Molecular switch in signal transduction: reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the gamma-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD-Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of alpha2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of alpha2 and facilitates the unwinding of a helical turn of alpha2 (residues 66-69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.     

A role for p21ras in the angiotensin II AT2 receptor transduction pathway

We have previously shown that the activation of the AT2 receptor of Ang II induced neurite outgrowth in NG108-15 cells. We also found that stimulation of NG108-15 cells with Ang II induced a rapid decrease in GTP-bound p21ras. In order to investigate the possible role of p21ras in Ang II-induced neuronal differentiation, we have established NG108-15 sublines which inducibly express a dominant inhibitory form of p21ras (p21N17Ras). We observed that IPTG-induced expression of p21N17Ras in these NG108-15 sublines induced the same morphological changes as does Ang II in control untransfected cells. Immunofluorescence labeling of beta-tubulin showed that expression of p21N17Ras induced neurite outgrowth and elongation. These observations were supported by Western blot analysis of the level of polymerized tubulin. These results strongly support the hypothesis that AT2 receptor-induced neuronal differentiation in NG108-15 cells is mediated by the inhibition of p21ras.     

Structural basis for the interaction of Ras with RalGDS

The Malthus-AT system provided a satisfactory method for examining the effects of permeabilizing agents on the activity of sub-inhibitory concentrations of antibacterial agents against Pseudomonas aeruginosa G48. Under this system, disodium edetate potentiated the activity of chlorhexidine diacetate (CHA), cetylpyridinium chloride, para-chlorometaxylenol and triclosan. Nitrilotriacetic acid enhanced the activity of some of the antibacterials tested, whereas sodium polyphosphate markedly reduced the efficacy of CHA.     

A role for p21ras/MAP kinase in TCR-mediated activation of LFA-1

LFA-1 is a beta2 integrin that plays well-characterized roles in adhesion of T lymphocytes to APC, T cell-mediated cytolysis, and leukocyte-endothelial cell interactions. Although it is clear that LFA-1 must undergo affinity or avidity changes to bind its cellular ligand ICAM-1, the intracellular signaling pathways involved are not well characterized. Here, we show that the Ras-mitogen-activated protein kinase (MAPK) signaling pathway is also involved in TCR-activated LFA-1 adhesion. Expression of a dominant negative form of p21ras in a thymocyte cell line inhibits, while constitutively active p21ras both enhances and sustains, subsequent TCR-triggered adhesion to isolated ICAM-1. However, the Ras/MAPK pathway alone is not sufficient for activating T cell LFA-1, as inhibition of both downstream MAPK/extracellular regulated kinase kinase (MEK) activity and phosphatidylinositol 3-kinase activity is required for complete inhibition of adhesion.     

v-K-ras leads to preferential farnesylation of p21(ras) in FRTL-5 cells: multiple interference with the isoprenoid pathway

The isoprenoid pathway in FRTL-5 thyroid cells was found to be deeply altered on transformation with v-K-ras. A dramatic overall reduction of protein prenylation was found in v-K-ras-transformed cells in comparison with the parent FRTL-5 cells, as shown by labeling cells with [3H]mevalonic acid. This phenomenon was accompanied by a relative increase of p21(ras) farnesylation and by a decrease of the ratio between the amounts of geranylgeraniol and farnesol bound to prenylated proteins. Analysis of protein prenylation in FRTL-5 cells transformed by a temperature-sensitive mutant of the v-K-ras oncogene indicated that these variations represent an early and specific marker of active K-ras. Conversely, FRTL-5 cells transformed with Harvey-ras showed a pattern of [3H]-mevalonate (MVA)-labeled proteins similar to that of nontransformed cells. The K-ras oncogene activation also resulted in an overall decrease of [3H]-MVA incorporation into isopentenyl-tRNA together with an increase of unprocessed [3H]-MVA and no alteration in [3H]-MVA uptake. The effects of v-K-ras on protein prenylation could be mimicked in FRTL-5 cells by lowering the concentration of exogenous [3H]-MVA whereas increasing the [3H]-MVA concentration did not revert the alterations observed in transformed cells. Accordingly, v-K-ras expression was found to: (i) down-regulate mevalonate kinase; (ii) induce farnesyl-pyrophosphate synthase expression; and (iii) augment protein farnesyltransferase but not protein geranylgeranyl-transferase-I activity. Among these events, mevalonate kinase down-regulation appeared to be related strictly to differential protein prenylation. This study represents an example of how expression of the v-K-ras oncogene, through multiple interferences with the isoprenoid metabolic pathway, may result in the preferential farnesylation of the ras oncogene product p21(ras).     

