Non-native interactions in protein folding intermediates: molecular dynamics simulations of hen lysozyme

Molecular dynamics simulations of protein denaturation can complement and extend experimental studies of protein folding by providing atomic-level structural information about conformational transitions and any conformational states along the unfolding pathway. Previous unfolding simulations of hen egg-white lysozyme have resulted in intermediate structures with an unfolded alpha-domain and a structured beta-domain, which is inconsistent with experiment. In contrast, the beta-domain unfolded first in the two simulations presented here leaving a structured alpha-domain. Following this, intermediate states were identified that differ with respect to the packing of the helices and the elements of non-native structure adopted. The non-native structure is critical for explaining many of the experimental observations. Overall, the pooled ensemble of these intermediates is in agreement with the experimental data for the major kinetic intermediate, suggesting that the kinetic intermediate may be made up of distinct, but rapidly interconverting, partially folded conformations distinguished primarily by differences in helix packing.     

Recent advances in molecular recognition and signal transduction of active peptides: receptors for opioid peptides

1. Opioid peptides are a family of structurally related neuromodulators which play a major role in the control of nociceptive pathways. These peptides act through membrane receptors of the nervous system, defined as mu, delta and kappa and endowed with overlapping but distinct pharmacological, anatomical and functional properties. 2. Recent cloning of an opioid receptor gene family has opened the way to the use of recombinant DNA technology at the receptor level. 3. This review focuses on the molecular cloning and functional characterization of opioid receptors and provides first insights into molecular aspects of opioid peptide recognition and signal transduction mechanisms, using the cloned receptors as investigation tools.     

Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells

Signal transduction initiated by transforming growth factor beta 1 (TGF beta 1) was studied in two sublines of the same colon carcinoma cell line, which respond in opposite ways to TGF beta 1, by proliferation or by growth inhibition. TGF beta 1 activates ras proteins within 5 min of addition when it acts to inhibit growth but not when it acts as a mitogen. In both cases TGF beta 1 also rapidly modulates the activities of three protein kinases, detected by their in gel kinase activity on the mitogen-activated protein kinase (MAP kinase) substrate, myelin basic protein (MBP). When TGF beta 1 acts as a mitogen for U9 cells, it increases the activity of MBP kinases of 57, 105, and 130 kDa within 10 min of the addition without detectably activating ras proteins. When TGF beta 1 inhibits the growth of HD3 cells, it activates ras proteins and the 57-kDa MBP kinase within 5 min but inhibits the activity of the 105- and 130-kDa MBP kinases. In HD3 cells ras activation occurred in two signal transduction pathways, one from TGF beta 1 leading to growth inhibition and one from epidermal growth factor (EGF) leading to proliferation. In addition to ras proteins, EGF activates a different set of MBP kinases in HD3 cells than does TGF beta 1, MBP kinases of 85, 57, and 44 kDa. The latter is likely to be the 44-kDa MAP kinase extracellular signal-regulated kinase (erk) 1, because EGF treatment of HD3 cells activates erk1 by increasing its phosphotyrosine level. Therefore, in two closely related epithelial cell lines TGF beta 1 activates two different signal transduction pathways, one ras-dependent and one ras-independent, and modulates the activities of a set of MBP kinases.     

Gene transfer of type 1 interleukin-1 receptor extracellular-domain complementary DNA into rabbit synovial cell line HIG-82 results in cellular blockade of interleukin-1 signal transduction

