Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.     

In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line

Abstract: Amyloid β-peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that Aβ25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25-35 were specific because Aβ1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1-40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25-35. Damage and death of ECs induced by Aβ25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.     

Changes associated with tyrosine phosphorylation during short-term hypoxia in retinal microvascular endothelial cells in vitro

The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKCα), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO₂ = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO₂ = 3%) in humidified, 5% CO₂, 37°C, tissue culture chambers, on a time-course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (M_r ～ 85 kDa). Protein tyrosine kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia-induced phosphotyrosine. The expression of HSP70, PKCα, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short-term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases. © 1995 Wiley-Liss, Inc.     

Post-ischemic administration of the acetylcholinesterase inhibitor ENA-713 prevents delayed neuronal death in the gerbil hippocampus

We examined by morphological methodology the effect of (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydrogentartrate (ENA-713), an acetylcholinesterase (AChE) inhibitor, on ischemia-induced neuronal death in the gerbil hippocampus due to a 5-min ligation of bilateral common carotid arteries after light ether anesthesia. Pyramidal cells had been decreased to 27% of sham-operated controls and the number of hypertrophic astrocytes expressing glial fibrillary acidic protein (GFAP) markedly increased in the hippocampal CA1 subfield 14 days after ischemia. However, post-ischemic administration of ENA-713 (three times 0.2 mg/kg, i.p.) significantly ameliorated this ischemia-induced decrease in the number of pyramidal cells by 47% of sham-operated controls, furthermore, it reduced the ischemia-induced accumulation of GFAP-positive astrocyte in the CA1 region. Together with previous results showing that ENA-713 protected against the ischemia-induced cholinergic abnormalities in the gerbil brain and improved cholinergic dysfunctions in the senescent rat brain, our present findings suggest that ENA-713 prove to be useful for treatment with senile dementia such as cerebrovascular dementia.     

Multiple modes of ligand recognition: Crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors,olomoucine and isopentenyladenine

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(δ2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties. © 1995 Wiley-Liss, Inc.     

Atomic and residue hydrophilicity in the context of folded protein structures

Water-protein interactions drive protein folding, stabilize the folded structure, and influence molecular recognition and catalysis. We analyzed the closest protein contacts of 10,837 water molecules in crystallographic structures to define a specific hydrophilicity scale reflecting specific rather than bulk solvent interactions. The tendencies of different atom and residue types to be the nearest protein neighbors of bound water molecules correlated with other hydrophobicity scales, verified the relevance of crystallographically determined water positions, and provided a direct experimental measure of water affinity in the context of the folded protein. This specific hydrophilicity was highly correlated with hydrogen-bonding capacity, and correlated better with experimental than computationally derived measures of partitioning between aqueous and organic phases. Atoms with related chemistry clustered with respect to the number of bound water molecules. Neutral and negatively charged oxygen atoms were the most hydrophilic, followed by positively-charged then neutral nitrogen atoms, followed by carbon and sulfur atoms. Agreement between observed side-chain specific hydrophilicity values and values derived from the atomic hydrophilicity scale showed that hydrophilicity values can be synthesized for different functional groups, such as unusual side or main chains, discontinuous epitopes, and drug molecules. Two methods of atomic hydrophilicity analysis provided a measure of complementarity in the interfaces of trypsin:pancreatic trypsin inhibitor and HIV protease:U-75875 inhibitor complexes. © 1995 Wiley-Liss, Inc.     

