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Physiological Substrate (physiological + substrate)
Selected AbstractsSensitivity of alveolar macrophages to substrate mechanical and adhesive propertiesCYTOSKELETON, Issue 6 2006Sophie Féréol Abstract In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (,0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Experimental validation of metabolic pathway modelingFEBS JOURNAL, Issue 13 2008An illustration with glycolytic segments from Entamoeba histolytica In the search for new drug targets in the human parasite Entamoeba histolytica, metabolic control analysis was applied to determine, experimentally, flux control distribution of amebal glycolysis. The first (hexokinase, hexose-6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase (PPi -PFK), aldolase and triose-phosphate isomerase) and final (3-phosphoglycerate mutase, enolase and pyruvate phosphate dikinase) glycolytic segments were reconstituted in vitro with recombinant enzymes under near-physiological conditions of pH, temperature and enzyme proportion. Flux control was determined by titrating flux with each enzyme component. In parallel, both glycolytic segments were also modeled by using the rate equations and kinetic parameters previously determined. Because the flux control distribution predicted by modeling and that determined by reconstitution were not similar, kinetic interactions among all the reconstituted components were experimentally revised to unravel the causes of the discrepancy. For the final segment, it was found that 3-phosphoglycerate was a weakly competitive inhibitor of enolase, whereas PPi was a moderate inhibitor of 3-phosphoglycerate mutase and enolase. For the first segment, PPi was both a strong inhibitor of aldolase and a nonessential mixed-type activator of amebal hexokinase; in addition, lower Vmax values for hexose-6-phosphate isomerase, PPi -PFK and aldolase were induced by PPi or ATP inhibition. It should be noted that PPi and other metabolites were absent from the 3-phosphoglycerate mutase and enolase or aldolase and hexokinase kinetics experiments, but present in reconstitution experiments. Only by incorporating these modifications in the rate equations, modeling predicted values of flux control distribution, flux rate and metabolite concentrations similar to those experimentally determined. The experimentally validated segment models allowed ,in silico experimentation' to be carried out, which is not easy to achieve in in vivo or in vitro systems. The results predicted a nonsignificant effect on flux rate and flux control distribution by adding parallel routes (pyruvate kinase for the final segment and ATP-dependent PFK for the first segment), because of the much lower activity of these enzymes in the ameba. Furthermore, modeling predicted full flux-control by 3-phosphoglycerate mutase and hexokinase, in the presence of low physiological substrate and product concentrations. It is concluded that the combination of in vitro pathway reconstitution with modeling and enzyme kinetics experimentation permits a more comprehensive understanding of the pathway behavior and control properties. [source] Protein kinase C , phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin-releasing hormoneFEBS JOURNAL, Issue 13 2007Yoshiko Akita Protein kinase C , (PKC,) is activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH4C1 cells. We analyzed the downstream mechanism after PKC, activation. Exposure of GH4C1 cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino-acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH-stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKC,-overexpressing clones, and purified recombinant PKC, directly phosphorylated K8 with a profile similar to that observed in TRH-stimulated cells. PKC, and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell,cell contact area after TRH stimulation. MS analyses of phospho-K8 and K8-synthesized peptide (amino acids 1,53) showed that PKC, phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH-stimulated cells and PKC,-overexpressing cells, as assessed by immunoblotting using antibodies to phospho-K8. These results suggest that K8 is a physiological substrate for PKC,, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH,PKC, signaling in pituitary cells. [source] Kisspeptin/GPR54 system as potential target for endocrine disruption of reproductive development and functionINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2010M. Tena-Sempere Summary Kisspeptins, the products of Kiss1 gene acting via G protein-coupled receptor 54 (also termed Kiss1R), have recently emerged as essential gatekeepers of puberty onset and fertility. Compelling evidence has now documented that expression and function of hypothalamic Kiss1 system is sensitive not only to the activational effects but also to the organizing actions of