Inhibition of parathyroid hormone responsiveness in clonal osteoblastic cells expressing a mutant form of 3',5'-cyclic adenosine monophosphate-dependent protein kinase

PTH activates multiple acute intracellular signals within responsive target cells, but the importance of cAMP vs. other second messenger signals in mediating different biological responses to PTH is not known. To address these questions, we developed a genetic approach to block activation of the cAMP-dependent protein kinase (PK-A) in PTH-responsive cell lines. Clonal rat osteosarcoma cells (UMR 106-01) were stably transfected with REV-I, a plasmid that directs synthesis of a mutant cAMP-resistant form of the type I regulatory subunit of PK-A. In the transfected bone cells, most of the catalytic subunits of PK-A were associated with the mutant regulatory subunit, and activation of PK-A by cAMP was correspondingly inhibited. We have characterized one such mutant (UMR 4-7) that expressed large amounts of mutant mRNA and exhibited inducible blockade of PK-A via the REV-1 metallothionein promoter. In the absence of metallothionein induction, these cells exhibited nearly normal PTH responsiveness, but after REV-1 induction by Zn2+, they were resistant to PTH-induced activation of PK-A and regulation of membrane phospholipid synthesis by both PTH and cAMP analogs. The mutant UMR 4-7 cell provides a model system in which the consequences of cAMP production by PTH or other agonists that activate adenylate cyclase in osteoblasts may be specifically inhibited by brief exposure to Zn2+. Such mutant cell lines will facilitate further investigation of the linkage between early signalling events and subsequent biological responses in the action of PTH and other agonists on target cells in bone.     

Allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2). Part 3: Identification and biological activity of indanone containing mGlu2 receptor potentiators

We have identified and synthesized a series of phenyl-tetrazolyl and 4-thiopyridyl indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation, as well as PK properties, led to the discovery of 28 (EC50=186 nM), which displayed activity in a rodent model for schizophrenia.     

Four human thiopurine s-methyltransferase alleles severely affect protein structure and dynamics

Thiopurine S-methyltransferase (TPMT) metabolizes cytotoxic thiopurine drugs used in the treatment of leukemia and inflammatory bowel disease. TPMT is a major pharmacogenomic target with 23 alleles identified to date. Several of these alleles cause rapid protein degradation and/or aggregation, making it extremely difficult to study the structural impact of the TPMT polymorphisms experimentally. We, therefore, have performed multiple molecular dynamics simulations of the four most common alleles [TPMT*2 (A80P), *3A (A154T/Y240C), *3B (A154T) and *3C (Y240C)] to investigate the molecular mechanism of TPMT inactivation at an atomic level. The A80P polymorphism in TPMT*2 disrupts helix α3 bordering the active site, which breaks several salt-bridge interactions and opens up a large cleft in the protein. The A154T polymorphism is located within the co-substrate binding site. The larger threonine alters the packing of substrate-binding residues (P68, L69, Y166), increasing the solvent exposure of the polymorphic site. This packing rearrangement may account for the complete lack of activity in the A154T mutant. The Y240C polymorphism is located in β-strand 9, distant from the active site. Side-chain contacts between residue 240 and helix α8 are lost in TPMT*3C. Residues 154 and 240 in TPMT*3A are connected through a hydrogen-bonding network. The dual polymorphisms result in a flattened, slightly distorted protein structure and an increase in the thiopurine-binding site solvent accessibility. The two variants that undergo the most rapid degradation in vivo, TPMT*2 and *3A, are also the most deformed in the simulations.     

Molecular dynamics simulations of small peptides: dependence on dielectric model and pH

