Modeling of Supramolecular Properties of Molecular Tweezers, Clips, and Bowls

The electrostatic potential surface (EPS) is calculated for molecular tweezers, clips, and bowls at different levels of theory (semiempirical AM1, ab initio HF/6-31G*, and density functional theory pBP/DN**). According to these calculations, the molecular electrostatic potential (MEP) on the concave side of the molecular tweezers and clips is suprisingly negative for hydrocarbons. This finding seems to be a general phenomenon in nonconjugated ?-electron systems with concave-convex topology and it explains the receptor properties of the molecular tweezers and clips. Analogous calculations performed for the conjugated aromatic molecular bowls show different results. The DFT calculations predict that in these systems the more negative MEP lies on the concave side similar to the findings for the nonconjugated molecular tweezer- and clip-systems, whereas the AM1 calculation leads to the opposite result that the MEP is more negative on convex side of the bowl-systems.     

Modulation of the transport of a lysosomal enzyme by PDGF

The major excreted protein (MEP) of transformed mouse fibroblasts is the lysosomal protease, cathepsin L. MEP is also secreted by untransformed mouse cells in response to growth factors and tumor promoters, and is thought to play a role in cell growth and transformation. To determine the relationship between MEP synthesis and MEP secretion, we have examined these events in PDGF-treated NIH 3T3 cells. PDGF enhanced MEP synthesis and caused the diversion of MEP from the lysosomal delivery pathway to a secretory pathway. These two effects were found to be regulated independently at various times after growth factor addition. Short PDGF treatments (0.5 or 1 h) resulted in quantitative secretion of MEP although synthesis was near the control level. High levels of both synthesis and secretion occurred between 2 and 14 h of PDGF treatment. Between 18 and 30 h, the amount of secreted MEP returned to the low control level even though synthesis remained elevated. The secretion was specific for MEP; other lysosomal enzymes were not found in the media from PDGF-treated cells. PDGF-induced secretion of MEP was inhibited 84% by cycloheximide, suggesting that protein synthesis is required to elicit this effect. PDGF also caused a time-dependent increase in mannose 6-phosphate (Man-6-P) receptor-mediated endocytosis. These data support a model in which PDGF alters the distribution of Man-6-P receptors such that the Golgi concentration of receptors becomes limiting, thereby causing the selective secretion of the low affinity ligand, MEP.     

GAME: A computer graphics method for calculating and displaying the molecular electrostatic potential

A new method for calculating and displaying the molecular electrostatic potential on the molecular surface is described. The main advantage of the method, besides some others, is its consistency. This means one only needs one data set for the surface and the MEP: a 3D field of the electron density from any source.     

Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated

The major excreted protein (MEP) of transformed mouse fibroblasts is a mannose 6-phosphate-containing glycoprotein whose synthesis and secretion are increased in malignantly transformed 3T3 cells and whose synthesis is increased by treatment of 3T3 cells with tumor promoters or growth factors. When pulse-labeled extracts from Kirsten virus-transformed NIH 3T3 (KNIH) cells were immunoprecipitated using an antibody against secreted MEP, one cellular protein was immunoprecipitated that had the same molecular weight and tryptic peptide map as the secreted protein. Pulse-chase labeling experiments showed that 50-60% of this 39,000-mol-wt form was secreted in transformed cells. Of the 40-50% remaining, approximately 5% was processed into two lower molecular weight forms (29,000 and 20,000) which are sequestered within the cell. Similar processing of these proteins was observed in the nontransformed parent NIH 3T3 (NIH) cells. However, in NIH cells, much less of the synthesized MEP was secreted. Measurements of steady-state levels of these three forms of cellular MEP by Western blot immunolocalization revealed approximately fourfold more MEP in KNIH cells than in NIH cells as well as differences in the relative distribution of MEP forms in transformed and nontransformed cells. Subcellular fractionation of KNIH cells on a Percoll gradient demonstrated a distribution of total MEP similar to that of several lysosomal enzymes. The light lysosomal/Golgi peak from these gradients contained both the precursor 39,000-mol-wt form of MEP and the 20,000-mol-wt form, whereas the heavy lysosomal peak was enriched in the 20,000-mol-wt form. The distribution of MEP forms was found to be similar in NIH cells except that the 29,000-mol-wt form was also seen to be enriched in the heavy lysosomal peak. This biochemical localization of MEP was confirmed by immunolocalization with light and electron microscopy. These data support the hypothesis that MEP is a lysosomal protein that is secreted by transformed cells.     