sex steroids during critical stages of development. Thus, studies in rodents have demonstrated that early exposures to androgens and oestrogens are crucial for proper sexual differentiation of the patterns of Kiss1 mRNA expression, whereas the actions of oestrogen along puberty are essential for the rise of hypothalamic kisspeptins during this period. This physiological substrate provides the basis for potential endocrine disruption of reproductive maturation and function by xeno-steroids acting on the kisspeptin system. Indeed, inappropriate exposures to synthetic oestrogenic compounds during early critical periods in rodents persistently decreased hypothalamic Kiss1 mRNA levels and kisspeptin fibre density in discrete hypothalamic nuclei, along with altered gonadotropin secretion and/or gonadotropin-releasing hormone neuronal activation. The functional relevance of this phenomenon is stressed by the fact that exogenous kisspeptin was able to rescue defective gonadotropin secretion in oestrogenized animals. Furthermore, early exposures to the environmentally-relevant oestrogen, bisphenol-A, altered the hypothalamic expression of Kiss1/kisspeptin in rats and mice. Likewise, maternal exposure to a complex cocktail of endocrine disruptors has been recently shown to disturb foetal hypothalamic Kiss1 mRNA expression in sheep. As a whole, these data document the sensitivity of Kiss1 system to changes in sex steroid milieu during critical periods of sexual maturation, and strongly suggest that alterations of endogenous kisspeptin tone induced by inappropriate (early) exposures to environmental compounds with sex steroid activity might be mechanistically relevant for disruption of puberty onset and gonadotropin secretion later in life. The potential interaction of xeno-hormones with other environmental modulators (e.g., nutritional state) of the Kiss1 system warrants further investigation. [source] Catalytic mechanism and substrate selectivity of aldo-keto reductases: Insights from structure-function studies of Candida tenuis xylose reductaseIUBMB LIFE, Issue 9 2006Regina Kratzer Abstract Aldo-keto reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)-dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure-function relationship studies for xylose reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the ,-NH3+ group of Lys is a key source of catalytic power of xylose reductase. A molecular-level analysis of the substrate binding pocket of xylose reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel reductases with improved specificities for biocatalytic applications. iubmb Life, 58: 499-507, 2006 [source] Separation and characterization of the 1,3-propanediol and glycerol dehydrogenase activities from Clostridium butyricum E5 wild-type and mutant DJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2001H. Malaoui H. MALAOUI AND R. MARCZAK. 2001. Aims:,Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e. propanediol and glycerol dehydrogenase. Methods and Results:,These two enzymes, which belong to the dha regulon, were separated by gel filtration. Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+. Both strains accumulate glycerol at high levels. Conclusion:,The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type. Significance and Impacts of the study: These properties make Cl. butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol. [source] Increased intracellular [dATP] enhances cardiac contraction in embryonic chick cardiomyocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008Brenda Schoffstall Abstract Although ATP is the physiological substrate for cardiac contraction, cardiac contractility is significantly enhanced in vitro when only 10% of ATP substrate is replaced with 2,-deoxy-ATP (dATP). To determine the functional effects of increased intracellular [dATP] ([dATP]i) within living cardiac cells, we used hypertonic loading with varying exogenous dATP/ATP ratios, but constant total nucleotide concentration, to elevate [dATP]i in contractile monolayers of embryonic chick cardiomyocytes. The increase in [dATP]i was estimated from dilution of dye added in parallel with dATP. Cell viability, average contractile amplitude, rates of contraction/relaxation, spontaneous beat frequency, and Ca2+ transient amplitude and kinetics were examined. At total [dATP]i above ,70 µM, spontaneous contractions ceased, and above ,100 µM [dATP]i, membrane blebbing was also observed, consistent with apoptosis. Interestingly, [dATP]i of ,60 µM (,40% increase over basal [dATP]i levels) enhanced both amplitude of contraction and the rates of contraction and relaxation without affecting beat frequency. With total [dATP]i of ,60 µM or less, we found no significant change in Ca2+ transients. These data indicate that there is an "optimal" concentration of exogenously loaded [dATP]i that under controlled conditions can enhance contractility in living cardiomyocytes without affecting beat frequency or Ca2+ transients. J. Cell. Biochem. 