OBJECTIVE: To produce, by means of expression cloning, a soluble type 1 interleukin-1 receptor (sIL-1R), and to assess its inhibitory properties on the IL-1 pathway. METHODS: High-affinity IL-1R sites were identified in a human chondrosarcoma cell line by means of 125I-IL-1beta binding. A 1-kilobase complementary DNA (cDNA) encoding the ligand-binding domain of the type 1 IL-1R was cloned by using polymerase chain reaction, and the cDNA was inserted into a mammalian expression vector pRc/CMV. The sIL-1R expression vector was transfected into a rabbit synovial cell line (HIG-82) and a stably transfected cell population was selected. The production of sIL-1R was confirmed in the medium of transfected cells using 125I-IL-1beta binding. 35S labeling of transfected cultures, followed by immunoprecipitation and gel electrophoresis, was used to characterize the size of the recombinant sIL-1R. Stromelysin and IL-1alpha steady-state messenger RNA (mRNA) levels were assessed by Northern blotting. Prostaglandin E2 (PGE2) release was measured by enzyme-linked immunosorbent assay. RESULTS: IL-1R on the surface of HIG-82 cells bound 125I-IL-1beta with an equilibrium dissociation constant (Kd) of 67.3 +/- 7.8 pM (mean +/- SD). Transfection of the sIL-1R expression vector into a synovial cell line in vitro resulted in the appearance of an sIL-1R protein that bound 125I-IL-1beta with high affinity in the medium (Kd = 108 +/- 5 pM). Two protein bands (Mr 42 kd and 47 kd) were immunoprecipitated with an antibody against type 1 T cell-derived sIL-1R. Expression of sIL-1R was accompanied by a marked decrease in both stromelysin and IL-1alpha steady-state mRNA levels. In conjunction, there was a significant inhibition of basal and IL-1-stimulated PGE2 released by sIL-1R-producing cells. CONCLUSION: The data suggest that gene transfer of type 1 sIL-1R into the synovium may be an effective means of inhibiting IL-1-induced metalloproteinase expression and inflammatory responses.     

Effect of structural analogs of platelet activating factor on the intracellular signal transduction in murine peritoneal neutrophils and macrophages of P388D1 line

Direct and modulate effects of platelet activating factor (PAF), its structural analogues and ATP on primary and second processes at peritoneal neutrophils and P388D1 cells activation has been studied. The effect of compounds was evaluated on changes in Ca2+ transport and generation of reactive oxygen species. It was shown, that the synthetic analogues of MS series interact with PAF receptor, mobilize Ca2+ from thapsigargin-dependent intracellular stores and inhibit Ca2+ response on PAF in both types of cells. Unlike PAF the analogues do not induce the formations of reactive oxygen species in neutrophils and inhibit the PMA-induced respiratory burst. The activation of pyrinoreceptor of P388D1 cells by exogenous ATP does not inhibit PAF induced Ca2+ rise in cytoplasm, though partly releases Ca2+ from the same store.     

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.     

FGF signal transduction in PC12 cells: comparison of the responses induced by endogenous and chimeric receptors

Loss of articular cartilage, which is the most important pathological lesion occurring in osteoarthritis, has been shown to be enzymatically mediated. The matrix metalloproteinases (MMPs) are a group of enzymes which have been implicated in this degradation of articular cartilage matrix. The use of pharmacological agents to inhibit this catabolic process in the joint is a potential route for therapeutic intervention. The gelatinase MMPs, MMPs-2 and 9, were purified by affinity chromatography from equine cell cultures. The ability of phenylbutazone, flunixin, betamethasone, dexamethasone, methylprednisolone acetate (MPA), hyaluronan, pentosan polysulphate and polysulphated glycosaminoglycan (PSGAG) to inhibit equine MMPs-2 and 9 were assessed by two degradation assays. Whilst some agents did have direct effects on MMP activity, these effects were only obtained at concentrations which were unlikely to be achieved for any length of time in vivo. It is improbable that any pharmacological agent, currently used in the horse, has a significant effect on gelatinase MMP activity.     

Characterization of the signal transduction pathway activated in human monocytes and dendritic cells by MPIF-1, a specific ligand for CC chemokine receptor 1

The receptor specificity and signal transduction pathway has been identified and characterized for a truncated form of myeloid progenitor inhibitory factor-1 (MPIF-1(24-99)). MPIF-1 binds specifically to sites, in particular CCR1, shared with macrophage inflammatory protein-1alpha (MIP-1alpha) on the surface of human monocytes and dendritic cells, as inferred by its ability to compete for [125I]MIP-1alpha, but not for [125I]MIP-1beta or [125I]monocyte chemotactic protein-1(MCP-1) binding to intact cells. Based on calcium flux, MPIF-1 is an agonist on CCR1-transfected HEK-293 cells, monocytes, and dendritic cells, but not on CCR5-, CCR8-, or CX3CR1-transfected cells. The inhibitory effect of guanosine 5'-O-(3-thio-triphosphate) (GTP-gammaS) or pertussis toxin pretreatment on MPIF-1 binding and calcium mobilization, respectively, indicates the involvement of G proteins in the interaction of MPIF-1 and its receptor(s). The increase in intracellular free calcium concentration following MPIF-1 treatment is mainly due to the influx of calcium from an extracellular pool. However, a portion of the intracellular free calcium concentration is derived from a phospholipase C inhibitor-sensitive intracellular pool. MPIF-1 induces a rapid dose-dependent release of [3H]arachidonic acid from monocytes that is dependent on extracellular calcium and is blocked by phospholipase A2 (PLA2) inhibitors. Furthermore, PLA2 activation is shown to be necessary for filamentous actin formation in monocytes. Thus, the MPIF-1 signal transduction pathway appears to include binding to CCR1; transduction by G proteins; effector function by phospholipase C, protein kinase C, calcium flux, and PLA2; and cytoskeletal remodeling.     

