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R Monaco

Publications and source records attributed to R Monaco.

At least 37 records · Page 2Linked to original sources
PubMed1997

Inhibition of oncogenic and activated wild-type ras-p21 protein-induced oocyte maturation by peptides from the ras-binding domain of the raf-p74 protein, identified from molecular dynamics calculations.

In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein-protein interaction interface, e.g., between residues 60 and 72, residues 97-110, and 111-121. Since the rap-1A-RBD complex is inactive, these three regions are implicated in ras-p21-induced activation of raf. We have therefore co-microinjected peptides corresponding to these three regions, 62-76, 97-110, and 111-121, into oocytes with oncogenic p21 and microinjected them into oocytes incubated in in insulin, which activates normal p21. All three peptides, but not a control peptide, strongly inhibit both oncogenic p21- and insulin-induced oocyte maturation. These findings corroborate our conclusions from the theoretical results that these three regions constitute raf effector domains. Since the 97-110 peptide is the strongest inhibitor of oncogenic p21, while the 111-121 peptide is the strongest inhibitor of insulin-induced oocyte maturation, the possibility exists that oncogenic and activated normal p21 proteins interact differently with the RBD of raf.

Amino Acid Sequence↗
PubMed1997

The usefulness of bone turnover in predicting the response to transdermal estrogen therapy in postmenopausal osteoporosis.

Transdermal estrogen therapy is now an accepted form of treatment for postmenopausal osteoporosis. Ninety postmenopausal osteoporotic women were randomized to receive either transdermal estrogen (0.05 mg/day 17 beta-estradiol) and calcium (n = 45) or calcium alone (n = 45). The study period was 2 years. Bone mineral density (BMD) at the lumbar spine (by dual-energy X-ray absorptiometry [DXA]) and markers of bone turnover (alkaline phosphatase, osteocalcin, hydroxyproline, pyridinoline cross-links) were assessed at baseline and after 1 and 2 years. In the estrogen-treated group, BMD showed a significant increase (p < 0.001) both after 1 and 2 years, with a reduction in biochemical markers. To investigate the effectiveness of estrogen treatment of postmenopausal osteoporosis in relation to bone turnover, we also divided the patients on the basis of bone turnover, as assessed by measurement of whole body retention (WBR) of 99mTc-methylene diphosphonate. WBR revealed that 26 patients had high bone turnover (HT) and 55 had low bone turnover (LT). The response to estrogen was greater in the HT patients than in the LT patients; in fact BMD increased by 5.7 and 6.6% in HT patients and by 2.6 and 2.7% in LT patients after 1 and 2 years, respectively. In conclusion, the present study demonstrates that, while the BMD decreases in the patients treated with calcium alone, 2-year treatment with transdermal estrogen increases axial BMD and that the response to estrogen treatment is influenced by bone turnover. Therefore, the evaluation of bone turnover may be useful to identify those postmenopausal osteoporotic women who may especially benefit from treatment with estrogen.

Administration, Cutaneous↗
PubMed1996

Prediction of the three-dimensional structure of the rap-1A protein from its homology to the ras-gene-encoded p21 protein.

rap-1A, an anti-oncogene-encoded protein, is a ras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32-47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A at ras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure of ras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 A from that of ras-p21. The effector binding domains from residues 32-47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115-126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.

Algorithms↗
PubMed1996

Computed three-dimensional structures for the ras-binding domain of the raf-p74 protein complexed with ras-p21 and with its suppressor protein, rap-1A.

The three-dimensional structures of the ras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to the ras-binding domain, RBD (residues 55-131), of the raf-p74 protein, a critical target protein of ras-p21 in the ras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of both ras-p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of the ras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains of ras-p21 and rap-1a, including residues 32-47, a domain that directly interacts with RBD, 60-66, 96-110, involved in the interaction of ras-p21 with jun kinase (JNK) and jun protein, and 115-126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its beta-2 binding loop (residues 63-71) and residues 89-91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK and jun.

GTP-Binding Proteins↗
PubMed1996

Intratumor heterogeneity of K-ras2 mutations in colorectal adenocarcinomas: association with degree of DNA aneuploidy.

