Potential role of human cytomegalovirus and p53 interaction in coronary restenosis.

A subset of patients who have undergone coronary angioplasty develop restenosis, a vessel renarrowing characterized by excessive proliferation of smooth muscle cells (SMCs). Of 60 human restenosis lesions examined, 23 (38 percent) were found to have accumulated high amounts of the tumor suppressor protein p53, and this correlated with the presence of human cytomegalovirus (HCMV) in the lesions. SMCs grown from the lesions expressed HCMV protein IE84 and high amounts of p53. HCMV infection of cultured SMCs enhanced p53 accumulation, which correlated temporally with IE84 expression. IE84 also bound to p53 and abolished its ability to transcriptionally activate a reporter gene. Thus, HCMV, and IE84-mediated inhibition of p53 function, may contribute to the development of restenosis.

Automatic and accurate method for analysis of proteins that undergo hinge-mediated domain and loop movements.

BACKGROUND: The structures of proteins that undergo significant main-chain conformational change are reported with increasing frequency. Three-dimensional atomic models are often available for two alternative conformational states of the same molecule. Inspection has shown these states to be varied in nature, arising by mechanisms that include hinge-facilitated closure between domains and smaller-scale loop motions within domains; these movements are often induced by metal ion binding or ligand binding. Polypeptides that display flexible segments are also observed in different crystal conformations or as alternatively packed subunits. Although subjective visual inspection has been previously used to compare structures and analyze conformational changes, there is a need for an objective method. RESULTS: We have developed a straightforward, robust, and objective algorithm that can locate the residues that mediate and participate in the changes between the two conformational states. Our method does not require initial superpositioning. We illustrate the method by considering several test cases. The first example is maltose binding protein, a polypeptide that exhibits rigid-body domain closure involving multiple hinges. The second is lactate dehydrogenase, which undergoes both loop and subdomain movement; we accurately describe the location and relative magnitude of these deformations. Finally, in the example of aspartate transcarbamoylase, both hinge-mediated domain movement and functionally relevant loop rearrangements are described. In the instances in which domain closure occurs, the residues that serve as hinges between the domains involved are accurately predicted. In addition, our technique successfully identifies the exact residues that undergo intra-domain loop movements, even for movements that are accompanied by larger scale inter-domain rearrangements. CONCLUSIONS: Our algorithm is successful in its comprehensive analysis and description of complex hinge-mediated domain motion for all structures displaying rigid-body movement and is accurate in identifying the location of any independent intra-domain rearrangements.

E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection.

The adenovirus immediate-early protein E1A activates the adenovirus E2 promoter and several cellular gene promoters through transcription factor E2F. The immediate-early proteins of human cytomegalovirus (HCMV) can complement an E1A-deficient adenovirus mutant and activate the adenovirus E2 promoter. HCMV also has been shown to activate the adenovirus E2 promoter. On the basis of these findings, we have investigated whether HCMV can activate the promoter of the cellular dihydrofolate reductase (DHFR) gene, which requires E2F binding for maximal promoter activity. We show that HCMV activates the DHFR promoter and that products of the HCMV major immediate-early gene region mediate the activation of the promoter specifically through the E2F site. We used gel mobility shift assays to search for potential molecular mechanisms for this activation and found an "infection-specific" multimeric complex that bound to the E2F sites in the DHFR and E2 promoters in extracts from HCMV-infected cells but not in extracts from uninfected cells. Several antibodies against HCMV immediate-early gene products had no effect on this infection-specific complex. Subsequently, the complex was found to contain E2F, cyclin A, p33cdk2, and p107 and to be similar to S-phase-specific complexes that recently have been identified in several cell types. A functional role for the binding of the cyclin A-p33cdk2 complex to cellular gene promoters has yet to be demonstrated; however, HCMV infection causes the induction of both cellular DNA replication and transcription of growth-related genes containing E2F sites in their promoters. The findings described above therefore may relate to both of these effects of HCMV infection. We also provide evidence that some of the molecular events associated with adenovirus infection are different from those associated with HCMV infection.

Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3).

Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.

Induction of NF-kappa B-like activity by platelet-derived growth factor in mouse fibroblasts.

