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1.
Physical interactions between d-phosphoglycerate dehydrogenase (EhPGDH) and phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica was observed by pull-down assay, gel filtration chromatography, chemical cross-linking, emission anisotropy, molecular docking and molecular dynamic simulations. The protein–protein complex had a 1:1 stochiometry with a dissociation constant of 3.453 × 10−7 M. Ionic interactions play a significant role in complex formation and stability. Analysis of the energy minimized average simulated model of the protein complex show that the nucleotide binding domain of EhPGDH specifically interacts with EhPSAT. Denaturation studies suggest that the nucleotide binding domain (Nbd) and substrate binding domain (Sbd) of EhPGDH are independent folding/unfolding units. Thus the Nbd-EhPGDH was separately cloned over-expressed and purified to homogeneity. Fluorescence anisotropy study show that the purified Nbd interacts with EhPSAT. Forward enzyme catalyzed reaction for the EhPGDH-PSAT complex showed efficient Km values for 3-phosphoglyceric acid as compared to only EhPGDH suggesting a possibility of substrate channelling in the protein complex. 相似文献
2.
We studied the role of protein kinase C (PKC) and protein threonine phosphorylation in the inhibition and stimulation of growth of the protozoan parasite Entamoeba histolytica. PKC was activated after serum deprivation in E. histolytica and during this period proteins became threonine phosphorylated. Conversely, on serum stimulation of serum-deprived cells, PKC activation was rapidly reversed and the threonine phosphorylation of proteins quickly declined. Growth of E. histolytica was not affected by either PKC inhibitors H-7 and GF109203X or by down-regulation of PKC by Phorbol 12-Myristate 13-Acetate (PMA). Interestingly, very low doses of PMA which caused activation of PKC and were unable to down-regulate PKC after 48 h of culture, negatively influenced the growth of E. histolytica. Serine/threonine phosphatase inhibitors Okadaic acid and Calyculin A drastically inhibited growth of E. histolytica. In conclusion, the growth of E. histolytica is not adversely affected by PKC down-regulation. On the contrary, growth inhibition of E. histolytica is associated with activation of Ca(2+), Diacylglycerol (DAG)-dependent PKC, and threo nine phosphorylation of proteins. 相似文献
3.
Majumder S Schmidt G Lohia A Aktories K 《Applied and environmental microbiology》2006,72(12):7842-7848
Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[14C]glucose. 相似文献
4.
Somlata Kamanna S Agrahari M Babuta M Bhattacharya S Bhattacharya A 《The Journal of biological chemistry》2012,287(14):10844-10852
The protozoan parasite Entamoeba histolytica can invade both intestinal and extra intestinal tissues resulting in amoebiasis. During the process of invasion E. histolytica ingests red blood and host cells using phagocytic processes. Though phagocytosis is considered to be a key virulence determinant, the mechanism is not very well understood in E. histolytica. We have recently demonstrated that a novel C2 domain-containing protein kinase, EhC2PK is involved in the initiation of erythrophagocytosis. Because cells overexpressing the kinase-dead mutant of EhC2PK displayed a reduction in erythrophagocytosis, it appears that kinase activity is necessary for initiation. Biochemical analysis showed that EhC2PK is an unusual Mn(2+)-dependent serine kinase. It has a trans-autophosphorylated site at Ser(428) as revealed by mass spectrometric and biochemical analysis. The autophosphorylation defective mutants (S428A, KDΔC) showed a reduction in auto and substrate phosphorylation. Time kinetics of in vitro kinase activity suggested two phases, an initial short slow phase followed by a rapid phase for wild type protein, whereas mutations in the autophosphorylation sites that cause defect (S428A) or conferred phosphomimetic property (S428E) displayed no distinct phases, suggesting that autophosphorylation may be controlling kinase activity through an autocatalytic mechanism. A reduction and delay in erythrophagocytosis was observed in E. histolytica cells overexpressing S428A and KDΔC proteins. These results indicate that enrichment of EhC2PK at the site of phagocytosis enhances the rate of trans-autophosphorylation, thereby increasing kinase activity and regulating the initiation of erythrophagocytosis in E. histolytica. 相似文献
5.
Genes encoding alpha- and beta-subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized. Deduced amino acid sequences of the alpha- and beta-subunit of E. histolytica FT (EhFT) were 298- and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively. They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms. Recombinant alpha- and beta-subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human H-Ras possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL. Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT. This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine. EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT. These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis. 相似文献
6.
Jang YS Song KJ Kim JY Lee YA Kim KA Lee SK Shin MH 《The Korean journal of parasitology》2011,49(2):177-180
Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and μ-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and μ-calpain may be involved in colon epithelial cell death induced by E. histolytica. 相似文献
7.