Detailed information about intratumor K-ras2 mutations in colorectal adenocarcinomas and a possible association with DNA content heterogeneity is still lacking. DNA diploid and aneuploid subclones, detected among multiple histologically selected primary sectors (57 superficial and 40 deep) and 9 lymph node metastases, were flow cytometrically sorted and separately submitted to codons 12-13 K-ras2 mutation spectrum analysis. DNA aneuploidy was absent among 20 near and 20 distant mucosa sites and present in 7/9 lymph node metastases and in 17/19 primary tumors (90%). Primary intratumor DNA multiclonality was approximately 50%. Degree of DNA aneuploidy (DNA Index) distribution was nonrandom and showed peaks at approximate mean DNA Index values 1.2, 1.5, and 1.8. K-ras2 mutations were detected in 0/20 mucosa cases, in 2/9 lymph node metastases, and in 9/19 adenocarcinomas (47%). No more than one mutation type per tumor was detected. Intratumor distribution of K-ras2 mutations was homogeneous in 6 and heterogeneous in 3 cases. Homogeneous distribution was associated with DNA near-diploid aneuploidy. K-ras2 mutations were strongly associated with DNA Index in the near-diploid region (83%) and almost absent (5%) among DNA near-triploid subclones (P = 0.0001). K-ras2 mutation intratumor heterogeneity indicates that sampling of the tumor may be a critical step and suggests that K-ras2 activation may be a late event in a subgroup of tumors. Our data also suggest the existence of an early process of the colorectal carcinogenesis that favors both K-ras2 mutations and DNA near-diploid aneuploidy. Onset of DNA near-triploid subclones appears, instead, to be independent from K-ras2 activation.

Adenocarcinoma↗
PubMed1995

Structural effects of the binding of GTP to the wild-type and oncogenic forms of the ras-gene-encoded p21 proteins.

Molecular dynamics calculations have been performed to determine the average structures of ras-gene-encoded p21 proteins bound to GTP, i.e., the normal (wild-type) protein and two oncogenic forms of this protein, the Val 12- and Leu 61-p21 proteins. We find that the average structures for all of these proteins exhibit low coordinate fluctuations (which are highest for the normal protein), indicating convergence to specific structures. From previous dynamics calculations of the average structures of these proteins bound to GDP, major regional differences were found among these proteins [Monaco et al. (1995), J. Protein Chem., in press]. We now find that the average structures of the oncogenic proteins are more similar to one another when the proteins are bound to GTP than when they are bound to GDP [Monaco et al. (1995), J. Protein Chem., in press]. However, they still differ in structure at specific amino acid residues rather than in whole regions, in contradistinction to the results found for the p21-GDP complexes. Two exceptions are the regions 25-32, in an alpha-helical region, and 97-110. The two oncogenic (Val 12- and Leu 61-) proteins have similar structures which differ significantly in the region of residues 97-110. This region has recently been identified as being critical in the interaction of p21 with kinase target proteins. The differences in structure between the oncogenic proteins suggest the existence of more than one oncogenic form of the p21 protein that can activate different signaling pathways.

Binding Sites↗
PubMed1995

Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of the ras-gene-encoded p21 protein with the structure of the normal (non-transforming) wild-type protein.

The ras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10-16, 32-47, 55-74, 85-89, 100-110, and 119-134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32-47 and 55-74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55-74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.

Cell Transformation, Neoplastic↗
PubMed1995

Conformation of the transmembrane domain of the c-erbB-2 oncogene-encoded protein in its monomeric and dimeric states.

The human c-erbB-2 oncogene is homologous to the rat neu oncogene, both encoding transmembrane growth factor receptors. Overexpression and point mutations in the transmembrane domain of the encoded proteins in both cases have been implicated in cell transformation and carcinogenesis. In the case of the neu protein, it has been proposed that these effects are mediated by conformational preferences for an alpha-helix in the transmembrane domain, which facilitates receptor dimerization, an important step in the signal transduction process. To examine whether this is the case for c-erbB-2 as well, we have used conformational energy analysis to determine the preferred three-dimensional structures for the transmembrane domain of the c-erbB-2 protein from residues 650 to 668 with Val (nontransforming) and Glu (transforming) at position 659. The global minimum energy conformation for the Val-659 peptide from the normal, nontransforming protein was found to contain several bends, whereas the global minimum energy conformation for Glu-659 peptide from the mutant, transforming protein was found to be alpha-helical. Thus, the difference in conformational preferences for these transmembrane domains may explain the difference in transforming ability of these proteins. The presence of higher-energy alpha-helical conformations for the transmembrane domain from the normal Val-659 protein may provide an explanation for the presence of a transforming effect from overexpression of c-erbB-2. In addition, docking of the oncogenic sequences in their alpha-helical and bend conformations shows that the all-alpha-helical dimer is clearly favored energetically over the bend dimer.