Nuclear factor kappa B (NF-kappa B) modulates the expression of numerous genes via interaction with a specific DNA sequence termed the kappa B site. Its activity is modulated by a cytosolic inhibitor protein termed I kappa B, and its activation occurs in response to a variety of agents in a variety of cell types, most notably B and T lymphocytes. Data presented here show that an activity (designated complex I) that binds specifically to the kappa B site is induced in density-arrested Balb/c-3T3 mouse fibroblasts by platelet-derived growth factor (PDGF), a potent mitogen for these cells. Increased levels of complex I, as evaluated by electrophoretic mobility shift assays of nuclear extracts, were observed in cells treated for 1-4 h (but not 15 min) with the BB isoform of PDGF. 12-O-tetradecanoylphorbol 13-acetate (TPA) and the AA isoform of PDGF also stimulated this response and both isoforms, but not TPA, were effective in cells depleted of protein kinase C. Complex I most likely is authentic NF-kappa B, a p50-p65 heterodimer, or a closely related factor because it exhibited properties characteristic of those previously described for NF-kappa B including inducibility by deoxycholate and cycloheximide and sensitivity to I kappa B. A second kappa B binding activity (complex II), which apparently contained p50 homodimers, displayed limited induction by PDGF, whereas a third complex (complex III) migrated faster than but behaved similarly to complex I. These studies suggest that NF-kappa B or an NF-kappa B-like factor may participate in the expression of PDGF-inducible genes.

Are predicted structures good enough to preserve functional sites?

BACKGROUND: A principal goal of structure prediction is the elucidation of function. We have studied the ability of computed models to preserve the microenvironments of functional sites. In particular, 653 model structures of a calcium-binding protein (generated using an ab initio folding protocol) were analyzed, and the degree to which calcium-binding sites were recognizable was assessed. RESULTS: While some model structures preserve the calcium-binding microenvironments, many others, including some with low root mean square deviations (rmsds) from the crystal structure of the native protein, do not. There is a very weak correlation between the overall rmsd of a structure and the preservation of calcium-binding sites. Only when the quality of the model structure is high (rmsd less than 2 A for atoms in the 7 A local neighborhood around calcium) does the modeling of the binding sites become reliable. CONCLUSIONS: Protein structure prediction methods need to be assessed in terms of their preservation of functional sites. High-resolution structures are necessary for identifying binding sites such as calcium-binding sites.

Human cytomegalovirus and herpes simplex type 2 virus in normal and adenocarcinomatous prostate glands.

Normal prostate, benign prostate hypertrophy (BHP), and prostate adenocarcinoma (ACP) biopsy specimens were analyzed for the presence of human cytomegalovirus (HCMV) and herpes simplex virus type 2 (HSV-2)-specific macromolecules. HCMV DNA homologous sequences were detected in 2 of 13 normal prostate, 2 of 9 BHP, and 3 of 10 ACP tissues, and HSV-2 DNA homologous sequences were found in 1 normal prostate tissue and 2 ACP tissues. In situ DNA-RNA hybridizations indicated HCMV-specific RNA in 3 of 8 BHP and 4 of 9 ACP tissues. No positive in situ hybridization for HCMV RNA was observed in normal prostate tissues. Parallel in situ DNA-RNA hybridization localized HSV-2-specific RNA in only 1 of 8 tumor sections. No HSV-2-specific RNA was observed in sections of normal and BHP tissues. Anticomplement immunofluorescence (ACIF) tests of BHP and ACP specimens showed specific HCMV immunofluorescence in 3 of 8 BHP and 4 of 9 ACP tissues. ACIF results correlate closely with in situ hybridization results and imply some degree of HCMV association with prostatic abnormality. The results also suggest that latent HCMV may be harbored by the human prostate gland.

Glioblastoma cell-specific expression of fibroblast growth factor receptor-1beta requires an intronic repressor of RNA splicing.

The fibroblast growth factor receptor-1 (FGFR-1) primary transcript is alternatively processed to produce receptors that vary in their ligand affinity and specificity. A high affinity form of this receptor--FGFR-1beta--that lacks the alpha exon is observed on the neoplastic transformation of glial cells. In this study, we have identified a 62-bp sequence located 97 bp downstream from the alpha exon that is required for the exclusion of this exon in a human glioblastoma cell line. Deletion or mutation of this sequence is sufficient to allow enhanced inclusion of the alpha exon or a heterologous exon in glioblastoma cells. Therefore, it would appear that this sequence element plays a key role in the glioblastoma-specific splicing to form FGFR-1beta mRNA.

Fibroblast growth factor receptor-1 alpha-exon exclusion and polypyrimidine tract-binding protein in glioblastoma multiforme tumors.

