Evaluation of the feasibility of and results of measuring health-status changes in patients undergoing surgical treatment for skeletal metastases.

The goal of treating patients with skeletal metastases is to decrease pain and improve or maintain physical function. Assessment of the effectiveness of treatment should therefore include evaluation of patient-rated measures of quality of life. The primary objective of the study was to determine the feasibility of studying the effect of surgical treatment of skeletal metastases on quality of life. The secondary objective was to provide data that begin to characterize this effect. The characteristics of patients with skeletal metastases are heterogeneous, patient enrollment in the study may be low, high attrition occurs secondary to death, and well accepted health-status measures (such as the Short Form-36) may be ineffective at detecting changes in health status; therefore, it is difficult to study these patients. High attrition and adjuvant treatment with radiation or chemotherapy made it impractical to draw firm conclusions about the effect of surgical treatment, but a trend toward improvement in selected health-status measures for both physical and mental health was noted. Analysis of patient-rated health-status scores as predictors of survival indicates that improvement in these scores 6 weeks after surgery is associated with an increase in the length of survival following surgery.

Molecular dynamics of the H-ras gene-encoded p21 protein; identification of flexible regions and possible effector domains.

We previously reported a complete computer-based three-dimensional structure for residues 1-171 of the Gly 12-containing ras-gene-encoded p21 protein complexed with GDP. This structure was subsequently shown to closely agree with a high-resolution x-ray crystallographic structure of p21. In this communication, we report a molecular dynamics stimulation of the modelled structure in an explicit shell of water molecules to identify domains within the protein that are unusually flexible. These domains represent regions which are most likely to undergo important conformational changes when the protein is activated by binding to GTP or by oncogenic amino acid substitutions such as Val for Gly 12. The starting structure was surrounded with water molecules, temperature-equilibrated and then followed over a 100 ps trajectory during which time the energy converged after about 50 ps. Regions of the protein that were found to have the largest coordinate fluctuations involved residues 12-16, 30-35, 40-52, 60-73, 85-89, 101-109, 119-123, and 127-131. Many of these sequences with high flexibility have been implicated in the functioning of this protein. Since the overall largest fluctuations were observed for residues 101-106 and 119-123, p21 peptides containing these residues (96-110 and 115-126) were synthesized and were found to inhibit strongly the effects of oncogenic p21 protein in an oocyte maturation assay. These results indicate that the flexible p21 sequences may constitute critical functional domains of the activated protein and that this general approach may be useful for identification of important functional domains in proteins.

Activated conformations of the ras-gene-encoded p21 protein. 2. Comparison of the computed and high-resolution x-ray crystallographic structures of Gly-12 p21.

The ras-oncogene-encoded p21 protein is a G-protein that has been shown to cause the malignant transformation of normal cells and has been implicated in causing human tumors. p21 is thought to be activated by the binding of GTP in place of GDP to the protein. We have previously constructed the three-dimensional structure of the p21 protein bound to GDP from an available alpha-carbon tracing of this protein using a combination of molecular dynamics and energy minimization (Dykes, et al., J. Biomol. Struct. Dynamics, 9:1025-1044). Until the recent publication of the all-heavy-atom x-ray crystallographic molecular coordinates of p21 residues 1-166 bound to a non-hydrolyzable GTP derivative (GppNp), no all-atom structure of the p21 protein has been available in the Brookhaven National Laboratories Protein Data Bank (PDB). In this communication we compare our computed structure for the p21-GDP complex to this x-ray crystal structure. We find that the two structures agree quite closely with one another, the overall RMS deviation for the backbone being 1.47 A and 2.71 A for all of the atoms. We have identified the regions of the protein that are responsible for the most significant deviations between the two structures, i.e., residues 32-40 and 61-74. We find that the main chain in the 32-40 segment deviates significantly from residue 32 to residue 36 and the side chain phenolic rings of residue 32 differ greatly between the two structures. The 61-74 region is the least-well defined region in the whole protein crystallographically having, by far, the highest temperature factor (B-factor). The backbone and side chain conformations in the 61-74 segment differ markedly, the overall RMS deviation being 3.1 A for the backbone and 5.7 A for all atoms. Both of these regions have been found in x-ray crystallographic studies of p21-GDP and p21-GTP complexes to undergo significant changes in conformation upon the binding of GTP in place of GDP to the protein. We have further compared our computed structure of the p21 protein with the x-ray crystal structure with regard to the conformations of individual segments, in particular, structurally conserved sequences (SCR), i.e., those sequences that have structural and sequence homology to corresponding sequences in the related G-protein, bacterial elongation factor Tu(EF-Tu), and variable loop regions.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms.

We have recently shown that a peptide (residues 35-47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41,251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13-259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.

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.

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)

Pathways for activation of the ras-oncogene-encoded p21 protein.

The ras-oncogene-encoded p21 protein is known to cause a large number of human tumors. This protein differs from its normal counterpart protein, which is present in all eukaryotic cells, in that it contains a single amino acid substitution at critical positions in the polypeptide chain, such as at Gly 12, Gly 13, Ala 59, and Gln 61. Using computer-based molecular modeling, it has been found that one region of this protein that is a candidate for interacting with other intracellular proteins is the region from residues 35 to 47. In oocyte microinjection experiments, it was found that this peptide strongly inhibits the mitogenic effects of oncogenic (Val 12-containing)p21 but does not inhibit the cellular effects of activation of normal p21 protein. Furthermore, it has been shown that the cellular effects of oncogenic p21 protein can be completely inhibited by selectively blocking protein kinase C (PKC) with a highly specific inhibitor of this protein, CGP 41 251, a staurosporine derivative. This inhibitor, however, only weakly inhibits the effects of normal cellular ras-p21 protein. In addition, a photoaffinity-labeled p21 protein has been microinjected into NIH 3T3 fibroblasts and have isolated intracellular proteins of MW 35, 43 and 61 kda covalently bound to it. The 43 kda protein is the major one and appears to be critical to the functioning of the p21 protein. Our results suggest that oncogenic and normal p21 proteins utilize overlapping but distinct pathways; the oncogenic pathway can be blocked selectively and requires the activation of PKC and the presence of the 43 kda protein.

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)

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.