Proteins, RNAs and chaperones in enzyme evolution: a folding perspective.

The present roles of RNA molecules as templates and of proteins as cellular catalysts may not have always been so clearly defined during evolution. Recent work on ribozymes shows that the catalytic activity of early RNAs may have been more general than previously thought. How did evolution select proteins, not RNA, to be the major biological catalysts? Why were chaperones necessary for the evolution of modern protein enzymes?

Salivary Genomics, Transcriptomics and Proteomics: The Emerging Concept of the Oral Ecosystem and their Use in the Early Diagnosis of Cancer and other Diseases.

There is an increasingly growing interest world-wide for the genomics, transcriptomics and proteomics of saliva and the oral cavity, since they provide a non-invasive source of unprecedently rich genetic information. The complexity of oral systems biology goes much beyond the human genome, transcriptome and proteome revealed by oral mucosal cells, gingival crevicular fluid, and saliva, and includes the complexity of the oral microbiota, the symbiotic assembly of bacterial, fungal and other microbial flora in the oral cavity. In our review we summarize the recent information on oral genomics, transcriptomics and proteomics, of both human and microbial origin. We also give an introduction and practical advice on sample collection, handling and storage for analysis. Finally, we show the usefulness of salivary and oral genomics in early diagnosis of cancer, as well as in uncovering other systemic diseases, infections and oral disorders. We close the review by highlighting a number of possible exploratory pathways in this emerging, hot research field.

Chaperone overload is a possible contributor to 'civilization diseases'.

Molecular chaperones dampen the effect of damaging mutations that would otherwise be removed from the population by natural selection. Here, I propose that the development of modern medical practice depressed this process, leading to a rise of phenotypically silent mutations in the genome. The background of misfolded proteins increases during ageing and, by competition, prevents the chaperone-mediated buffering of silent mutations. Phenotypically exposed mutations contribute to a more-abundant manifestation of multigene-diseases. This 'chaperone overload' hypothesis emphasizes the need for efficient ways to enhance chaperone capacity in ageing subjects, and will hopefully lead to the identification and 'repair' of silent mutations.

Aging cellular networks: chaperones as major participants.

We increasingly rely on the network approach to understand the complexity of cellular functions. Chaperones (heat shock proteins) are key "networkers", which sequester and repair damaged proteins. In order to link the network approach and chaperones with the aging process, we first summarize the properties of aging networks suggesting a "weak link theory of aging". This theory suggests that age-related random damage primarily affects the overwhelming majority of the low affinity, transient interactions (weak links) in cellular networks leading to increased noise, destabilization and diversity. These processes may be further amplified by age-specific network remodelling and by the sequestration of weakly linked cellular proteins to protein aggregates of aging cells. Chaperones are weakly linked hubs (i.e., network elements with a large number of connections) and inter-modular bridge elements of protein-protein interaction, signalling and mitochondrial networks. As aging proceeds, the increased overload of damaged proteins is an especially important element contributing to cellular disintegration and destabilization. Additionally, chaperone overload may contribute to the increase of "noise" in aging cells, which leads to an increased stochastic resonance resulting in a deficient discrimination between signals and noise. Chaperone- and other multi-target therapies, which restore the missing weak links in aging cellular networks, may emerge as important anti-aging interventions.

Chemical chaperones: mechanisms of action and potential use.

An increasing number of studies indicate that low-molecular-weight compounds can help correct conformational diseases by inhibiting the aggregation or enable the mutant proteins to escape the quality control systems, and thus their function can be rescued. The small molecules were named chemical chaperones and it is thought that they nonselectively stabilize the mutant proteins and facilitate their folding. Chemical chaperones are usually osmotically active, such as DMSO, glycerol, or deuterated water, but other compounds, such as 4-phenylbutiric acid, are also members of the chemical chaperone group. More recently, compounds such as receptor ligands or enzyme inhibitors, which selectively recognize the mutant proteins, were also found to rescue conformational mutants and were termed pharmacological chaperones. An increasing amount of evidence suggests that the action of pharmacological chaperones could be generalized to a large number of misfolded proteins, representing new therapeutic possibilities for the treatment of conformational diseases. A new and exciting strategy has recently been developed, leading to the new chemical group called folding agonist. These small molecules are designed to bind proteins and thus restore their native conformation.