There has been much interest recently in the structure of small peptides in solution. A recent study by Bradley and co-workers [(1989) in Techniques of Protein Chemistry, Hugli, T.E., Ed., Academic Press, Orlando, FL, pp. 531-546; (1990) Journal of Molecular Biology, 215, pp. 607-622] describes a 17-residue peptide that is stable as a monomeric helix in aqueous solution at low pH, as determined by two-dimensional nmr and CD spectroscopy. They also have determined the helix content of the peptide as a function of pH using CD. We performed molecular dynamics simulations, with an empirical force field, of this peptide at low pH, with three different dielectric models: a linear distance-dependent dielectric function (epsilon = R); a modified form [J. Ramstein and R. Lavery (1988) Proceedings of the National Academy of Science, USA, Vol. 85, pp. 7231-7235] of the sigmoidal distance-dependent dielectric function of Hingerty and co-workers [(1985) Biopolymers, Vol. 24, pp. 427-439]; and epsilon = 1 with the peptide immersed in a bath of water molecules. We found that simulations with the sigmoidal dielectric function and the model with explicit water molecules resulted in average distances for particular interactions that were consistent with the experimental nmr results, with the sigmoidal function best representing the data. However, these models exhibited very different helix-stabilizing interactions. We also performed simulations using the sigmoidal function at moderate and high pH to compare to experimental determinations of the pH dependence of helix content. Helix content did not decrease with increases in pH, as shown experimentally. We did, however, observe changes in a specific side chain-helix dipole interaction that was implicated in determining the pH-dependent behavior of this peptide. Overall, the sigmoidal dielectric function was a reasonable alternative to adding explicit water molecules. In comparing 100 ps molecular dynamics simulations, the sigmoidal function was much less computer intensive and sampled more of conformational space than the treatment using explicit water molecules. Sampling is especially important for this system since the peptide has been shown experimentally to populate both helical and nonhelical conformations.     

Metal interactions with beef heart mitochondrial ATPase

Atomic absorption and electron paramagnetic resonance spectroscopy were used to study the metal binding sites of beef heart mitochondrial ATPase (F1). Quantitative and qualitative properties of these sites are described. Two different separation techniques were able to distinguish two very tight sites from one tight (easily exchangeable) metal binding site on F1. Of these sites, two are specific for magnesium while one can be substituted with Mn2+, Co2+, or Zn2+. When MgAMP-PNP was incubated with F1, a fourth metal was bound to the enzyme. The carboxyl group modified by dicyclohexylcarbodiimide is shown not to be involved in binding of any of the tightly bound metals. Qualitative properties of the metal binding sites using the Mn2+-enzyme complex as a probe were ascertained using EPR at pH 6.8 and 8.0. CrATP and Mn2+ appear to bind to different metal sites on F1. The possible role of the metals in regulation of catalysis, and their relation to nucleotide binding is discussed.     

Mus Spretus Line-1 Sequences Detected in the Mus Musculus Inbred Strain C57bl/6j Using Line-1 DNA Probes

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.     

The early steps in the unfolding of azurin

High-temperature molecular dynamics simulations were used to gain insight into the early steps in the unfolding pathway of azurin, a blue copper protein with a beta-barrel structure formed by two sheets arranged in a Greek key folding topology. The results reveal that unfolding of the beta-barrel in azurin is associated with dislocation of its unique alpha-helix with respect to the protein scaffold. Exposure of the hydrophobic core to solvent precedes complete disruption of secondary and tertiary structure, and modifications in the region around the active site are directly connected with this event. Denaturation of the protein initiates from the sheet coordinating the copper ion, and the other sheet maintains its topology. Results derived from the simulation were compared with experimental data obtained with different techniques, showing excellent agreement and providing a framework to understand the process of disruption and formation of the beta-barrel in azurin.     

The structure of the major transition state for folding of an FF domain from experiment and simulation

We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Phi-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (PhiF=0.9-1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (PhiF=0-0.2). The hydrophobic core and other parts of the protein displayed intermediate Phi-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Phi-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation-condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Phi-values.     

Cadherin-mediated differential cell adhesion controls slow muscle cell migration in the developing zebrafish myotome

Slow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. Here we examine the cellular and molecular basis for slow-twitch muscle cell migration. We show that slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, which correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development.     

Side-chain dynamics are critical for water permeation through aquaporin-1

Molecular dynamics simulations of aquaporin-1 embedded in a solvated lipid bilayer were carried out to investigate the mechanism of water permeation. The 2.2 Å resolution crystal structure of the bovine protein was used for five independent trajectories. During the equilibration and preparatory steps in which the protein was held fixed, water molecules inside the water channel adopted the same positions as observed in the crystal structure but they did not pass through the channel, suggesting that the dynamic motion of the protein is critical for water permeation. When the protein atoms were allowed to move, the side chains of the two asparagines in the two conserved Asn-Pro-Ala motifs near the center of the channel formed hydrogen bonds with water and helped water molecules move through the channel by actively aligning them for transport. The main-chain oxygen atoms, which were exposed to the pore surface in the crystal structure, also contributed to water transfer. Besides the constriction region observed in the crystal structure (Arg¹⁹⁷, Phe⁵⁸, His¹⁸², and Cys¹⁹¹), we found that His⁷⁶ and Val¹⁵⁵ act as a valve by dynamically blocking water permeation and helping control flow.