Prevention of autoimmune diabetes by linomide in nonobese diabetic (NOD) mice is associated with up-regulation of the TCR-mediated activation of p21(ras)

Oral therapy with linomide protects prediabetic nonobese diabetic (NOD) mice from insulin-dependent diabetes mellitus. The mechanisms by which linomide exerts its protective effect are not fully understood. A decreased TCR-mediated activity of the GTP-GDP binding p21(ras) proto-oncogene is associated with prediabetes in NOD mice. However, the role of this signal transduction defect in the pathogenesis of autoimmune diabetes is not known. The TCR-mediated and protein kinase C-induced activations of p21(ras) were determined in mononuclear cells from lymph nodes of linomide-treated and untreated prediabetic NOD mice. TCR cross-linking by Con A induced an increase of 13 +/- 6.8% and a decrease of 0.8 +/- 1.8% in p21(ras) activity in the linomide-treated group and the untreated controls, respectively. Cell stimulation with PMA resulted in a 15 +/- 2% increase in p21(ras) activity in the linomide-treated mice and a 10 +/- 11.4% decrease in the untreated mice. Protein levels of p21(ras) and its regulatory elements, the GTPase-activating protein and the guanine nucleotide-releasing factor, mSOS, were comparable in both groups. We, therefore, conclude that prevention of autoimmune diabetes by linomide is associated with up-regulation of the p21(ras) T cell signal transduction defect in NOD mice.     

Structural basis for the pathophysiology of lipoprotein(a) in the athero-thrombotic process

Lipoprotein Lp(a) is a major and independent genetic risk factor for atherosclerosis and cardiovascular disease. The essential difference between Lp(a) and low density lipoproteins (LDL) is apolipoprotein apo(a), a glycoprotein structurally similar to plasminogen, the precursor of plasmin, the fibrinolytic enzyme. This structural homology endows Lp(a) with the capacity to bind to fibrin and to membrane proteins of endothelial cells and monocytes, and thereby to inhibit plasminogen binding and plasmin generation. The inhibition of plasmin generation and the accumulation of Lp(a) on the surface of fibrin and cell membranes favor fibrin and cholesterol deposition at sites of vascular injury. Moreover, insufficient activation of TGF-beta due to low plasmin activity may result in migration and proliferation of smooth muscle cells into the vascular intima. These mechanisms may constitute the basis of the athero-thrombogenic mode of action of Lp(a). It is currently accepted that this effect of Lp(a) is linked to its concentration in plasma. An inverse relationship between Lp(a) concentration and apo(a) isoform size, which is under genetic control, has been documented. Recently, it has been shown that inhibition of plasminogen binding to fibrin by apo(a) is also inversely associated with isoform size. Specific point mutations may also affect the lysine-binding function of apo(a). These results support the existence of functional heterogeneity in apolipoprotein(a) isoforms and suggest that the predictive value of Lp(a) as a risk factor for vascular occlusive disease would depend on the relative concentration of the isoform with the highest affinity for fibrin.     

Rac binding to p67(phox). Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase

Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b558 with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Herein, the interaction between Rac and p67(phox) is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67(phox) was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67(phox) but not p47(phox), and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67(phox) with a 1:1 stoichiometry and with Kd values of 120 and 60 nM, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431-13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801) previously identified two regions in Rac1 that are important for activity: the "effector region" (residues 26-45) and the "insert region" (residues 124-135). Proteins mutated in the effector region (Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in both the Kd and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67(phox). Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67(phox). The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67(phox), the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b558.     