Pharmacological modulation of the heat shock response.

Life presents a continuous series of stresses. Increasing the adaptation capacity of the organism is a long-term survival factor of various organisms and has become an attractive field of intensive therapeutic research. Induction of the heat shock response promotes survival after a wide variety of environmental stresses. Preclinical studies have proven that physiological and pharmacological chaperone inducers and co-inducers are an efficient therapeutic approach in different acute and chronic diseases. In this chapter, we summarize current knowledge of the current state of chaperone modulation and give a comprehensive list of the main drug candidates.

Lipocortin I is not accessible for protein kinase C bound to the cytoplasmic surface of the plasma membrane in streptolysin-O-permeabilized pig granulocytes.

We previously observed a 38 kDa protein that was a major protein component of the cytosolic extract of pig granulocytes and the dominant substrate of protein kinase C at supra-physiological Ca2+ concentrations. The purified 38 kDa protein itself required Ca2+ to be phosphorylated by protein kinase C. Now we demonstrate that this protein, which is also present in human granulocytes, is identical to lipocortin I. The identification is based on the chromatographic properties and immunoblot of the purified protein which is also a good substrate for tissue transglutaminase. Phosphorylation of lipocortin I by protein kinase C was investigated in granulocytes permeabilized with streptolysin-O. At physiological intracellular Ca2+ concentrations lipocortin I was not phosphorylated at all. At supra-physiological Ca2+ concentrations (0.5 mM), lipocortin I was also not phosphorylated when protein kinase C was translocated to the membrane by treatment of the cells with phorbol myristate acetate. Its phosphorylation was detectable only in control experiments when protein kinase C was activated in the cytosol by the addition of dioleoylglycerol and phosphatidylserine to the permeabilized cells. The data presented show that, in permeabilized granulocytes, Ca(2+)-lipocortin is not formed at physiological Ca2+ concentrations, and at supra-physiological Ca2+ concentrations the Ca(2+)-lipocortin I is not accessible to protein kinase C bound to the cytoplasmic surface of the plasma membrane.

Alterations in the properties and isoform ratios of brain Na+/K(+)-ATPase in streptozotocin diabetic rats.

In this study we analysed the changes in the properties of rat cerebral cortex Na+K(+)-ATPase in streptozotocin induced diabetes (STZ-diabetes). Special attempt was made to determine whether insulin treatment of diabetic animals could restore the altered parameters of this enzyme. Na+/K(+)-ATPase activity was found to be decreased by 15% after 2 weeks, and by 37% after 4 weeks in diabetic rat brains with a parallel decrease in maximal capacity of low affinity ouabain binding sites. There was no significant change in the high affinity ouabain binding sites. The Kd values did not change significantly. Western blot analysis of brain Na+/K(+)-ATPase isoforms indicated a 61 +/- 5.8% and 20 +/- 2.8% decrease of the alpha 1 and alpha 3 isoforms, respectively in 4 weeks diabetic animals. Change in the amount of the alpha 2 isoform proved to be less characteristic. Both types of beta subunit isoform showed a significant decrease in four weeks diabetic rats. Our data indicate a good correlation in diabetic rats between changes in Na-/K(+)-ATPase activity, low affinity ouabain binding capacity and the level of alpha 1 isoform. While insulin treatment of diabetic animals restored the blood glucose level to normal, a complete reversal of diabetes induced changes in Na+/K(+)-ATPase activity, ouabain binding capacity and Na+/K(+)-ATPase isoform composition could not be achieved.

Effect of chemical modification on the crystallization of Ca2+-ATPase in sarcoplasmic reticulum.