104: 2217,2227, 2008. © 2008 Wiley-Liss, Inc. [source] Arabidopsis mitogen-activated protein kinase MPK12 interacts with the MAPK phosphatase IBR5 and regulates auxin signalingTHE PLANT JOURNAL, Issue 6 2009Jin Suk Lee Summary Mitogen-activated protein kinase (MAPK) phosphatases are important negative regulators in the MAPK signaling pathways responsible for many essential processes in plants, including development, stress management and hormonal responses. A mutation in INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5), which is predicted to encode a dual-specificity MAPK phosphatase, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis roots. To further characterize IBR5, and to understand how it might help integrate MAPK cascades with hormone signaling, we searched for IBR5-interacting MAPKs. Yeast two-hybrid assays, in vitro binding assays and in vivo protein co-immunoprecipitation studies demonstrated that MPK12 and IBR5 are physically coupled. The C-terminus of MPK12 appears to be essential for its interaction with IBR5, and in vitro dephosphorylation and immunocomplex kinase assays indicated that activated MPK12 is efficiently dephosphorylated and inactivated by IBR5. MPK12 and IBR5 mRNAs are both widely expressed across Arabidopsis tissues, and at the subcellular level each protein is predominantly localized in the nucleus. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, but shows normal ABA sensitivity. MPK12 suppression in an ibr5 background partially complements the ibr5 auxin-insensitivity phenotype. Our results demonstrate that IBR5 is a bona fide MAPK phosphatase, and suggest that MPK12 is both a physiological substrate of IBR5 and a novel negative regulator of auxin signaling in Arabidopsis. [source] Crystallization and preliminary X-ray analysis of RecG, a replication-fork reversal helicase from Thermotoga maritima complexed with a three-way DNA junctionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001Martin R. Singleton The monomeric 3,-5, helicase RecG from the thermophilic bacterium Thermotoga maritima has been crystallized in complex with a three-way DNA junction, the preferred physiological substrate. The crystals were obtained by hanging-drop vapour diffusion. The crystals belong to space group C2, with unit-cell parameters a = 133.7, b = 144.6, c = 84.0,Å, , = 113.8°. Native data to a resolution of 3.25,Å were collected from crystals flash-cooled to 100,K. [source] Ion channel activity of transmembrane segment 6 of Escherichia coli proton-dependent manganese transporterBIOPOLYMERS, Issue 8 2010uková Abstract Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure,function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His211 were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure,function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 718,726, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprinFEBS JOURNAL, Issue 18 2008Daniel Ambort In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin,Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin,Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unkown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively. [source] Characterization of membrane-bound prolyl endopeptidase from brainFEBS JOURNAL, Issue 17 2008Jofre Tenorio-Laranga Prolyl oligopeptidase (POP) is a serine protease that cleaves small peptides at the carboxyl side of an internal proline residue. Substance P, arginine,vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. POP has been implicated in a variety of brain processes, including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension, and psychiatric disorders. Although POP has been considered to be a soluble cytoplasmic peptidase, significant levels of activity have been detected in membranes and in extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and urine, suggesting the existence of noncytoplasmic forms. Furthermore, a closely associated membrane prolyl endopeptidase (PE) activity has been previously detected in synaptosomes and shown to be different from the cytoplasmic POP activity. Here we isolated, purified and characterized this membrane-bound PE, herein referred to as mPOP. Although, when attached to membranes, mPOP presents certain features that distinguish it from the classical POP, our results indicate that this protein has the same amino acid sequence as POP except for the possible addition of a hydrophobic membrane anchor. The kinetic properties of detergent-soluble mPOP are fully comparable to those of POP; however, when attached to the membranes in its natural conformation, mPOP is significantly less active and, moreover, it migrates anomalously in SDS/PAGE. Our results are the first to show that membrane-bound and cytoplasmic POP are encoded by