Structural basis for molecular recognition between nuclear transport factor 2 (NTF2) and the GDP-bound form of the Ras-family GTPase Ran

Nuclear transport factor 2 (NTF2) and the Ras-family GTPase Ran are two soluble components of the nuclear protein import machinery. NTF2 binds GDP-Ran selectively and this interaction is important for efficient nuclear protein import in vivo. We have used X-ray crystallography to determine the structure of the macromolecular complex formed between GDP-Ran and nuclear transport factor 2 (NTF2) at 2.5 A resolution. The interaction interface involves primarily the putative switch II loop of Ran (residues 65 to 78) and the hydrophobic cavity and surrounding surface of NTF2. The major contribution to the interaction made by the switch II loop accounts for the ability of NTF2 to discriminate between GDP and GTP-bound forms of Ran. The aromatic side-chain of Ran Phe72 inserts into the NTF2 cavity and accounts for 22% of the surface area buried by the interaction interface, while salt bridges are formed between Lys71 and Arg76 of Ran with Asp92/Asp94 and Glu42 of NTF2, respectively. These salt bridges account for the inhibition of the Ran-NTF2 interaction by NTF2 mutants such as E42 K and D92/94N in which the negatively charged residues surrounding the cavity were altered. Because the interaction interface maintains the positions of key Ran residues involved in binding MgGDP, NTF2 binding may help stabilize the switch state of Ran, possibly in the context of targeting it to other components of the nuclear protein import machinery to specify directionality of transport. The binding of GDP-Ran at the NTF2 cavity raises the possibility that this interaction might be modulated by a metabolite or small molecule substrate for NTF2's putative enzymatic activity.     

High frequency (139.5 GHz) electron paramagnetic resonance spectroscopy of the GTP form of p21 ras with selective 17O labeling of threonine

Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(beta, gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [17O gamma]threonine to a final enrichment of 30%. A Mn(II)-17O hyperfine interaction was observed, but the value of the coupling constant, 0.11 +/- 0.04 mT, is the smallest such value yet reported. Ab initio calculations indicate that this value is consistent with direct coordination of the threonine hydroxyl group and provide an estimate for the Mn(II)-17O bond length of 2.7 A. The measured hyperfine coupling constant and associated bond length starkly contrast with typical values for Mn(II)-17O coordination complexes, namely, approximately 0.25 mT and approximately 2.2 A, respectively. This contrast underscores the peculiar weakness of this Mn(II)-O interaction in p21 and persuasively argues that the nucleotide-induced conformational change, which is known to encompass the region of p21 involving Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.     

Molecular basis for interprotein complex-dependent effects on the redox properties of amicyanin

The quinoprotein methylamine dehydrogenase (MADH), type I copper protein amicyanin, and cytochrome c-551i form a complex within which interprotein electron transfer occurs. It was known that complex formation significantly lowered the oxidation-reduction midpoint potential (Em) value of amicyanin, which facilitated an otherwise thermodynamically unfavorable electron transfer to cytochrome c-551i. Structural, mutagenesis, and potentiometric studies have elucidated the basis for this complex-dependent change in redox properties. Positively charged amino acid residues on the surface of amicyanin are known to stabilize complex formation with MADH and influence the ionic strength dependence of complex formation via electrostatic interactions. Altering the charges of these residues by site-directed mutagenesis had no effect on the Em value of amicyanin, ruling out charge neutralization as the basis for the complex-dependent changes in redox properties. The Em value of free amicyanin varies with pH and exhibits a pKa value for the reduced form of 7.5. The crystal structure of reduced amicyanin at pH 4.4 reveals that His95, which serves as a ligand for Cu2+, has rotated by 180 degrees about the Cbeta-Cgamma bond relative to its position in oxidized amicyanin and is no longer in the copper coordination sphere. At pH 7.7, the crystal structure of reduced amicyanin contains an approximately equal distribution of two active-site conformers. One is very similar to the structure of reduced amicyanin at pH 4.4, and the other is very similar to the structure of oxidized amicyanin at pH 4.8. Potentiometric analysis of amicyanin in complex with MADH indicates that its Em value is not pH-dependent from pH 6.5 to 8.5, and exhibits an Em value similar to that of free amicyanin at high pH. The structure of reduced amicyanin at pH 4.4, with His95 protonated and "flipped", was modeled into the structure of the complex of oxidized amicyanin with MADH. This showed that in the complex, the redox-linked pH-dependent rotation of His95 is hindered because it would cause an overlap of van der Waals' radii with residues of MADH. These results demonstrate that protein-protein interactions profoundly affect the redox properties of this type I copper protein by restricting a pH-dependent, redox-linked conformational change of one of the copper ligands.     

Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin

OBJECTIVES: Prostate cancer recurrence, evidenced by rising prostate-specific antigen (PSA) levels after radical prostatectomy, is an increasingly prevalent clinical problem in need of new treatment options. Preclinical studies have suggested that for tumors in general, settings of minimal cancer volume may be uniquely suitable for recombinant vaccine therapy targeting tumor-associated antigens. A clinical study was undertaken to evaluate the safety and biologic effects of vaccinia-PSA (PROSTVAC) administered to subjects with postprostatectomy recurrence of prostate cancer and to assess the feasibility of interrupted androgen deprivation as a tool for modulating expression of the vaccine target antigen, as well as detecting vaccine bioactivity in vivo. METHODS: A limited Phase I clinical trial was conducted to evaluate the safety and biologic effects of vaccinia-PSA administered in 6 patients with androgen-modulated recurrence of prostate cancer after radical prostatectomy. End points included toxicity, serum PSA rise related to serum testosterone restoration, and immunologic effects measured by Western blot analysis for anti-PSA antibody induction. RESULTS: Toxicity was minimal, and dose-limiting toxicity was not observed. Noteworthy variability in time required for testosterone restoration (after interruption of androgen deprivation therapy) was observed. One subject showed continued undetectable serum PSA (less than 0.2 ng/mL) for over 8 months after testosterone restoration, an interval longer than those reported in previous androgen deprivation interruption studies. Primary anti-PSA IgG antibody activity was induced after vaccinia-PSA immunization in 1 subject, although such antibodies were detectable in several subjects at baseline. CONCLUSIONS: Interrupted androgen deprivation may be a useful tool for modulating prostate cancer bioactivity in clinical trials developing novel biologic therapies. Immune responses against PSA may be present among some patients with prostate cancer at baseline and may be induced in others through vaccinia-PSA immunization.     

Structural determinants required for the interaction between Rho GTPase and the GTPase-activating domain of p190

The Rho family small GTP-binding proteins are subjected to regulation by Rho GTPase-activating proteins (GAPs) in the course of transmitting diverse intracellular signals. To understand the mechanism of GAP-catalyzed GTP hydrolysis of Rho GTPases, we have studied the interaction between RhoA and p190, the RasGAP binding phosphoprotein which has been implicated as a Rho-specific GAP, by delineating the structural determinants of RhoA and p190 GAP domain (p190GD) that are involved in their functional coupling. Besides the conserved residues Tyr34, Thr37, and Phe39 in the switch I region of RhoA which are required for p190GD interaction, chimeras made between RhoA and Cdc42, a close relative of RhoA with which p190GD interacts 50-fold less efficiently, revealed that residues outside the switch I and neighboring regions of RhoA, residues 85-122 in particular, contain the major p190GD-specifying determinant(s). Mutation of the unique Asp90 of RhoA in this region mostly abolished p190GD stimulation, whereas the corresponding reverse mutation of Cdc42 (S88D) was able to respond to p190GD-catalysis similarly as RhoA. Further kinetic analysis of these mutants provided evidence that Asp90 of RhoA contributes primarily to the specific binding interaction with p190GD. On the other hand, two charged residues of p190GD, Arg1283 and Lys1321, which are located in the putative G-protein binding helix pocket of GAP domain, were found to be involved in different aspects of interaction with RhoA. The R1283L mutant of p190GD lost GAP activity but retained the ability to bind to RhoA, while K1321A failed to stimulate and to bind to RhoA. These results indicate that residue Asp90 constitutes the second GAP-interactive site in RhoA which is mostly responsible for conferring p190GD-specificity, and suggest that the role of p190GD in the GTPase reaction of RhoA is in part to supply active site residue Arg1283 for efficient catalysis.     