CC-chemokines enhance the replication of T-tropic strains of HIV-1 in CD4(+) T cells: role of signal transduction

This study demonstrates that several CC-chemokines, including those that inhibit entry and replication of macrophage-tropic strains of HIV, increase the replication of T cell (T)-tropic strains in CD4(+) T cells. Enhancement of T-tropic HIV replication is observed at early stages of replication, requires signaling through inhibitory guanine nucleotide-binding regulatory (Gi) proteins, and is associated with increased cell surface colocalization of CD4 and the T-tropic HIV coreceptor CXCR4. These findings may further our understanding of the factors that influence the replication and spread of T-tropic strains of HIV in vivo and suggest that the use of cell signaling CC-chemokines as therapeutic agents for the purpose of limiting HIV replication in vivo should be approached with caution.     

No evidence for mediation of ischemic preconditioning by alpha 1-adrenergic signal transduction pathway or protein kinase C in the isolated rat heart

A model of vertical signal flow across a layered cortical structure is presented and analyzed. Neurons communicate through spikes, which evoke an excitatory or inhibitory postsynaptic potential (spike response model). The layers incorporate two anatomical features-dendritic and axonal arborization patterns and distance-dependent time delays. The vertical signal flow through the network is discussed for various stimulus conditions using two different, but typical, axonal arborization patterns. We find stationary as well as oscillatory response, but the oscillatory response may be restricted to a single layer. Confronted with conflicting stimuli the network separates the patterns through phase-shifted oscillations. We also discuss two hypothetical animals, to be called "cat" and "mouse." These have different axonal arborizations, which give rise to a different oscillatory response (if any) of the various layers.     

Thy-1 and AvGp50 signal transduction complex in the avian nervous system: c-Fyn and G alpha i protein association and activation of signalling pathways

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called 'catch'. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of approximately 600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the approximately 600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of approximately 600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the approximately 600 kDa protein regulates catch.     

Mutagenesis of the human 5-HT1B receptor: differences from the closely related 5-HT1A receptor and the role of residue F331 in signal transduction

We have used a combination of sequence comparisons, computer-based modeling and site-directed mutagenesis to investigate the molecular interactions involved in ligand binding and signal transduction of the human 5-HT1B receptor. Two amino acid residues, S212 in transmembrane region (TM) V and F331 in TM VI, were replaced by alanines. These amino acids are conserved in many G protein-coupled receptors and therefore likely to be important for receptor function. The mutant receptors were expressed in Chinese hamster ovary cells. The 5-HT-like agonist 5-carboxamido-tryptamine (5-CT) bound with 15-fold lower affinity to the S212A mutant as compared to wild-type receptor and the antagonist methiothepin bound with 17-fold lower affinity to the F331A mutant. No reduction in the affinity of 5-HT was seen for the S212A mutant, although an equivalent mutation in the 5-HT1A receptor resulted in a 100-fold reduction of 5-HT binding. The inhibition of forskolin-stimulated cyclic AMP production by 5-HT was significantly reduced in cells expressing the F331A mutant, even though the endogenous ligand 5-HT bound with somewhat increased affinity. Methiothepin acted as an inverse agonist and increased the forskolin-stimulated cyclic AMP production at both the wild-type receptor and the mutants, and the effect was stronger on the F331A mutant. These results suggest that F331 is involved in the conformational changes necessary for signal transduction.     

Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase

Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.     