The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca2+-ATPase forms monomer-type (P1) type crystals in the E1 and dimer-type (P2) crystals in the E2 conformation. The P1 type crystals are induced by Ca2+ or lanthanides; P2 type crystals are observed in Ca2+-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca2+-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca2+-ATPase in the E2 conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A2, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5'-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca2+-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form.

The nonapeptide leucinostatin A acts as a weak ionophore and as an immunosuppressant on T lymphocytes.

Earlier studies have disclosed that leucinostatin A, a hydrophobic nonapeptide antibiotic, assumes an alpha-helical secondary structure in nonpolar environments. The present report demonstrates that the peptide acts as a weak ionophore facilitating the transport of mono-and divalent cations through the plasma membrane of T lymphocytes and through artificial membranes. Leucinostatin A does not change the thymidine uptake of both resting mouse thymocytes and peripheral blood lymphocytes but dose-dependently prevents the activation of T lymphocytes by tetradecanoyl-phorbol-acetate and by anti-T cell receptor antibody.

The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review.

The 90-kDa molecular chaperone family (which comprises, among other proteins, the 90-kDa heat-shock protein, hsp90 and the 94-kDa glucose-regulated protein, grp94, major molecular chaperones of the cytosol and of the endoplasmic reticulum, respectively) has become an increasingly active subject of research in the past couple of years. These ubiquitous, well-conserved proteins account for 1-2% of all cellular proteins in most cells. However, their precise function is still far from being elucidated. Their involvement in the aetiology of several autoimmune diseases, in various infections, in recognition of malignant cells, and in antigen-presentation already demonstrates the essential role they likely will play in clinical practice of the next decade. The present review summarizes our current knowledge about the cellular functions, expression, and clinical implications of the 90-kDa molecular chaperone family and some approaches for future research.

Steroid receptor and molecular chaperone encounters in the nucleus.

Steroid hormone receptors interact with several different molecular chaperones. DeFranco and Csermely discuss how the molecular chaperones p23 and Hsp90 may serve to regulate the activity of the ligand-bound steroid receptors within the nucleus. The authors hypothesize that these chaperone proteins may have a proactive role in promoting recycling of receptors once they have interacted with chromatin and in allowing rebinding of ligand once the receptors have been recycled.

The somatostatin analogue TT-232 induces apoptosis in A431 cells: sustained activation of stress-activated kinases and inhibition of signalling to extracellular signal-regulated kinases.

TT-232 is a somatostatin analogue containing a five-residue ring structure. The present report describes TT-232-induced signalling events in A431 cells, where a 4-h preincubation with the peptide irreversibly induced a cell death program, which involves DNA-laddering and the appearance of shrunken nuclei, but is unrelated to somatostatin signalling. Early intracellular signals of TT-232 include a transient two-fold activation of the extracellular signal-regulated kinase (ERK2) and a strong and sustained activation of the stress-activated protein kinases c-Jun NH(2)-terminal kinase (JNK)/SAPK and p38MAPK. Blocking the signalling to ERK or p38MAPK activation had no effect on the TT-232-induced cell killing. At the commitment time for inducing cell death, TT-232 decreased EGFR-tyrosine phosphorylation and prevented epidermial growth factor (EGF)-induced events like cRaf-1 and ERK2 activation. Signalling to ERK activation by FCS, phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF) was similarly blocked. Our data suggest that TT-232 triggers an apoptotic type of cell death, concomitant with a strong activation of JNK and a blockade of cellular ERK2 activation pathways.

Regulation of insulin receptor, insulin receptor substrate-1 and phosphatidylinositol 3-kinase in 3T3-F442A adipocytes. Effects of differentiation, insulin, and dexamethasone.