Neoplastic transformation of glial cells alters inclusion of the alpha exon in human fibroblast growth factor receptor-1 (FGFR-1) mRNA transcripts. Although normal cells predominantly include the alpha exon, this exon is excluded in most glioblastoma cell transcripts, creating a high-affinity receptor form. In this study, we identified polypyrimidine tract-binding protein (PTB) as a regulator of FGFR-1 splicing. PTB interacted in a sequence-specific manner with the ISS-1 regulatory element in the intron upstream of the a exon. PTB expression was also strongly increased in seven malignant glioblastoma multiforme tumors relative to adjacent normal tissue, but not in a low-grade astrocytoma. These results suggest that increased expression of PTB may contribute to glial cell malignancy.

Recognizing native folds by the arrangement of hydrophobic and polar residues.

Central to the ab initio protein folding problem is the development of an energy function for which the correct native structure has a lower energy than all other conformations. Existing potentials of mean force typically rely extensively on database-derived contact frequencies or knowledge of three-dimensional structural information in order to be successful in the problem of recognizing the native fold for a given sequence from a set of decoy backbone conformations. Is the detailed statistical information or sophisticated analysis used by these knowledge-based potentials needed to achieve the observed degree of success in fold recognition? Here we introduce a novel pairwise energy function that enumerates contacts between hydrophobic residues while weighting their sum by the total number of residues surrounding these hydrophobic residues. Thus it effectively selects compact folds with the desired structural feature of a buried, intact core. This approach represents an advance over using pairwise terms whose energies of interaction that are independent of the position in the protein and greatly improves the discrimination capability of an energy function. Our results show that 85% of a set of 195 representative native folds were recognized correctly. The 29 exceptions were lipophilic proteins, small proteins with prosthetic groups or disulfide bonds, and oligomeric proteins. Overall, our method separates the native fold from incorrect folds by a larger margin (measured in standard deviation units) than has been previously demonstrated by more sophisticated methods. The arrangement of hydrophobic and polar residues alone as evaluated by our novel scoring scheme, is unexpectedly effective at recognizing native folds in general. It is surprising that a simple binary pattern of hydrophobic and polar residues apparently selects a give unique fold topology.

Factors affecting the ability of energy functions to discriminate correct from incorrect folds.

Eighteen low and medium resolution empirical energy functions were tested for their ability to distinguish correct from incorrect folds from three test sets of decoy protein conformations. The energy functions included 13 pairwise potentials of mean force, covering a wide range of functional forms and methods of parameterization, four potentials that attempt to detect properly formed hydrophobic cores, and one environment-based potential. the first of the three test sets consists of large ensembles of plausible conformations for eight small proteins, all of which have correct native secondary structure and are reasonably compact. The second is the set of all subconformations in a database of known protein structures applied to the sequences in that database (ungapped threading). The third is a set of ensembles of 1000 conformations each for seven small proteins taken from molecular dynamics simulations at 298 K and 498 K. Our results show that there are functions effective for each challenge set; moreover, success in one test is no guarantee of success in another. We examine the factors that seem to be important for accurate discrimination of correct structures in each of the test sets, and note that extremely simple functions are often as effective as more complex functions.

Ab initio fold prediction of small helical proteins using distance geometry and knowledge-based scoring functions.

The problem of protein tertiary structure prediction from primary sequence can be separated into two subproblems: generation of a library of possible folds and specification of a best fold given the library. A distance geometry procedure based on random pairwise metrization with good sampling properties was used to generate a library of 500 possible structures for each of 11 small helical proteins. The input to distance geometry consisted of sets of restraints to enforce predicted helical secondary structure and a generic range of 5 to 11 A between predicted contact residues on all pairs of helices. For each of the 11 targets, the resulting library contained structures with low RMSD versus the native structure. Near-native sampling was enhanced by at least three orders of magnitude compared to a random sampling of compact folds. All library members were scored with a combination of an all-atom distance-dependent function, a residue pair-potential, and a hydrophobicity function. In six of the 11 cases, the best-ranking fold was considered to be near native. Each library was also reduced to a final ab initio prediction via consensus distance geometry performed over the 50 best-ranking structures from the full set of 500. The consensus results were of generally higher quality, yielding six predictions within 6.5 A of the native fold. These favorable predictions corresponded to those for which the correlation between the RMSD and the scoring function were highest. The advantage of the reported methodology is its extreme simplicity and potential for including other types of structural restraints.

Ab initio construction of protein tertiary structures using a hierarchical approach.

We present a hierarchical method to predict protein tertiary structure models from sequence. We start with complete enumeration of conformations using a simple tetrahedral lattice model. We then build conformations with increasing detail, and at each step select a subset of conformations using empirical energy functions with increasing complexity. After enumeration on lattice, we select a subset of low energy conformations using a statistical residue-residue contact energy function, and generate all-atom models using predicted secondary structure. A combined knowledge-based atomic level energy function is then used to select subsets of the all-atom models. The final predictions are generated using a consensus distance geometry procedure. We test the feasibility of the procedure on a set of 12 small proteins covering a wide range of protein topologies. A rigorous double-blind test of our method was made under the auspices of the CASP3 experiment, where we did ab initio structure predictions for 12 proteins using this approach. The performance of our methodology at CASP3 is reasonably good and completely consistent with our initial tests.

