Mechanism of thrombin clearance by human astrocytoma cells

Astroglial cells secrete a variety of factors that contribute to the regulation of neurite initiation and continued outgrowth, among them proteases and protease inhibitors. An alteration in the balance between these proteins has been implicated in Alzheimer's disease, resulting in an accumulation of thrombin:protease nexin 1 (PN1) complexes in the brains of these patients. This report aims at providing a biochemical explanation for this phenomenon. We show that human astrocytoma cells bind and internalize thrombin and thrombin:PN1 complexes efficiently by a PN1-dependent mechanism. Binding was potently inhibited by soluble heparin and did not occur with the mutant PN1 (K7E) deficient in heparin binding. Receptor-associated protein, an antagonist of the low-density lipoprotein receptor-related protein (LRP), inhibited internalization of thrombin by the astrocytoma cells, but did not affect cell-surface binding. The results are consistent with a mechanism by which astrocytoma cells clear thrombin in a sequential manner: thrombin is first complexed with PN1, then bound to cell-surface heparins, and finally internalized by LRP. This mechanism provides a link between the neuronal growth regulators thrombin and PN1 and proteins genetically associated with Alzheimer's disease, such as LRP.     

Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein

The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.     

Importing statistical measures into Artemis enhances gene identification in the <Emphasis Type="Italic">Leishmania</Emphasis> genome project

Background

Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.

Results

Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.

Conclusion

An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

     

Electrostatic dependence of the thrombin-thrombomodulin interaction

The rate constants for the binding interaction between thrombin and a fully active fragment of its anticoagulant cofactor, thrombomodulin, have been determined by surface plasmon resonance. At physiological ionic strength, the k(a) was 6.7x10(6) M(-1) s(-1 )and the dissociation rate constant was 0.033 s(-1). These extremely fast association and dissociation rates resulted in an overall binding equilibrium constant of 4.9 nM, which is similar to previously reported values. Changing the ionic strength from 100 mM to 250 mM NaCl caused a tenfold decrease in the association rate while the dissociation rate did not change significantly. A similar effect was observed with tetramethylammonium chloride. A Debye-Hückel plot of the data had a slope of -6 and an intercept at 0 ionic strength of 10(9) M(-1) s(-1). The same slope and intercept were obtained for data that was collected in the presence of glycerol to slow the association rates. These results show that the thrombin-TM456 interaction is extremely rapid and nearly completely electrostatically steered. An association model is presented in which TM456 approaches thrombin along the direction of the thrombin molecular dipole.     

Disulfide bond plasticity in epidermal growth factor

Epidermal growth factor (EGF) has a (1-3,2-4,5-6) disulfide-bonding pattern. This pattern is found in nearly all EGF-like domains, despite wide variation in sequences. Biological data from EGF and at least one EGF-like domain show that disulfide bond isomers have significant bioactivity and suggests that the EGF fold can accommodate alternate disulfide-bonding patterns. The disulfide bonds in murine EGF were altered to seven different patterns and structures were calculated incorporating all the restraints from the highest resolution restraint set available (Tejero et al., 1996). Results showed that besides the native (1-3,2-4,5-6), two other disulfide-bonding patterns: (1-2,3-4,5-6) and (1-3,2-5,4-6) satisfied the restraints as well as the native. The results for these two patterns were indistinguishable from the native on the basis of distance and dihedral violations, XPLOR energies, Procheck statistics, and RMSDs of the final set of structures. Two other disulfide bond patterns, (1-2,3-5, 4-6) and (1-4,2-3,5-6) were able to satisfy all the distance restraints but had one or more cysteine dihedral violations. For all seven isomers, the final calculated structures were highly similar to EGF with all-atom RMSD's in the 1. 5-2 A range. These results suggest that the EGF backbone fold has the unique property of accommodating several different disulfide-bonding patterns.     

Induced fit, folding, and recognition of the NF-kappaB-nuclear localization signals by IkappaBalpha and IkappaBbeta

Protein structure prediction codes based on the associative memory Hamiltonian were used to probe the binding modes between the nuclear localization signal (NLS) polypeptide of NF-kappaB and the inhibitors IkappaBalpha and IkappaBbeta. Experimentally, it is known that the NLS polypeptide is unstructured in the NF-kappaB complex with DNA but it forms an extended helical structure with the NLS (residues 301-304) between the two helices in the NF-kappaB/IkappaBalpha complex. The simulations included the NF-kappaB(p65) and (p50) NLS polypeptides and various mutants alone and in the presence of IkappaBalpha and IkappaBbeta. The simulations predict that the NLS polypeptide by itself binds tightly to IkappaBalpha and IkappaBbeta. In the NF-kappaB (p50/p65) heterodimer, the p50 NLS is predicted to remain free to bind to importin alpha. In the interaction with IkappaBalpha, both p65 NLSs are predicted to be bound. In IkappaBbeta, the NLS polypeptide binds to two binding sites, as seen in the crystal structure, with one site heavily favored for stable binding.     

R-subunit isoform specificity in protein kinase A: distinct features of protein interfaces in PKA types I and II by amide H/2H exchange mass spectrometry

The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/²H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA.     

Mechanism of oxidation of pi bonds by cytochrome P-450. Electronic requirements of the transition state in the turnover of phenylacetylenes

Substituted phenylacetylenes are oxidized by purified reconstituted rat liver cytochrome P-450b to the corresponding 2-arylacetic acids. A linear free energy correlation with a rho value of -2.2 exists between the rates of formation of the arylacetic acids and the substituent sigma constants. The enzyme is simultaneously inactivated but the inactivation rates, in contrast to those of metabolite formation, are essentially identical for all the phenylacetylenes. Inactivation is thus insensitive to the electronic nature of the substituents. The metabolite: inactivation ratio consequently decreases from 38 for p-methylphenylacetylene to 4 for p-nitrophenylacetylene. Replacement of the terminal proton of phenylacetylene with deuterium gives rise to an isotope effect of 1.7 on metabolite formation without altering enzyme inactivation. The differential effect of substituents on heme alkylation and metabolite formation requires the two pathways to diverge early in the catalytic process and argues against mechanisms that involve radical cation intermediates.