Amino Acid Sequence↗
PubMed1995

Asthma management and mode of acquisition of inhaled bronchodilators.

BACKGROUND: Controversy has existed about the benefits and disadvantages associated with the availability of inhaled bronchodilators over the counter (OTC). AIMS: To compare sociodemographic features, use of preventive medications, use of medical services and control of asthma in adults who purchased inhaled bronchodilators OTC with those who purchased on prescription (script). METHODS: A cross-sectional telephone survey of 772 adults 18 years and over who used inhaled bronchodilators for their asthma. Symptoms, asthma medications, and management practices were determined by a structured questionnaire administered by trained telephone interviewers. RESULTS: Two hundred and thirty adults purchased their bronchodilator OTC and 542 on script. OTC purchasers were more likely to be male (OR: 1.5), have had tertiary education (1.5) and be in paid employment (2.8); they were less likely to report frequent symptoms (wheeze, nocturnal symptoms or EIA more than once a week) (0.71); were less likely to use preventive medications more than twice a day (0.57) and were less likely to have consulted a general practitioner in the previous year (0.38). However, poor control of asthma symptoms was evident in both OTC and script groups. Forty per cent of the OTC group who had symptoms more than two to three times a week and 34% of the script group were not using preventive medication. CONCLUSIONS: Undertreatment and suboptimal management of asthma were apparent in both OTC and script groups.

Adult↗
PubMed1994

Conformational effects of environmentally induced, cancer-related mutations in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. A considerable number of environmentally induced, cancer-related p53 mutations in human tumors have been found in a highly conserved proline-rich sequence of the p53 protein encompassed by amino acid residues 147-158. Using conformational energy analysis based on ECEPP (Empirical Conformational Energy for Peptides Program), we have determined the low-energy three-dimensional structures for this dodecapeptide sequence for the human wild-type p53 protein and three environmentally induced, cancer-related mutant p53 proteins with His-151, Ser-152, and Val-154, respectively. The results suggest that the wild-type sequence adopts a well-defined low-energy conformation and that the mutant peptides adopt well-defined conformations that are distinctly different from the conformation of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of these environmentally induced, cancer-related, mutant p53 proteins may be mediated by distinct local conformational changes in the protein.

Amino Acid Sequence↗
PubMed1994

Conformation of the transmembrane domain of the epidermal growth factor receptor.

The transmembrane domain of growth factor receptors, such as the epidermal growth factor receptor (EGFR) and the related c-erbB-2/neu oncogene protein, has been implicated in the process of receptor dimerization and mitogenic signal transduction, and hence in cellular transformation and oncogenesis. Amino acid substitutions in the transmembrane domain of the c-erbB-2/neu protein that cause a transforming effect may exert this effect through a conformational change from a bend conformation to an alpha-helical structure in this region of the protein, but similar amino acid substitutions at homologous positions in the transmembrane domain of the EGFR (e.g., Val-->Glu at position 627) fail to have a transforming effect. To examine whether this failure may be due to structural effects, we have used conformational energy analysis to determine the preferred three-dimensional structures for the nonapeptide sequence of the transmembrane domain of the EGFR from residues 623-631 with Val or Glu at position 627. The global minimum energy conformations of both nonapeptides were found to be non-alpha-helical with bends at positions 624-625 and 627-628. The failure of the Val-->Glu substitution to produce a conformational change to an alpha-helix in this region may be responsible for its lack of transforming effect. However, the presence of higher energy alpha-helical conformations for the nonapeptide from the normal EGFR may provide an explanation for the presence of a transforming effect from overexpression of the EGFR.

Amino Acid Sequence↗