Using a hydrophobic contact potential to evaluate native and near-native folds generated by molecular dynamics simulations.

There are several knowledge-based energy functions that can distinguish the native fold from a pool of grossly misfolded decoys for a given sequence of amino acids. These decoys, which are typically generated by mounting, or "threading", the sequence onto the backbones of unrelated protein structures, tend to be non-compact and quite different from the native structure: the root-mean-squared (RMS) deviations from the native are commonly in the range of 15 to 20 angstroms. Effective energy functions should also demonstrate a similar recognition capability when presented with compact decoys that depart only slightly in conformation from the correct structure (i.e. those with RMS deviations of approximately 5 angstroms or less). Recently, we developed a simple yet powerful method for native fold recognition based on the tendency for native folds to form hydrophobic cores. Our energy measure, which we call the hydrophobic fitness score, is challenged to recognize the native fold from 2000 near-native structures generated for each of five small monomeric proteins. First, 1000 conformations for each protein were generated by molecular dynamics simulation at room temperature. The average RMS deviation of this set of 5000 was 1.5 angstroms. A total of 323 decoys had energies lower than native; however, none of these had RMS deviations greater than 2 angstroms. Another 1000 structures were generated for each at high temperature, in which a greater range of conformational space was explored (4.3 angstroms RMS deviation). Out of this set, only seven decoys were misrecognized. The hydrophobic fitness energy of a conformation is strongly dependent upon the RMS deviation. On average our potential yields energy values which are lowest for the population of structures generated at room temperature, intermediate for those produced at high temperature and highest for those constructed by threading methods. In general, the lowest energy decoy conformations have backbones very close to native structure. The possible utility of our method for screening backbone candidates for the purpose of modelling by side-chain packing optimization is discussed.

1,25-Dihydroxyvitamin D3 silences 3',5'-cyclic adenosine monophosphate enhancement of somatostatin gene transcription in human thyroid C cells.

Treatment of the somatostatin (SRIF)-producing TT cell line with 1,25 dihydroxyvitamin D3 (1,25 D3) lowered intracellular SRIF mRNA and peptide concentration. In separate experiments, the cAMP analog 8-(4-chlorophenylthio)-cAMP stimulated a rapid increase in SRIF mRNA content of the TT cells and SRIF peptide secretion. To determine whether 1,25 D3 could inhibit either the transcriptional or secretory effects of cAMP, TT cells were pretreated with 1,25 D3 for 4 days followed by treatment with the cAMP analog. Pretreatment with 1,25 D3 inhibited the cAMP-mediated increase of SRIF mRNA, but had no effect on the secretory response. We conclude that the ability of 1,25 D3 to silence SRIF gene expression is more potent than cAMP enhancer activity but that 1,25 D3 has no effect on that portion of the cAMP-dependent pathway which regulates peptide secretion.

Distance geometry generates native-like folds for small helical proteins using the consensus distances of predicted protein structures.

For successful ab initio protein structure prediction, a method is needed to identify native-like structures from a set containing both native and non-native protein-like conformations. In this regard, the use of distance geometry has shown promise when accurate inter-residue distances are available. We describe a method by which distance geometry restraints are culled from sets of 500 protein-like conformations for four small helical proteins generated by the method of Simons et al. (1997). A consensus-based approach was applied in which every inter-Calpha distance was measured, and the most frequently occurring distances were used as input restraints for distance geometry. For each protein, a structure with lower coordinate root-mean-square (RMS) error than the mean of the original set was constructed; in three cases the topology of the fold resembled that of the native protein. When the fold sets were filtered for the best scoring conformations with respect to an all-atom knowledge-based scoring function, the remaining subset of 50 structures yielded restraints of higher accuracy. A second round of distance geometry using these restraints resulted in an average coordinate RMS error of 4.38 A.

Estrogenic and antiestrogenic effects of enclomiphene and zuclomiphene on gonadotropin secretion by ovine pituitary cells in culture.