Insulin rapidly stimulates tyrosine kinase activity of its receptor resulting in phosphorylation of its cytosolic substrate insulin receptor substrate 1 (IRS-1), which in turn associates with and activates the enzyme phosphatidylinositol 3-kinase (PI 3-kinase). In the present study we have examined these three initial steps in insulin action during the differentiation of 3T3-F442A adipocytes and after treatment with dexamethasone or insulin. The differentiation of 3T3-F442A cells was characterized by a 13-fold increase in insulin receptor protein, a 9-fold increase in IRS-1, and a 10- and 4.5-fold increase in their insulin-stimulated phosphorylation, respectively. The mRNA expression of these two proteins showed a similar 8-fold increase during differentiation. In addition there was a 3.5-fold increase in PI 3-kinase protein [85 kilodalton (kDa) subunit] and a 16-fold increase in IRS-1-associated PI 3-kinase activity between day 0 and day 8 of differentiation. Dexamethasone (1 microM) treatment of differentiated cells induced a further 48% (P < 0.05) increase in insulin receptor level, but the autophosphorylation of the receptor was decreased by 31 +/- 1% (P < 0.02). At the same time there was a decrease by 56 +/- 4% (P < 0.005) in IRS-1 protein and by 31 +/- 1% (P < 0.001) in IRS-1 phosphorylation. The expression of insulin receptor mRNA was unchanged, but the expression of IRS-1 mRNA was decreased by approximately 75% after dexamethasone. By contrast, dexamethasone induced a 69% increase in the level of PI 3-kinase as determined by immunoblotting. The combined effect of decreased IRS-1 phosphorylation and increased PI 3-kinase protein was a minimal change (15% decrease) in the association/activation between IRS-1 and PI 3-kinase. Chronic treatment with 100 nM insulin induced a time- and dose-dependent decrease in insulin receptor and IRS-1 protein levels reaching a nadir of 34 +/- 5% (P < 0.005) and 39 +/- 5% (P < 0.01) of control levels after 24 h, respectively. There was an even more marked decrease in the phosphorylation level of these proteins. Chronic insulin treatment also produced a 30% decrease in PI 3-kinase protein levels and a approximately 50% decrease in the association/activation between IRS-1/PI 3-kinase. The expression of insulin receptor and IRS-1 mRNA was unchanged during chronic insulin treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Defensins purified from human granulocytes bind C1q and activate the classical complement pathway like the transmembrane glycoprotein gp41 of HIV-1.

The transmembrane glycoprotein gp41 of HIV-1 contains a C1q binding domain (HIVenv 583-610) and activates the human complement system through the classical pathway. Based on structural and functional similarities between human defensins (human neutrophil peptide, HNP 1-3) and synthetic peptides representing the env 583-610 region of HIV-1, we found it interesting to investigate the C1q binding and complement activating ability of human defensins. Human defensins were purified and characterized by size exclusion chromatography, ultrafiltration, gel electrophoresis and HPLC. The complement activating ability of the purified peptides was assessed in a solid-phase immunoassay. Defensins, fixed to an ELISA plate, were able to bind the C1q subcomponent of the first complement component (C1), triggering the classical pathway of complement activation which led to C4b binding to the plate. Reduction and subsequent alkylation of disulfide bridges of defensins greatly decreased the C1q binding ability but complement activation (C4b binding) remained high. Further acetylation of the reduced defensin peptide resulted in a molecule which bound very little or no C1q but still activated the complement cascade. These phenomena indicate that defensins interact with the complement system via C1q-dependent and C1q-independent mechanisms, and extend the number of functional similarities between defensins and gp41 of HIV-1 to include C1q binding and complement activation.

Insulin-induced phosphorylation of a 38 kDa DNA-binding protein in ventricular cardiomyocytes: possible implication of nuclear protein phosphatase activity.