The computer program MolPot: a useful tool in chemical reactivity analysis

MolPot is a fast program for producing reactivity diagrams of molecular compounds, particularly for drugs. These diagrams are characteristic of the molecule envelope and the Molecular Electrostatic Potential (MEP) of the molecule surface (van der Waals surface or the portion of this surface that is solvent accessible). The input data includes only the atom names and their Cartesian coordinates.     

Design of novel ligands of CDP‐methylerythritol kinase by mimicking direct protein‐protein and solvent‐mediated interactions

The methylerythritol 4‐phosphate (MEP) pathway for the biosynthesis of the isoprenoid universal building blocks (isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) is present in most of human pathogens and is absent in animals, turning it into a promising therapeutic druggable pathway. Two different strategies, a pharmacophore‐directed virtual screening and a protein‐protein interaction (PPI)‐mimicking cyclic peptide were used to search for compounds that bind to the PPI surface of the 4‐(cytidine 5‐diphospho)‐2C‐methyl‐D‐erythritol kinase (CMK), which catalyzes the fourth step of the MEP pathway. A significant part of the pharmacophore hypothesis used in this study was designed by mimicking water‐mediated PPI relevant in the CMK homodimer complex stabilization. After database search and with the aid of docking and molecular dynamics (MD) simulations, a 7H‐furo[3,2‐g]chromen‐7‐one derivative and a cyclic peptide were chosen as candidates to be ligands of CMK. Their binding affinities were measured using surface plasmon resonance (SPR) technology. Copyright © 2010 John Wiley & Sons, Ltd.     

Heparin modulates the secretion of a major excreted protein-like molecule by vascular smooth muscle cells

Previous work from our laboratory has shown that heparin specifically induces the release of a pair of proteins of approximately 35,000 and 37,000 Da into the culture medium of vascular smooth muscle cells (SMC). In this report, we demonstrate that the previously identified 37,000-Da smooth muscle protein is composed of two protein species with very similar molecular weights based on migration patterns in SDS-polyacrylamide gels. The larger molecular weight species in this doublet has a similar molecular weight and shares antigenic determinants with major excreted protein (MEP), a lysosomal proteinase previously shown to be secreted by normal and transformed fibroblasts and epidermal cells. Antisera to MEP precipitated the higher molecular weight band from the doublet; preimmune serum was not reactive with the smooth muscle protein. Exposure of smooth muscle cells to heparin resulted in decreased amounts of immunoprecipitable protein released into the medium. Thus, it now appears that three proteins in the 35,000-38,000 molecular weight range are modulated by heparin, and that the largest of the heparin-modulated vascular SMC proteins has a similar molecular weight and is immunologically related to MEP. The release of MEP-like protein from SMC is decreased by heparin, while the remaining two heparin-modulated proteins are increased in the presence of heparin.     

A geometry optimization and molecular electrostatic potential mapping study of structure-activity relationship for some anti-Alzheimer agents.

Molecular geometries of some substituted (pyrroloamino)pyridines which possess anti-Alzheimer activity were optimized and potential-derived CHelpG point charges were computed using ab initio SCF molecular orbital approach employing the 3-21G basis set. AM1 molecular orbital calculations were performed using these optimized geometries and thus optimized Hybridization. Displacement Charges (HDC) combined with L?wdin charges continuously distributed in three dimension were obtained. Molecular electrostatic potential (MEP) maps of the molecules were obtained in two ways: (i) using the HDC-based model with the help of which MEP minima near the molecules were located, and (ii) using the CHelpG point charges, MEP values on the van der Waals surfaces of the molecules were computed. The MEP maps computed using both the methods have negative MEP regions near the pyridine nitrogen atom which appears to be the main binding site of the molecules with the appropriate receptor. Both electrostatic interaction and lipophilic association between these molecules and the receptor appear to contribute to biological activity.     