17 beta-Estradiol (E2) alters gonadotropin secretions in ovine pituitary cell cultures by 1) augmenting the LH response to LHRH and 2) inhibiting the basal secretion of FSH. These responses were used to study the estrogenic and antiestrogenic effects of enclomiphene, zuclomiphene, and their commercial mixture, Clomid. In terms of the LH response to LHRH, Clomid and enclomiphene (10(-6) M) acted as E2 antagonists because they blocked the action of E2 (10(-10) M). By themselves they did not alter the LHRH response. Previous studies using rat pituitary cultures showed opposite results, since both enclomiphene and Clomid acted as estrogens in rat cultures. Apparently, species differences are involved. One conclusion from these data, therefore, is that it would be unwise to predict how Clomid acts in any species without direct experimentation. Zuclomiphene (10(-7)--10(-5) M) acted as a E2 agonist, in terms of the LH response to LHRH, because it sensitized cultures to LHRH. Surprisingly, all of the clomiphenes, including zuclomiphene, acted primarily as E2 antagonists when FSH secretion was observed. At a concentration of 10(-6) M, they blocked the inhibitory effects of E2 (10(-10 M) on FSH secretion, but enclomiphene and Clomid also exhibited minor estrogenic action when administered alone. The clear but unexpected role of zuclomiphene as both E2 agonist (as measured by LH secretion) and antagonist (as measured by FSH secretion) suggests that zuclomiphene may be an especially useful compound for dissociating the effects of E2 on FSH and LH in vivo, at least in sheep. Of more theoretical interest is the fact that zuclomiphene can affect two E2-responsive systems oppositely. Both systems appear to reside in the same subset of pituitary cells.

Antiestrogens and ovine gonadotrophs: antagonism of estrogen-induced changes in gonadotropin secretions.

17 beta-Estradiol (E2) was found to affect gonadotrophs in long term ovine pituitary cell culture in several ways which were easily measured. It inhibited basal FSH secretion and, concomitantly, stimulated LH secretion. High physiological levels of E2 (10(-10) M; 27 pg/ml) were able to elicit about 70% of the maximal responses, and the effects were developed fully between 6-24 h after the addition of E2. In culture, the antiestrogens (AEs) tamoxifen, nafoxidine, and CI-680 antagonized both the inhibitory and stimulatory effects of E2 (10(-10) M) on gonadotropin secretion in a dose-dependent manner, and 10(-6) M AE was fully effective. At concentrations of 10(-6) M or lower, the AEs showed no significant intrinsic estrogenicity. At higher concentrations, the AEs progressively altered gonadotropin secretion in an apparently nonspecific fashion. Preincubations of cultures with AEs (3.3 X 10(-6) M) antagonized E2 action (10(-10) M) for approximately 18-24 h after the removal of AEs. Tamoxifen appeared to be the most potent AE tested. In conclusion, long term ovine pituitary cell cultures have provided a useful in vitro system for testing the biological potencies of AE analogs. AEs appeared to act as pure estrogen antagonists in ovine gonadotrophs.

Secretion of ovine luteinizing hormone in vitro: differential positive control by 17 beta-estradiol and a preparation of porcine ovarian inhibin.

17 beta-Estradiol (E2) and an enriched preparation of porcine ovarian inhibin can positively regulate LH secretion in two different ways in ovine pituitary cell cultures. One major effect of these agents is to increase the sensitivity of cultures to low levels of LHRH or LHRH analogs. The second effect is to increase culture responsiveness to high levels of LHRH or its analogs. Prolonged treatment with 1 nM E2, for instance, increased pituitary sensitivity to D-Lys6-LHRH by 10-fold, since it lowered the ED50 of D-Lys6-LHRH from approximately 400 pM to about 40 pM; the maximum LH secretory response to D-Lys6-LHRH (1-100 nM) was not changed, however. In contrast, short term treatment with an E2-free preparation of porcine ovarian inhibin could increase, by 350%, normal LH responsiveness to high levels of D-Lys6-LHRH (1-100 nM), but had no effect on sensitivity; the ED50 remained unchanged. Chronic treatment with inhibin eventually increased sensitivity, and interestingly, a short treatment with E2 (6 h) caused a major increase in both sensitivity and responsiveness, but the responsiveness component usually disappeared between 6 and 48 h of chronic E2 treatment. Treatment with both E2 and the inhibin preparation caused the greatest increase in pituitary sensitivity to D-Lys6-LHRH (ED50 decreased to approximately 30 pM), but the increased responsiveness to D-Lys6-LHRH (1-100 nM) declined with time as with E2-only treatment. The different actions of E2 and the ovarian inhibin preparation seen to perturb different aspects of LH secretion. Therefore, study of these phenomena may uncover novel molecular details governing LHRH-stimulated LH release. Furthermore, these two types of LH secretory control may be physiologically important, at least in sheep, and perhaps in other animals as well.