Biomedical subjects

M R Pincus

Publications and source records attributed to M R Pincus.

At least 73 records · Page 4Linked to original sources

PubMed1992

Evidence that the ras oncogene-encoded p21 protein induces oocyte maturation via activation of protein kinase C.

The ras oncogene-encoded p21 protein is known to induce cell maturation of Xenopus laevis oocytes and malignant transformation of NIH 3T3 mouse fibroblasts. The pathways involved in oocytes and NIH 3T3 cells appear to be similar to one another. For example, in both cases, the ras p21-induced cellular events involve increased intracellular levels of the second messengers diacylglycerol and inositol phosphates, the former of which activates protein kinase C (PKC). To investigate the pathway of ras-induced oocyte maturation, we have explored the relationship between p21 protein and PKC. We show that the maturation signal from oncogenic p21 microinjected into Xenopus oocytes is completely blocked by the relatively specific PKC inhibitor CGP 41251, a staurosporine analogue that selectively inhibits PKC, but not by an inactive analogue of staurosporine, CGP 42700. Microinjection of purified PKC or of phorbol ester induces maturation of oocytes. PKC-induced maturation is inhibited by CGP 41251 but not by CGP 42700. Maturation induced by microinjected PKC is also not inhibited by two specific anti-p21 agents, the inactivating anti-p21 monoclonal antibody Y13-259 and the amino acid derivative azatyrosine. Both of these agents block p21-induced cell maturation. These results suggest that ras effects depend upon the action of PKC, whose activation is an event that occurs downstream of p21 in the maturation signal pathway.

Alanine↗

PubMed1992

Identification of structured peptide segments in folding proteins.

Prediction of the structures of long polypeptides and small proteins has been carried out using conformational energy calculations. These calculations can be applied to large proteins if structured regions of their sequences can be identified. Three different approaches to identifying such sequences are presented. First, sequences of five or more contiguous hydrophobic residues tend to nucleate alpha-helices. Second, peptide sequences from parent proteins that have the same biological activities as the parent proteins are highly structured. Third, structured synthetic peptide segments from proteins inhibit the folding of the parent proteins by competing with the corresponding segment of the protein chain for associating with complementary regions. Examples of each of these approaches are presented.

Amino Acid Sequence↗

PubMed1992

Enkephalin is a competitive antagonist of cholecystokinin in the gastrointestinal tract, as predicted from prior conformational analysis.

Prior calculations based on ECEPP (Empirical Conformational Energies for Peptides Program) of the low energy minima for cholecystokinin (CCK) and Met-enkephalin have demonstrated that significant structural features of these two peptides are identical. This result suggested the possibility that Met-enkephalin, as well as other enkephalin analogues of similar structure, could associate with receptors for CCK. To test this theoretical result, we examined the ability of Met-enkephalin and its analogues to bind to peripheral CCK receptors in the rat gastrointestinal tract; in particular, we measured the ability of the opiate peptide to inhibit the effects of CCK in a physiological assay system which we have previously characterized: CCK-induced contraction of the isolated rat pyloric sphincter. We find that Met-enkephalin is an antagonist of the CCK-8-induced contraction, with a IC50 of 110 nM. Furthermore, antibodies against CCK were found to cross-react with Met-enkephalin and its analogues in a manner which suggests a distinct structure-activity relationship. These experimental results strongly support the theoretical results of conformational analysis showing structural similarity between enkephalin and CCK. They further suggest that enkephalins could modulate the response of CCK systems under physiological conditions.

Amino Acid Sequence↗

PubMed1992

Correlation of the conformation of a modified ribonuclease octapeptide, homologous to peptide T, with its ability to induce CD4-dependent monocyte chemotaxis.

Peptide T, from the human immunodeficiency virus (HIV), whose sequence is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, has been shown to inhibit attachment of this virus to T cells and neural cells bearing the CD4 receptor. This peptide shares extensive homology with the 19-26 segment of ribonuclease A (RNase A), whose sequence is Ala-Ala-Ser-Ser-Ser-Asn-Tyr-Cys. Based on comparison of the structures of peptides occurring in proteins of known structure that are homologous to peptide T, viz, RNase A and endothiapepsin and on conformational energy calculations, we predicted that peptide T adopts a structure much like that for residues 19-26 in RNase A. A critical feature is a bend involving residues Thr 4-Asn 7 in peptide T corresponding to Ser 22-Tyr 25 in the RNase A peptide. Our proposed structure for peptide T has recently been confirmed by Cotelle et al. (Biochem. Biophys. Res. Commun. 171, 596-602). We now show directly that the RNase A peptide, with Met replacing Cys 26 to prevent disulfide exchange reactions, strongly induces monocyte-chemotaxis that is blocked by anti-CD4 monoclonal antibody. Both peptide T and RNase A fail to induce chemotaxis, however, in neutrophils which do not express surface CD4 receptors. These results suggest that both peptides interact with the CD4 receptor in inducing monocyte chemotaxis. We have also prepared cyclo-RNase A peptide with Met 26. Using molecular dynamics and conformational energy calculations, we find that the cyclic peptide cannot form a bend structure involving Ser 22-Tyr 25 that is superimposable on the RNase A bend.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Activated conformations of the ras-gene-encoded p21 protein. 1. An energy-refined structure for the normal p21 protein complexed with GDP.