A possible molecular basis for the effect of gastric anti-ulcerogenic drugs

The exact mechanism of action of the anti-ulcerogenic drugs is still under debate. According to the literature, under normal conditions the cAMP:cGMP ratio in the rat gastric mucosa is approximately 8:10. Following prostacyclin administration, this ratio transiently decreases but later shows a strong elevation, indicating profound changes in the intracellular cyclic nucleotide balance. There is evidence that this elevation or 'shift' in the cAMP:cGMP ratio is linked, on a cellular or molecular level, to the anti-ulcerogenic, cytoprotective processes in the stomach. Cimetidine and ranitidine (widely used H2 receptor-blocking drugs) administered at doses that are too low to interfere with gastric acid secretion, cause an elevation in the cAMP:cGMP ratio, an effect that is also observed with other prostaglandin derivatives and anti-ulcerogenic drugs. In this article, Gabor A. Balint discusses these data and how the elevation of the gastric mucosal cAMP:cGMP ratio is a useful molecular marker that could provide insights into the effects of anti-ulcerogenic drugs.     

Molecular basis for the interaction between human IgM and staphylococcal protein A

To examine the relationship between VH gene usage and reactivity of immunoglobulins, we cloned B cells from peripheral blood from adults and from human neonatal cord blood by EBV transformation. Nearly one-third of the B cell clones from both sources produced IgM reactive with staphylococcal protein A (SPA). None of such IgM reacted with other antigens, except for the crude extract of Staphylococcus aureus. All of 22 B cell clones producing IgM reactive with SPA expressed VH3 genes, while none of the control 15 clones secreting IgM nonreactive with SPA expressed VH3. The IgM proteins reactive with SPA could be clearly divided into two subjects based on the differential binding avidity to solid-phase SPA. Both kappa and lambda light chains were used in each subset of SPA-reactive IgM. Sequence analysis of the PCR products from seven VH3-IgM clones revealed that the VH3 genes were used in nearly germline configuration. The D and J gene usage was diverse. Comparison of amino acid sequences between antibodies with high and low avidity to SPA suggests that the differential avidity is related to amino acid sequence differences in the complementarity determining region 2 and framework region 3. The high frequencies of B cells committed to the production of SPA-reactive IgM in normal blood and the restricted use of VH3 heavy chain genes in nearly germline configuration in these cells support the notion that SPA behaves like a superantigen toward human B cells.     

Imagery-perception interaction depends on the shape of the image: A reply to Farah (1989).

M. J. Farah (see record 1989-31784-001) reported that point threshold stimuli are detected better if they appear at spatial locations in the visual field that are covered by an image. By replicating her experiment with 3 instead of the original 2 images, it was found that the effect depends on the shape of the mentally projected image. A 2nd experiment with 9 different shapes revealed that the effect is modulated by the compactness and the size of the image; it is enhanced with increasing compactness and attenuated with increasing size. These findings do not unequivocally support the idea that imagery and perception interact because both processes share the same representational medium. Rather, they suggest that imagery can cause a figure–ground segregation in the visual field and that the shape of the figure may determine the amount of attention that is allocated to different sections in the visual field. (PsycINFO Database Record (c) 2010 APA, all rights reserved)