Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions are influenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow) form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemical synthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acids and displaying up to approximately 2700-fold more potent antithrombin activity, comparable to that of full-length hirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe the structural and energetic properties of the S3 site of thrombin through perturbations introduced in the structure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacement of Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobic and steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge (p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity of fragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation, and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin. In this study, we have also identified the key features that are responsible for the preferential binding of hirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr --> Ala exchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier side chain (e.g., beta-naphthylalanine) improves binding preferentially to the fast form. These results provide strong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open and accessible conformation with respect to the less forgiving structure it acquires in the slow form. This view is also supported by the results of molecular dynamics simulations conducted for 18 ns on free thrombin in full explicit water, showing that after approximately 5 ns thrombin undergoes a significant conformational transition, from a more open conformation (which we propose can be related to the fast form) to a more compact and closed one (which we propose can be related to the slow form). This transition mainly involves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site, the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicate that the S3 site of the enzyme is less accessible to water in the putative slow form. This structural picture provides a reasonable molecular explanation for the fact that physiological substrates related to the procoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky side chain into the S3 site of the enzyme. Taken together, our results can have important implications for the design of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.     

NCS-1 inhibits insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes through a phosphatidylinositol 4-kinase-dependent pathway

Expression of NCS-1 (neuronal calcium sensor-1, also termed frequenin) in 3T3L1 adipocytes strongly inhibited insulin-stimulated translocation of GLUT4 and insulin-responsive aminopeptidase. The effect of NCS-1 was specific for GLUT4 and the insulin-responsive aminopeptidase translocation as there was no effect on the trafficking of the cation-independent mannose 6-phosphate receptor or the GLUT1 glucose transporter isoform. Moreover, NCS-1 showed partial colocalization with GLUT4-EGFP in the perinuclear region. The inhibitory action of NCS-1 was independent of calcium sequestration since neither treatment with ionomycin nor endothelin-1, both of which elevated the intracellular calcium concentration, restored insulin-stimulated GLUT4 translocation. Furthermore, NCS-1 did not alter the insulin-stimulated protein kinase B (PKB/Akt) phosphorylation or the recruitment of Cbl to the plasma membrane. In contrast, expression of the NCS-1 effector phosphatidylinositol 4-kinase (PI 4-kinase) inhibited insulin-stimulated GLUT4 translocation, whereas co-transfection with an inactive PI 4-kinase mutant prevented the NCS-1-induced inhibition. These data demonstrate that PI 4-kinase functions to negatively regulate GLUT4 translocation through its interaction with NCS-1.     

Timing the origin of New World monkeys

The origin of New World monkeys (Infraorder Platyrrhini) has been an extensively debated issue. In this study, we analyzed mitochondrial genomes from Cebus (Platyrrhini), Homo, Hylobates, Pan, Pongo (Hominoids), Macaca, Papio (Cercopithecoids), and Tarsius (outgroup) to investigate this matter. Two distinct methodologies were employed on mitochondrial genes to estimate divergence times: the traditional likelihood ratio test performed in ML analyses of individual and concatenated gene sequences and the recent multigene Bayesian approach. Using the Cercopithecoid-Hominoid split as calibration point (25 MYA), our results show consistently that Platyrrhines split from Catarrhines at around 35 MYA. Although the main focus of the study is New World monkey origins, we have also estimated other primate divergence times: Homo-Pan at 5-7 MYA; Pongo-(Homo/Pan) at 13-16 MYA; Hylobates-(Pongo/Homo/Pan) at 15-19 MYA; and Macaca-Papio at 10-12 MYA. Our estimate for the origin of New World monkeys is in agreement with the hypothesis of a transatlantic journey from Africa to South America, as suggested by the fossil record.     

The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with lipophilic sulfonamides

A series of new compounds was obtained by reaction of aromatic/heterocyclic sulfonamides incorporating amino groups with N,N-diphenylcarbamoyl chloride and diphenylacetyl chloride. These sulfonamides were assayed for the inhibition of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic CA I and CA II, and the transmembrane, cancer-associated isozyme CA IX. Good inhibitors against all these isoforms were detected, and the inhibition profile of the newly investigated isozyme IX was observed to be different from that of the cytosolic isozymes, I and II. This may lead to the development of novel anticancer therapies based on the selective inhibition of CA IX.     