Ventricular cardiomyocytes isolated from adult rat heart were used to analyze the effect of insulin on the phosphorylation of DNA-binding nuclear proteins and to elucidate the potential involvement of protein phosphatase-1 (PP-1) and PP-2A in this hormonal action. Cells were labelled with [33P]orthophosphate, stimulated with insulin (1.7 x 10(-7) M) and processed for the isolation of nuclei and extraction of DNA-binding proteins. Insulin was found to induce a rapid and constant increase in the serine/threonine phosphorylation of a 38 kDa DNA-binding protein, reaching 150% of control after 15 min and 180% after 150 min. Immunoprecipitation and Western blotting experiments revealed the presence of phosphorylated numatrin in the nuclear extract, however, insulin did not modify its phosphorylation state. Treatment of cardiomyocytes with okadaic acid (1 microM) resulted in a large increase (246 +/- 30%) in the phosphorylation of the 38 kDa protein. Using 32P-labelled phosphorylase as a substrate, we observed a significant inhibition of nuclear PP-1 activity to 38.5 +/- 7% (n = 3) of control after incubation of cardiomyocytes with insulin for 15 min. PP-2A, which corresponds to about 25% of total phosphatase activity, was also inhibited to the same extent. These data show the presence of an insulin-responsive 38 kDa DNA-binding phosphoprotein in the nucleus of cardiomyocytes, which is at least partly regulated by nuclear phosphatase activity. It is suggested that inhibition of nuclear PP-1 and PP-2A represents a possible mechanism of insulin signalling to the nucleus of target cells.

The Hsp90-specific inhibitor geldanamycin selectively disrupts kinase-mediated signaling events of T-lymphocyte activation.

The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone of eukaryotic cells. Its chaperone function in folding nascent proteins seems to be restricted to a subset of proteins including major components of signal transduction pathways (eg, nuclear hormone receptors, transcription factors, and protein kinases). Improper function of these proteins can be induced by selective disruption of their complexes with Hsp90 using the benzoquinonoid ansamycin geldanamycin. In this study, we demonstrate that geldanamycin treatment blocks interleukin (IL)-2 secretion, IL-2 receptor expression, and proliferation of stimulated T-lymphocytes. Moreover, geldanamycin decreases the amount and phosphorylation of Lck and Raf-1 kinases and prevents activation of the extracellular signal regulated kinase (ERK)-2 kinase. Geldanamycin also disrupts the T-cell receptor-mediated activation of nuclear factor of activated T-cells (NF-AT). Treatment with geldanamycin, however, does not affect the activation of lysophosphatide acyltransferase, which is a plasma membrane enzyme coupled to the T-cell receptor after T-cell stimulation. Through demonstrating the selective inhibition of kinase-related T-lymphocyte responses by geldanamycin, our results emphasize the substantial role of Hsp90-kinase complexes in T-cell activation.

The possible pitfalls of the measurement of intracellular calcium concentration of lymphocytes with the fluorescent indicator quin2.

The measurement of intracellular calcium concentration of small cells became feasible and relatively easily accomplishable following the development of the fluorescent calcium chelating dye, quin2, by R. Y. Tsien and his co-workers. The present paper gives an experimental survey of the major possible pitfalls of the method such as: 1) the improper choosing of the concentration of quin2/AM, 2) the misinterpretation of the calibration procedure, 3) the accidental heavy metal traces in the medium, and 4) the uneven distribution of quin2 among the individual cells or within a certain cell. We report some original data on: 1) the distorting effect of heavy metal ions on the measurement and the use of chelex 100 resin to eliminate heavy metal traces from the mediums, 2) the negligible contribution of dead cells to the overall fluorescence signal demonstrated by flow-cytometry, and 3) fluorescence polarization of quin2 in lymphocytes.

Binding affinity of proteins to hsp90 correlates with both hydrophobicity and positive charges. A surface plasmon resonance study.

The 90 kDa heat shock protein (hsp90) is a major cytoplasmic molecular chaperone associating with numerous other proteins including steroid receptors. Here we provide the first numerical analysis of hsp90-target associations using surface plasmon resonance. Binding affinities of histones, the "native molten globule", casein and calmodulin to hsp90 decrease in the order of Kd = 70 +/- 24, 220 +/- 70 and 1800 +/- 600 nM, respectively. Analysis of the structure of binding proteins revealed that their binding affinity depends on both hydrophobicity and positive charges making the discriminative features of hsp90 similar to those of other molecular chaperones.