variants of the same gene. [source] Role of the MRP1/ABCC1 Multidrug Transporter Protein in CancerIUBMB LIFE, Issue 12 2007Marcia Munoz Abstract Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistance-associated protein 1 (MRP1) transports a wide range of therapeutic agents as well as diverse physiological substrates and may play a role in the development of drug resistance in several cancers including those of the lung, breast and prostate, as well as childhood neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence that MRP1 is a MYCN target gene involved in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease. [source] Separation and characterization of the 1,3-propanediol and glycerol dehydrogenase activities from Clostridium butyricum E5 wild-type and mutant DJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2001H. Malaoui H. MALAOUI AND R. MARCZAK. 2001. Aims:,Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e. propanediol and glycerol dehydrogenase. Methods and Results:,These two enzymes, which belong to the dha regulon, were separated by gel filtration. Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+. Both strains accumulate glycerol at high levels. Conclusion:,The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type. Significance and Impacts of the study: These properties make Cl. butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol. [source] Pharmaceutical and pharmacological importance of peptide transportersJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2008Matthias Brandsch Peptide transport is currently a prominent topic in membrane research. The transport proteins involved are under intense investigation because of their physiological importance in protein absorption and also because peptide transporters are possible vehicles for drug delivery. Moreover, in many tissues peptide carriers transduce peptidic signals across membranes that are relevant in information processing. The focus of this review is on the pharmaceutical relevance of the human peptide transporters PEPT1 and PEPT2. In addition to their physiological substrates, both carriers transport many ,-lactam antibiotics, valaciclovir and other drugs and prodrugs because of their sterical resemblance to di- and tripeptides. The primary structure, tissue distribution and substrate specificity of PEPT1 and PEPT2 have been well characterized. However, there is a dearth of knowledge on the substrate binding sites and the three-dimensional structure of these proteins. Until this pivotal information becomes available by X-ray crystallography, the development of new drug substrates relies on classical transport studies combined with molecular modelling. In more than thirty years of research, data on the interaction of well over 700 di- and tripeptides, amino acid and peptide derivatives, drugs and prodrugs with peptide transporters have been gathered. The aim of this review is to put the reports on peptide transporter-mediated drug uptake into perspective. We also review the current knowledge on pharmacogenomics and clinical relevance of human peptide transporters. Finally, the reader's attention is drawn to other known or proposed human peptide-transporting proteins. [source] Inhibition of proprotein convertases: Approaches to block squamous carcinoma development and progressionMOLECULAR CARCINOGENESIS, Issue 8 2007Ricardo López de Cicco Abstract Most proprotein convertase (PC) inhibitors are compounds that act as competitive inhibitors. All of them contain the general cleavage motif RXK/RR that binds to the PC's active site impairing further interactions with their physiological substrates. The first inhibitors synthesized were the acyl-peptidyl-chloromethyl ketones that bind to the PC's active site through its peptidyl group and are able to transverse the plasma membrane due to the acyl moiety. For instance, one of the members of this family that exhibits reduced toxicity and has been widely used as an effective general PCs inhbitor is the derivative decanoyl-RVKR-chloromethylketone (CMK). Another approach to PC inhibition is based on proteins that contain either a natural or a bioengineered PC cleavage consensus site. In this context, the bioengineered serpin, alpha-1-antitrypsin Portland (alpha 1-PDX or PDX), proved to be a potent inhibitor of furin, the most studied of the cancer-related PCs. Both PDX and CMK were able to inhibit invasiveness of squamous cell carcinoma cell lines by blocking activation of cancer-associated PC substrates such as MT-MMPs, IGF-1R, and VEGF-C. A similar effect was produced by inhibiting PC-mediated processing using furin prosegment. PDX and CMK have also been assayed in vivo using skin carcinogenesis models. Newer promising small molecules and RNA interference approaches are also being developed to inhibit PCs. © 2007 Wiley-Liss, Inc. 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