Protein conformational landscapes: Energy minimization and clustering of a long molecular dynamics trajectory

Using energy minimization and cluster analysis, we have analyzed a 1020 ps molecular dynamics trajectory of solvated bovine pancreatic trypsin inhibitor. Elucidation of conformational sub states in this way both illustrates the degree of conformational convergence in the simulation and reduces the structural data to a tractable subset. The relative movement of structures upon energy minimization was used to estimate the sizes of features on the protein potential energy surface. The structures were analyzed using their pairwise root-mean-square C_α deviations, which gave a global measure of conformational changes that would not be apparent by monitoring single degrees of freedom. At time scales of 0.1 ps, energy minimization detected sharp transitions between energy minima separated by 0.1 Å rms deviation. Larger conformational clusters containing these smaller minima and separated by 0.25 Å were seen at 1 ps time scales. Both of these small features of the conformational landscape were characterized by movements in loop regions associated with small, correlated backbone dihedral angle shifts. On a nanosecond time scale, the main features of the protein energy landscape were clusters separated by over 0.7 Å rms deviation, with only seven of these sub states visited over the 1 ns trajectory. These substates, discernible both before and after energy minimization, differ mainly in a monotonic pivot of the loop residues 11–18 over the course of the simulation. This loop contains lysine 17, which specifically binds to trypsin in the active site. The trajectory did not return to previously visited clusters, indicating that this trajectory has not been shown to have completely sampled the conformational substates available to it. Because the apparent convergence to a single region of conformation space depends on both the time scale of observation and the size of the conformational features examined, convergence must be operationally defined within the context of the simulation. © 1995 Wiley-Liss, Inc.     

Dynamic modelling of a helical peptide in solution using NMR data: Multiple conformations and multi-spin effects

Summary Nuclear Overhauser effect (NOE) measurements on molecules in solution provide information about only the ensemble-averaged properties of these molecules. An algorithm is presented that uses a list of NOEs to produce an ensemble of molecules that on average agrees with these NOEs, taking into account the effect of surrounding spins on the buildup of each NOE (spin diffusion). A simplified molecular dynamics simulation on several copies of the molecule in parallel is restrained by forces that are derived directly from differences between calculated and measured NOEs. The algorithm is tested on experimental NOE data of a helical peptide derived from bovine pancreatic trypsin inhibitor.     

Molecular dynamics simulations of phenylimidazole inhibitor complexes of cytochrome P450 cam

Molecular dynamics simulations have been performed on three phenylimidazole inhibitor complexes ofP450 _cam, utilizing the X-ray structures and the AMBER suite of programs. Compared to their corresponding optimized X-ray structures, very similar features were observed for the 1-phenylimidazole (1-PI) and 2-phenylimidazole (2-PI) complexes during a 100 ps MD simulation. The 1-PI inhibitor binds as a Type II complex with the imidazole nitrogen as a ligand of the heme iron. Analysis of the inhibitor-enzyme interctions during the MD simulations reveals that electrostatic interactions of the imidazole with the heme and van der Waals interactions of the phenyl ring with nearby hydrophobic residues are dominant. By contrast, 2-PI binds as a Type I inhibitor in the substrate binding pocket, but not as a ligand of the iron. The interactions of this inhibitor are qualitatively different from that of the Type II 1-PI, being mainly electrostatic/H-bonding interactions with a bound water and polar residues. Although the third compound, 4-PI, in common with 1-PI, also binds as a Type II inhibitor, with one nitrogen of the imidazole as a ligand to the iron, the MD average binding orientation deviates significantly from the X-ray structure. The most important changes observed include: (1) the rotation of the imidazole ring of this inhibitor by about 90° to enhance electrostatic interactions of the imidazole NH group with the carbonyl group of LEU244, and (2) the rotation of the carbonyl group of ASP251 to form a H-bond with VAL254. An analysis of the H-bonding network surrounding this substrate in the optimized crystal structure revealed that there is no H-bonding partner either for the free polar NH group in the imidazole ring of 4-phenylimidazole or for the polar carbonyl group of the nearby ASP251 residue. The deviation of the dynamically averaged inhibitor-enzyme structure of the 4-PI complex from the optimized crystal structure can therefore be rationalized as a consequence of the optimization of the electrostatic interactions among the polar groups.     

Crystal structure of human pepsin and its complex with pepstatin.

The three-dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X-ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 A resolution to an R-factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 A resolution to an R-factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position.