A theoretical study of some new analogues of the anti-cancer drug camptothecin

The enzyme topoisomerase I (topo I), which is essential for cell replication, transiently causes a DNA single strand break and makes a complex with it. The anti-cancer agent camptothecin (CPT) binds to the topo I–DNA complex and stabilizes it, preventing resealing of the broken DNA strand and cell growth. Considering the structural factors of CPT that are believed to be involved in stabilizing the topo I–DNA complex via hydrogen bonding and stacking interactions, designs of two new analogues of CPT (topo I inhibitors) have been suggested. The molecular geometries of CPT, two of its analogues and certain other related molecules included in the study were fully optimized in both gas phase and aqueous media at the B3LYP/6-311++G(d,p) level of density functional theory. Solvation effects of aqueous media were treated using the polarizable continuum model (PCM). Net CHelpG charges and surface molecular electrostatic potentials (MEP) near the atomic sites of the molecules were studied. Structural analogy and surface MEP values suggests that the two new CPT analogues studied here would be potent topoisomerase I inhibitors. Figure Optimized structures of CPT and two of its new analogues, 10 and 11.     

Central nervous system antigen P84 can serve as a substrate for neurite outgrowth

Neurite outgrowth promoting properties of neural cell surface proteins can be assessed by immobilizing isolated membrane proteins on nitrocellulose-coated petri dishes. Using this method, we have identified a unique cell surface antigen, designated P84, as a new neural cell adhesion molecule. Immunoaffinity purified P84 contains three polypeptides with molecular weights of 167, 85, and 66 kDa. When spotted onto nitrocellulose-coated plates, P84 supports adhesion of mouse cerebellar neurons and neurite outgrowth. Glial cell attachment was also observed. Intact monoclonal antibodies directed against P84 inhibit adhesion and outgrowth on a P84 substrate. This antigen is found on the surfaces of neurons in cultures of cerebellar cells. It is also found on a subclass of unidentified flat cells. P84 is not found on oligodendrocytes or GFAP-positive astrocytes. As early as E9, P84 could be detected in the floor plate region of the spinal cord. This pattern persists throughout embryonic development. Postnatally, widespread expression of P84 is observed in a variety of CNS tissues.     

Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor.

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.     

Molecular cloning of rat NK target structure--the possibility of CD44 involvement in NK cell-mediated lysis

The nature of target molecules of natural killer (NK) cell-mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor-associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H-ras oncogene. 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell-mediated lysis against W31 and W14. Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44. Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti-rat CD44 mAb OX-50 recognize the same protein of W31 cell lysates with an 86 kDa molecular size. CD44 was suggested to be a target structure of NK cell-mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up-regulation of target cell susceptibility to NK cell-mediated lysis. Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.     

植物MEP途径的代谢调控机制

萜类代谢途径是植物中最重要的次生代谢途径之一,对其有效的调控决定着植物的生长发育、抗性及品质等各个方面。植物中类萜合成的前体物在质体中是由2-C-甲基-D-赤藓糖醇-4-磷酸(2-C-Methyl-D-Erythritol-4-Phosphate,MEP)途径合成的,MEP途径中的许多基因除了受到多基因编码和转录水平的调节外,还受到转录后调节机制的调节,而转录后调节是一种新发现的调节方式,其机制还不是很清楚。该文重点对近年来国内外有关植物MEP途径的多种调节方式,尤其是转录后调节的调节机制及其可能参与的信号分子方面的研究进展进行综述,为植物的MEP途径的代谢调控提供参考。     

Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus

Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.     

Biosynthesis of isoprenoids: characterization of a functionally active recombinant 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.     

Tumor promoters and Kirsten sarcoma virus increase synthesis of a secreted glycoprotein by regulating levels of translatable mRNA

We have studied the effect of tumor promoters and viral transformation on the synthesis and secretion of a glycoprotein of molecular weight 35,000 by mouse fibroblasts in culture. The secretion of this protein (known as MEP) in large amounts by transformed cells has been previously described (Gottesman, 1978). We show in this paper that NIH/3T3 cells either transformed by the Kirsten sarcoma virus or treated with biologically active phorbol esters synthesize and secrete increased amounts of MEP. This is due to increased levels of translatable RNA for MEP, since translation in a reticulocyte lysate of total RNA purified from virus-transformed cells or from normal cells treated for 6 hr with 4-o-tetradecanoyl phorbol-13-acetate (TPA) reveals a dramatic increase in the synthesis in vitro of MEP when compared to untreated cells. Our evidence indicates that a tumor virus and a tumor promoter both regulate expression of a transformation-related function at a pretranslational level.