A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gln 61, bound to GDP, has been constructed in four stages using the available alpha-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 61 likewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the alpha-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no alpha-carbon coordinates for residues 60-65 have been determined, these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factor Tu (EF-Tu) to which p21 bears an overall 40% sequence homology, were determined from their corresponding positions in a high-resolution structure of EF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the alpha-carbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the non-homologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1992

Conformational effects of selected cancer-related amino acid substitutions in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Cancer-related mutations in the human p53 protein tend to cluster in four of the five highly conserved domains of the protein, and, in particular, in the central region of domain IV from residues 241 to 253. Using conformational energy analysis based on ECEPP (Empirical Conformational Energies for Polypeptides Program), we have determined the preferred three dimensional structures for this tridecapeptide sequence for the human wild-type p53 protein and four cancer-related mutant p53 proteins (Ala 245, Ile 246, Trp 248, Ser 249). The results show that the mutant peptides adopt conformations that are distinctly different from that of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in murine wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of human mutant p53 proteins may be mediated by distinct local conformational changes in the protein.

Amino Acid Sequence↗

PubMed1991

A peptide from the GAP-binding domain of the ras-p21 protein as well as azatyrosine block ras-induced maturation of Xenopus oocytes.

The ras-oncogene-encoded p21 protein is known to produce malignant transformation of NIH 3T3 cells as well as maturation of Xenopus oocytes when microinjected into these cells. p21 protein is known to bind a GTPase activating protein (GAP) intracellularly; residues 32-45 have been implicated in interacting with GAP. We demonstrate here that a peptide corresponding to residues 35-47 of p21 as well as the antibiotic azatyrosine inhibit the ras-induced maturation of Xenopus oocytes in a dose-related manner upon microinjection. We have previously shown that this p21 peptide and azatyrosine could inhibit the effects of p21 protein on cell transformation and pinocytosis in NIH 3T3 cells. In the present study, in which we have extended these results to the oocyte system, we also demonstrate that both partially inhibit insulin-induced oocyte maturation, a process which is thought to involve activation of endogenous p21 protein; on the other hand, both agents fail to inhibit oocyte maturation induced by progesterone, which is known not to act through p21 protein activation. Control studies with other peptides and tyrosine analogues support the selective nature of these events. These results suggest that both the p21-related peptide and azatyrosine have potent anti-ras effects intracellularly.

Alanine↗

PubMed1991

Conformational energy analysis of the substitution of Val for Gly 233 in a functional region of platelet GPIb alpha in platelet-type von Willebrand disease.

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patient platelets exhibit an abnormally increased binding of circulating von Willebrand factor (vWF). We have recently shown that this abnormality is associated with a point mutation resulting in substitution of Val for Gly 233 in platelet membrane glycoprotein Ib alpha (GPIb alpha), a major component of the platelet GPIb/IX receptor for vWF. To investigate the effect of this substitution on the three-dimensional structure of this region of the protein, we have generated the allowed (low energy) conformations of the region of the GPI alpha protein containing residues 228-238 (with 5 residues on either side of the critical residue 233) with Gly 233 (wild type) and Val 233 (PT-vWD) using the computer program ECEPP (Empirical Conformational Energies of Peptides Program). The wild-type sequence is Tyr-Val-Trp-Lys-Gln-Gly-Val-Asp-Val-Lys-Ala. We find that the Gly 233-containing peptide can exist in two low energy conformers. The lowest energy conformer is a structure containing a beta-turn at Gln 232-Gly 233 while the alternative conformation is an amphipathic helical structure. Only the amphipathic helical structure is allowed for the Val 233-containing peptide which contains a hydrophobic 'face' consisting of Val 229, Val 233 and Val 236 and another hydrophilic surface composed of such residues as Lys 231 and Asp 235. No such surfaces exist for the lowest energy bend conformer for the Gly 233-containing peptide, but do exist in the higher energy helical structure. The amphipathic surfaces in the 228-238 region of the Val 233-containing GPIb alpha protein may associate strongly with complementary surfaces during vWF binding to the GPIb/IX receptor complex and may help explain heightened association of vWF with this receptor in PT-vWD.