Intrinsic Regulation of Brain Inflammatory Responses

It is now well accepted that inflammatory responses in brain contribute to the genesis and evolution of damage in neurological diseases, trauma, and infection. Inflammatory mediators including cytokines, cell adhesion molecules, and reactive oxygen species including NO are detected in human brain and its animal models, and interventions that reduce levels or expression of these agents provide therapeutic benefit in many cases. Although in some cases, the causes of central inflammatory responses are clear—for example those due to viral infection in AIDS dementia, or those due to the secretion of proinflammatory substances by activated lymphocytes in multiple sclerosis—in other conditions the factors that allow the initiation of brain inflammation are not well understood; nor is it well known why brain inflammatory activation is not as well restricted as it is in the periphery. The concept is emerging that perturbation of endogenous regulatory mechanisms could be an important factor for initiation, maintenance, and lack of resolution of brain inflammation. Conversely, activation of intrinsic regulatory neuronal pathways could provide protection in neuroinflammatory conditions. This concept is the extension of the principle of central neurogenic neuroprotection formulated by Donald Reis and colleagues, which contends the existence of neuronal circuits that protect the brain against the damage initiated by excitotoxic injury. In this paper we will review work initiated in the Reis laboratory establishing that activation of endogenous neural circuits can exert anti-inflammatory actions in brain, present data suggesting that these effects could be mediated by noradrenaline, and summarize recent studies suggesting that loss of noradrenergic locus ceruleus neurons contributes to inflammatory activation in Alzheimer's disease.     

Geographic and breed distribution of an Msp I PCR-RFLP in the bovine growth hormone (bGH) gene

Information is presented on the frequency of the Msp I (-) allele in the third intron of the bovine growth hormone gene in a large number of cattle breeds. Consideration of the breed frequencies in relation to their geographic origin shows a low frequency for breeds originating in Northern Europe, moderate frequencies for breeds originating in Eastern Europe or the countries surrounding the Mediterranean basin, and very high frequencies for breeds originating in the Indian subcontinent. Consideration of breed frequencies in relation to breed type, shows low to moderate frequencies for the humpless breeds, high frequencies for the humped breeds. Various explanations for this distribution are discussed, among them the possibility that the Msp I (-) allele originated in the Bos indicus breeds of the Indian subcontinent, from which it diffused through the humpless Bos taurus breeds of Eastern Europe, the Mediterranean basin, eventually reaching Western, Northern Europe, Western Africa in low frequencies.     

The mouse Kell blood group gene (Kel): cDNA sequence, genomic organization, expression, and enzymatic function

The human Kell blood group system is important in transfusion medicine, since Kell is a polymorphic protein and some of its antigens can cause severe reactions if mismatched blood is transfused, while maternal alloimmunization may lead to fetal and neonatal anemia. In humans, Kell is an Mr 93,000 type II membrane glycoprotein with endothelin-3-converting enzyme activity that is linked by a single disulfide bond to another protein, XK, that spans the membrane ten times. An absence of XK leads to clinical symptoms termed the McLeod syndrome. We determined the cDNA sequence of the mouse Kell homologue, the organization of the gene, expression of the protein and its enzymatic function on red cells. Comparison of human and mouse Kell cDNA showed 80% nucleotide and 74% amino acid sequence identity. Notable differences are that the mouse Kell protein has eight probable N-linked carbohydrate side chains, compared to five for human Kell, and that the mouse homologue has one more extracellular cysteine than human Kell protein. The mouse Kell gene (Kel), like its human counterpart, is similarly organized into 19 exons. Kel was located to proximal Chromosome 6. Northern blot analysis showed high expression in spleen and weaker levels in testis and heart. Western blot analysis of red cell membrane proteins demonstrated that mouse Kell glycoprotein has an apparent Mr of 110,000 and, on removal of N-linked sugars, 80,000. As in human red cells, Kell is disulfide-linked to XK and mouse red cells have endothelin-3-converting enzyme activity.     

A knowledge model for analysis and simulation of regulatory networks

MOTIVATION: In order to aid in hypothesis-driven experimental gene discovery, we are designing a computer application for the automatic retrieval of signal transduction data from electronic versions of scientific publications using natural language processing (NLP) techniques, as well as for visualizing and editing representations of regulatory systems. These systems describe both signal transduction and biochemical pathways within complex multicellular organisms, yeast, and bacteria. This computer application in turn requires the development of a domain-specific ontology, or knowledge model. RESULTS: We introduce an ontological model for the representation of biological knowledge related to regulatory networks in vertebrates. We outline a taxonomy of the concepts, define their 'whole-to-part' relationships, describe the properties of major concepts, and outline a set of the most important axioms. The ontology is partially realized in a computer system designed to aid researchers in biology and medicine in visualizing and editing a representation of a signal transduction system.