Chemomechanical transduction in the actomyosin molecular motor by 2',3'-dideoxydidehydro-ATP and characterization of its interaction with myosin subfragment 1 in the presence and absence of actin

The effect of torsional freedom about the N-glycoside bond of ATP in the ability of the nucleoside triphosphate to support chemomechanical transduction (Takenaka et al., 1978) has been investigated by examining the ability of the nucleotide analogue 2',3'-dideoxy-2',3'-didehydro-ATP (1b, enf-ATP) to act as a substrate for myosin subfragment 1 in the presence and absence of actin and to support actin sliding in the standard in vitro motility assay. By converting the ribosyl ring of the natural substrate to the rigid and almost planar enofuranosyl ring, effects on torsional freedom about the N-glycoside bond due to changes in ribosyl ring pucker and/or by steric interferences of the protons attached to the 2' and 3' carbons are eliminated allowing for increased torsional freedom about the N-glycoside bond. The data indicate that this enofuranosyl analogue is an excellent substrate for subfragment 1 and actosubfragment 1 and produces actin sliding velocities which are twice as fast as those observed with ATP in the standard in vitro motility assay. The analogue diphosphate is trapped in S1 by the common P(i) analogues, but the rate of formation of the ternary complex formed with Vi is very slow compared to that observed with MgADP. Similar conformations of S1 are formed with Mg.enf-ATP and MgATP under steady-state conditions, but S1 with bound Mg.enf-ADP differs significantly from that observed with MgADP.     

Molecular dynamics simulations of HLA-DR4 (DRB1*0405) complexed with analogue peptide: conformational changes in the putative T-cell receptor binding regions

The specific recognition of foreign peptide bound to the major histocompatibility complex (MHC) molecule by T-cell receptor (TCR) leads to T-cell activation. We found that analogue peptides containing single amino acid substitutions at the third amino acid position (p3), p5, p7 and p8 of the index peptide (YWALEAAAD) induced different response patterns of T cell clones specific for the index peptide in the context of the human MHC class II molecule HLA-DR4. Analogue peptides were classified into three types, agonists, antagonists or null peptides (non-agonistic and non-antagonistic peptides). A molecular basis for how these slight changes lead to such different consequences for T cells has not been described. To explore the mechanistic basis of these observations, molecular dynamics simulations at 300 K of 300 ps duration were carried out for the DR4-index peptide, DR4-agonist, and DR4-antagonist complexes. The simulations showed that the DR4-antagonist complexes were distinguished from the DR4-index peptide and DR4-agonist complexes by relatively higher deviations of C(alpha) atoms in proposed TCR-binding regions, suggesting that subtle changes of the exposed framework of the peptide binding groove by the antagonist peptides could induce the TCR antagonistic activities.     

Insulin-like growth factor I (IGF-I)-stimulated pancreatic beta-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells

Nutrients and certain growth factors stimulate pancreatic beta-cell mitogenesis, however, the appropriate mitogenic signal transduction pathways have not been defined. In the glucose-sensitive pancreatic beta-cell line, INS-1, it was found that glucose (6-18 mM) independently increased INS-1 cell proliferation (>20-fold at 15 mM glucose). Insulin-like growth factor I (IGF-I)-induced INS-1 cell proliferation was glucose-dependent only in the physiologically relevant concentration range (6-18 mM glucose). The combination of IGF-I and glucose was synergistic, increasing INS-1 cell proliferation >50-fold at 15 mM glucose + 10 nM IGF-I. Glucose metabolism and phosphatidylinositol 3'-kinase (PI 3'-kinase) activation were necessary for both glucose and IGF-I-stimulated INS-1 cell proliferation. IGF-I and 15 mM glucose increased tyrosine phosphorylation mediated recruitment of Grb2/mSOS and PI 3'-kinase to IRS-2 and pp60. Glucose and IGF-I also induced Shc association with Grb2/mSOS. Glucose (3-18 mM) and IGF-I, independently of glucose, activated mitogen-activated protein kinase but this did not correlate with IGF-I-induced beta-cell proliferation. In contrast, p70(S6K) was activated with increasing glucose concentration (between 6 and 18 mM), and potentiated by IGF-I in the same glucose concentration range which correlated with INS-1 cell proliferation rate. Thus, glucose and IGF-I-induced beta-cell proliferation were mediated via a signaling mechanism that was facilitated by mitogen-activated protein kinase but dependent on IRS-mediated induction of PI 3'-kinase activity and downstream activation of p70(S6K). The glucose dependence of IGF-I mediated INS-1 cell proliferation emphasizes beta-cell signaling mechanisms are rather unique in being tightly linked to glycolytic metabolic flux.