium from the intracellular stores. Our findings emphasize the influence of the expression system on characteristics of the response of VR1 to its ligands and the need for caution in extrapolating such results to other settings.

Serum human epididymis protein 4 (HE4) as a tumor marker in men with lung cancer

Abstract Background: Human epididymis protein 4 (HE4) is a reliable tumor marker for ovarian cancer, but only limited data are available on HE4 levels in lung malignancies. Methods: HE4 levels were measured at diagnosis in 98 men with lung cancer at different stages of the disease, and these results were compared to an age-matched healthy male cohort (n=98). The concentrations of classical tumor markers were also determined, and their efficacy was compared to that of HE4. Results: Compared to healthy controls, patients with lung neoplasm showed significantly higher HE4 levels [118.2 (80.6–150.1) pmol/L vs. 62.2 (47.2–76.1) pmol/L; p<0.001]. Although age and smoking modulated HE4 levels in the healthy cohort, no such effect was observed in the patient population. The area under the receiver operating characteristic curve (ROC-AUC) for HE4 was 0.848 (95% CI 0.792–0.904) for differentiating lung cancer patients from healthy controls, with a cut-off value of 97.6 pmol/L (sensitivity: 64.3%, specificity: 95.9%). HE4 levels were significantly elevated in all stages of lung cancer, and even in patients without clinical symptoms (p<0.05), but no difference was found between the different histological subgroups. A significant correlation was found between HE4 values and the tumor size determined by CT/MRI (Spearman’s ρ=0.227, p=0.030). The combination of HE4 with CEA and CA 125 considerably enhanced the diagnostic efficacy [ROC-AUC: 0.963 (95% CI 0.937–0.990), sensitivity: 91.8%, specificity: 92.8%]. Conclusions: Our data suggest that serum HE4, especially in combination with CEA and CA 125, qualifies as a surrogate diagnostic marker in men with lung cancer.

N-4-t-Butylbenzyl 2-(4-Methylsulfonylaminophenyl) Propanamide TRPV1 Antagonists : Structure Activity Relationships in the A-region

Structure-activity relationships for the A-region in a series of N-4-t-butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamides as TRPV1 antagonists have been investigated. Among them, the 3-fluoro analogue 54 showed high binding affinity and potent antagonism for both rTRPV1 and hTRPV1 in CHO cells. Its stereospecific activity was demonstrated with marked selectivity for the (S)-configuration (54S versus 54R). A docking study of 54S with our hTRPV1 homology model highlighted crucial hydrogen bonds between the ligand and the receptor contributing to its potency.

Phorbol ester treatment inhibits proliferation and differentiation of cultured human skeletal muscle satellite cells by differentially acting on protein kinase C isoforms

Abstract. We have previously shown that cultured human skeletal muscle cells express five protein kinase C (PKC) isoforms (PKCα, -γ, -η, -θ, and -ζ) and that expression levels of various PKC isozymes differentially change during differentiation. In this study we investigated the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on differentiation and on PKC isozymes of human skeletal muscle satellite cells. PMA inhibited the growth and fusion of cultured human myoblasts in a dose-dependent manner. In addition, prolonged treatment of cells with PMA suppressed the expression of the myogenic differentiation marker desmin showing similar dose-response characteristics. Furthermore, PMA also induced the intracellular translocation of PKCγ, -η, and -θ, whereas cellular localization of PKCα and -ζ were not altered. These changes in subcellular localization patterns were of great importance since only those PKC isoforms were translocated that possessed alterations in their expression levels during differentiation. Our findings, therefore, suggest that the PMA-induced inhibition of differentiation of human skeletal muscle cells is mediated by certain PKC isoforms. Moreover, these data strongly argue for differential and isozyme-specific roles of various PKC isoforms in these processes.

Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery

mAb proteomics, a reversed biomarker discovery approach, is a novel methodology to recognize the proteins of biomarker potential, but requires subsequent antigen identification steps. While in case of high‐abundant proteins, it generally does not represent a problem, for medium or lower abundant proteins, the identification step requires a large amount of sample to assure the proper amount of antigen for the ID process. In this article, we report on the use of combined chromatographic and precipitation techniques to generate a large set of fractions representing the human plasma proteome, referred to as the Analyte Library, with the goal to use the relevant library fractions for antigen identification in conjunction with mAb proteomics. Starting from 500u2009mL normal pooled human plasma, this process resulted in 783 fractions with the average protein concentration of 1u2009mg/mL. First, the serum albumin and immunoglobulins were depleted followed by prefractionation by ammonium sulfate precipitation steps. Each precipitate was then separated by size exclusion chromatography, followed by cation and anion exchange chromatography. The 20 most concentrated ion exchange chromatography fractions were further separated by hydrophobic interaction chromatography. All chromatography and precipitation steps were carefully designed aiming to maintain the native forms of the intact proteins throughout the fractionation process. The separation route of vitamin D‐binding protein (an antibody proteomics lead) was followed in all major fractionation levels by dot blot assay in order to identify the library fraction it accumulated in and the identity of the antigen was verified by Western blot.