Amino Acid Sequence↗

PubMed1991

An activating amino acid substitution in the c-abl oncogene protein fails to produce a local conformational change.

The bcr-abl chimeric gene of Philadelphia chromosome positive chronic myelogenous leukemias is only weakly transforming. This transformation activity is greatly enhanced by a Lys-for-Glu substitution at position 832 in the c-abl gene, as occurs in the highly transforming v-abl genes. It has been suggested that this mutation results in a significant structural change in the encoded protein product. Using conformational energy analysis, we have determined the allowed low-energy conformations for residues 828-836 of this protein with Lys and Glu at position 832. In both cases, the overwhelmingly preferred conformation for this region is a bend-helix motif. The helix terminates at residue 836, and there are no discernible differences in conformation between the Lys- and Glu-containing sequences. These results suggest that the activating amino acid substitution at position 832 in the c-abl protein product does not produce its effect via a local conformational change.

Amino Acid Sequence↗

PubMed1990

Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure.

The predicted conformation and position of the central transforming region (residues 55-67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1-166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an alpha-helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.

Computer Simulation↗

PubMed1990

The structure of the carboxyl terminus of the p21 protein. Structural relationship to the nucleotide-binding/transforming regions of the protein.

The carboxyl-terminal region of the ras oncogene-encoded p21 protein is critical to the protein's function, since membrane binding through the C-terminus is necessary for its cellular activity. X-ray crystal structures for truncated p21 proteins are available, but none of these include the C-terminal region of the protein (from residues 172-189). Using conformational energy analysis, we determined the preferred three-dimensional structures for this C-terminal octadecapeptide of the H-ras oncogene p21 protein and generated these structures onto the crystal structure of the remainder of the protein. The results indicate that, like other membrane-associated proteins, the membrane-binding C-terminus of p21 assumes a helical hairpin conformation. In several low-energy orientations, the C-terminal structure is in close proximity to other critical locales of p21. These include the central transforming region (around Gln 61) and the amino terminal transforming region (around Gly 12), indicating that extracellular signals can be transduced through the C-terminal helical hairpin to the effector regions of the protein. This finding is consistent with the results of recent genetic experiments.

Amino Acid Sequence↗

PubMed1990

Correlation of the structure of the transmembrane domain of the neu oncogene-encoded p185 protein with its function.

The human homologue of the neu oncogene is frequently found in human tumors. Certain amino acid substitutions at position 664 in the transmembrane domain of the neu oncogene-encoded p185 protein product are known to cause malignant transformation of cells. Using conformational energy analysis based on ECEPP (empirical conformational energies for polypeptides program), we have previously determined the preferred three-dimensional structures for the transmembrane domain of the p185 protein with a transforming (glutamic acid) and a nontransforming (valine) substitution at the critical position 664 and found that the global minimum-energy conformation of this region in the nontransforming protein contains a sharp bend, whereas the global minimum-energy conformation for this region from the transforming protein is entirely alpha-helical. We now demonstrate that this result holds for other known nontransforming (glycine, histidine, tyrosine, and lysine) and transforming (glutamine) substitutions at position 664. Furthermore, a simple statistical thermodynamic analysis of the results indicates that approximately 85% of each of the nontransforming sequences exist with the bend at positions 664 and 665, while approximately 90% of each of the transforming sequences exist as an alpha-helix. About 9% of the nontransforming sequences exist as the alpha-helix. These results suggest that if the intracellular concentration of the normal protein is increased at least 10-fold, thereby increasing the alpha-helical form by this factor, cell transformation should result. This conclusion is directly supported by genetic experiments in which this level of overexpression of the normal protein was achieved with attendant cell transformation.

Amino Acid Sequence↗

PubMed1990

Comparisons between the three-dimensional structures of the chemotactic protein CheY and the normal Gly 12-p21 protein.

The three-dimensional structure of a chemotactic protein CheY from Salmonella typhimurium has recently been determined by X-ray crystallography. The structure of this small protein, containing 129 amino acid residues, shows a domain consisting of a central beta-pleated sheet surrounded on both sides by alpha-helices. We have examined the sequence and the arrangement of the structural domains of the CheY protein and have compared them with other nucleotide binding protein sequences and structures. We find that the CheY protein has significant sequence homology to the ras-gene encoded p21 protein. In addition, the structural domains of the two proteins are arranged in a fundamentally similar manner, including the phosphate-binding site (both proteins bind phosphate-containing ligands). The striking similarity in the arrangement of the structural domains of the two proteins suggests that both may serve similar functions as signal transducers.

