An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

<p>Abstract</p> <p>Background</p> <p>The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of <it>B. stearothermophilus </it>GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step.</p> <p>Results</p> <p>We determined the crystal structure of <it>Cryptosporidium parvum </it>GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The <it>C. parvum </it>GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2Å resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits.</p> <p>Conclusion</p> <p>The structures of the <it>C. parvum </it>GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.</p

Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells

Helicobacter pylori Perturbs Iron Trafficking in the Epithelium to Grow on the Cell Surface

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche

Lipophilic Antifolate Trimetrexate Is a Potent Inhibitor of Trypanosoma cruzi: Prospect for Chemotherapy of Chagas' Disease

Trypanosoma cruzi, a protozoan parasite, is the causative agent for Chagas' disease, which poses serious public health problem in Latin America. The two drugs available for the treatment of this disease are effective only against recent infections and are toxic. Dihydrofolate reductase (DHFR) has a proven track record as a drug target. The lipophilic antifolate trimetrexate (TMQ), which is an FDA-approved drug for the treatment of Pneumocystis carinii infection in AIDS patients, is a potent inhibitor of T. cruzi DHFR activity, with an inhibitory constant of 6.6 nM. The compound is also highly effective in killing T. cruzi parasites. The 50 and 90% lethal dose values against the trypomastigote are 19 and 36 nM, and the corresponding values for the amastigote form are 26 and 72 nM, respectively. However, as TMQ is also a good inhibitor of human DHFR, further improvement of the selectivity of this drug would be preferable. Identification of a novel antifolate selective against T. cruzi would open up new therapeutic avenues for treatment of Chagas' disease

A tots ens porta consol: Carner, Ferrater

Conferència a càrrec de Josep M. Fonolleras, escriptor, que des de la perspectiva del lector es refereix a la lectura que en Joan i Gabriel Ferrater fan d'en Josep Carne

Neural network decision support system about lending

Обсуждаются различные подходы к анализу информации о клиентах банка с целью принятия решения о выдаче кредита. Исследованы различные способы моделирования данной задачи, оценена их точность и время получения результатов. The possibilities of various approaches to analyzing information about bank customers with the aim of making a decision on granting a loan are discussed. Various methods of modeling this problem are investigated; their accuracy and time of obtaining results are estimated

Comparison of CpPyK with LmPyK.

<p>(A) Superposition of A monomers of CpPyK (red), <i>L. mexicana</i> PyK without sulfate ion in the active site (1PKL, green), and <i>L. mexicana</i> PyK with sulfate ion in the active site (3E0V, blue). Sulfate ions bound at the effector binding site (labeled E) in all three structures are shown as stick models: CpPyK (yellow and red), 1PKL (green) and 3E0V (blue). Sulfate ions in the active site area (labeled A) in 3E0V are also shown in blue. One additional sulfate in the CpPyK structure at the interface of the C and A domains is shown in magenta. The glycerol molecule in the active site of CpPyK is shown as a ball and stick model. The two areas of the protein structures affected by the insertions in <i>L. mexicana</i> and <i>C. parvum</i> sequences are labeled. (B) Unwinding of helix α6’ in both monomers of CpPyK (red and yellow) as compared to 3E0V (cyan) and 1PKL (green). Residues 293 and 302 for CpPyK are labeled.</p

Crystal Structure of <em>Cryptosporidium parvum</em> Pyruvate Kinase

<div><p>Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of <em>Cryptosporidium parvum</em> is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from <em>C. parvum</em> has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in <em>C. parvum</em> pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the <em>C. parvum</em> pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to <em>C. parvum</em>, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.</p> </div

Cartoon drawing of monomers A (light pink) and B (light cyan) showing the orientation of the Arg342 side chain from monomer. A.

<p>Unwinding of the α6’ helix of monomer B results in the movement of the main chain carbonyl group of Gly295 too far away for interaction with Arg342 of monomer A. Arg294, Thr328 and Gln329 of the B monomer are also shown.</p