Amino Acid Sequence↗

PubMed1990

Enthalpic and entropic determinants for the specificity of alkylation of the histidine-12 residue of ribonuclease A by four bromoacetamido nucleoside affinity labels and bromoacetamide.

The binding and alkylation rate constants for the reaction of four bromoacetamido pyrimidine nucleosides and bromoacetamide with bovine pancreatic ribonuclease A (RNase A) have been determined as a function of temperature. The four nucleoside derivatives react exclusively or preferentially with the NE2 atom of histidine-12 and include 2'-bromoacetamido-2'-deoxyuridine, 2'-bromoacetamido-2'deoxyxylofuranosyluracil, 3'-bromoacetamido-3'-deoxythymidine and 3'-bromoacetamido-3'-deoxyarabinofuranosyluracil. Transition-state parameters, delta H++ and delta S++, reveal that nucleosides with "up" OH groups experience relative rate enhancements which have been attributed to contacts between these groups and the enzyme in the transition state (1). Variations in alkylation rates are explained in terms of different degrees of entropic destabilization (2) of the nucleosides in the enzyme.affinity label complex.

Acetamides↗

PubMed1990

Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein.

The GTP-binding p21 protein, encoded by the ras-oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate-binding loop regions, Tyr 4-Thr 20, herein designated as segment 1, and Ile 55-Met 67, herein designated, as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF-tu) (Val 12-Thr 28 corresponds to segment 1; His 78-Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9-Cys 25 corresponds to segment 1 and Tyr 95-Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF-tu and ADK are similar to one another and that the conformation of the segment 2 region of EF-tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF-tu is strikingly similar to that of the two segments in ADK. In the originally proposed X-ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Kinase↗

PubMed1990

Octapeptide segments from the amino terminus of glycophorin A contain the antigenic determinants of the M and N blood groups systems.

Human red blood cells with phenotype N/N and M/M were tested in an agglutination assay with anti-N and anti-M antibodies, respectively. After incubation of the synthesized octapeptide (Leu-Ser-Thr-Thr-Glu-Val-Ala-Met) from the N-amino terminus of glycophorin A, with anti-N antibody, there was significant inhibition of the agglutination of the N-positive cells. There was also inhibition of the agglutination of the M-positive cells with anti-M antibody by the synthesized octapeptide (Ser-Ser-Thr-Thr-Gly-Val-Ala-Met) from the M-amino terminus of glycophorin A. There was no inhibition, however, of the agglutination of M-positive cells with anti-M antibodies by the N-amino terminal octapeptide. Likewise, the M-amino terminal octapeptide did not inhibit agglutination of N-positive cells with anti-N. Because the synthesized octapeptides contained no carbohydrate, the anti-N and anti-M specificity appears to be determined principally by the peptides themselves. Further studies with the use of chimeric peptides indicate that the amino terminal amino acid leucine of N-glycophorin A is a primary determinant of the N antigen, whereas the amino terminal serine of M-glycophorin A is a primary determinant for the M antigen.

Amino Acid Sequence↗

PubMed1989

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of the neu oncogene-encoded p185 protein.

The neu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in the neu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658-667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an alpha-helix with a sharp bend (CD conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all alpha-helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

Conformational effects of amino acid substitutions at positions 10, 12, and 13 in the P21 protein.

Substitutions of amino acids for Gly 12 or Gly 13 in the ras oncogene-encoded P21 proteins have been demonstrated to produce unique structural changes in these proteins that correlate with their ability to produce cell transformation. For example, the P21 proteins with Arg 12 or Val 13 are both known to be actively transforming. Recent site-specific mutagenesis experiments on the transforming Arg 12 protein have found that the substitution of Val for Gly 10 has no effect on transforming activity whereas the substitution of Val for Gly 13 led to a loss of transforming activity. In this study, we examine the structural effects of these substitutions on the amino terminal hydrophobic decapeptide (Leu 6-Gly 15) of P21 using conformational energy analysis. The results show that the transforming proteins with Gly 10 and Arg 12 or Val 10 and Arg 12 can both adopt the putative malignancy-causing conformation, whereas, for the nontransforming protein with Arg 12 and Val 13, this conformation is energetically disallowed. These results further support the theory that due to structural changes the transforming P21 proteins are unable to bind to some regulatory cellular element which may be the recently identified binding protein responsible for the induction of increased GTPase activity in normal P21 compared with transforming mutants.

Amino Acids↗