Phagocytosis is the important virulent determinant of the enteric protozoan parasite Entamoeba histolytica. We compared the kinetics of phagosome maturation of attenuated and highly-virulent strains of E. histolytica using video microscopy. Phagosomes of attenuated strains were acidified rapidly within 2 min after phagosome formation (at the rate of 0.96 pH/min), persisted at pH 4.46+/-0.13, and degraded ingested GFP-Leishmania very efficiently (90-94% GFP fluorescence was lost in 30 min), while phagosomes of highly-virulent strains were acidified slowly (0.69 pH/min), persisted at 5.11+/-0.23, and degraded GFP less efficiently (60-71% decrease). These results suggest that efficiency of phagosome maturation is most probably inversely correlated with apparent virulence. 相似文献
8.
Stanley SL Reed SL 《American journal of physiology. Gastrointestinal and liver physiology》2001,280(6):G1049-G1054
The protozoan intestinal parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. E. histolytica causes two major clinical syndromes, amebic colitis and amebic liver abscess. Recent advances in the development of in vitro and in vivo models of disease, new genetic approaches, the identification of key E. histolytica virulence factors, and the recognition of crucial elements of the host response to infection have led to significant insights into the pathogenesis of amebic infection. E. histolytica virulence factors include 1) a surface galactose binding lectin that mediates E. histolytica binding to host cells and may contribute to amebic resistance to complement, 2) amebapores, small peptides capable of lysing cells, which may play a role in killing intestinal epithelial cells, hepatocytes, and host defense cells, and 3) a family of secreted cysteine proteinases that play a key role in E. histolytica tissue invasion, evasion of host defenses, and parasite induction of gut inflammation. Amebae can both lyse host cells and induce their suicide through programmed cell death. The host response is also an important factor in the outcome of infection, and neutrophils may play a key role in contributing to the tissue damage seen in amebiasis and in controlling amebic infection. 相似文献
9.
The protozoan parasite Entamoeba histolytica is the etiologic agent of amebiasis, a major global public health problem, particularly in developing countries. There is an effective anti-amoebic drug available, however its long term use produces undesirable side effects. As E. histolytica is a micro-aerophilic organism, it is sensitive to high levels of oxygen and the enzymes that are involved in protecting against oxygen-stress are crucial for its survival. Therefore serine acetyltransferase, an enzyme involved in cysteine biosynthesis, was used as a target for identifying potential inhibitors. Virtual screening with Escherichia coli serine acetyltransferase was carried out against the National Cancer Institute chemical database utilizing molecular docking tools such as GOLD and FlexX. The initial analysis yielded 11 molecules of which three compounds were procured and tested for biological activity. The results showed that these compounds partially block activity of the E. coli enzyme and the growth of E. histolytica trophozoites but not mammalian cells. 相似文献
10.
J R Okita S J Robertson R T Okita 《Prostaglandins, leukotrienes, and essential fatty acids》1992,46(2):93-98
The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
An active conformation of phosphoglycerate dehydrogenase (PGDH) from Escherichia coli has been obtained using X-ray crystallography. The X-ray crystal structure is used to examine the potential intermediates for V(max) regulation, for the redox reaction, and for cooperative effects of serine binding. The crystal structure at 2.2 A resolution contains bound NAD(+) cofactor, either sulfate or phosphate anions, and alpha-ketoglutarate, a nonphysiological substrate. A PGDH subunit is formed from three distinct domains: regulatory (RBD), substrate (SBD), and nucleotide binding (NBD). The crystal conformation of the homotetramer points to the fact that, in the absence of serine, coordinated movement of the RBD-SBD domains occurs relative to the NBD. The result is a conformational change involving the steric relationships of both the domains and the subunits. Within the active site of each subunit is a bound molecule of alpha-ketoglutarate and the coenzyme, NAD. The catalytic or active site cleft is changed slightly although it is still solvent exposed; therefore, the catalytic reaction probably involves additional conformational changes. By comparing the inhibited with the uninhibited complex, it is possible to describe changes in conformation that are involved in the inhibitory signal transduction of serine. 相似文献
12.
Variyam EP 《Trends in parasitology》2007,23(3):108-111
Most humans infected with the virulent protozoan parasite Entamoeba histolytica do not develop invasive disease. Available evidence indicates that beneficial bacteria and the mucus gel layer in the colon lumen protect the host mucosa. Glycosidases produced by some normal colonic bacteria and luminal proteases degrade the key adherence lectin on E. histolytica trophozoites and decrease their adherence to epithelial cells. The mucus gel layer prevents those trophozoites that escape the hydrolases from reaching the epithelial cells. Trophozoite mucosal invasion is triggered only when both protective mechanisms are lost, as might occur during an unrelated pathogenic enteric bacterial infection. A newly developed gnotobiotic model of intestinal amebiasis should enable testing of this hypothesis and provide clues to help design practical studies in humans. 相似文献
13.
We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ~5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica. 相似文献
14.
Stanley SL 《Trends in parasitology》2001,17(6):280-285
Few organisms are more aptly named than Entamoeba histolytica, an intestinal protozoan parasite that can lyse and destroy human tissue. Within the past four years, new models of E. histolytica infection have begun to illuminate how amoebic trophozoites cause intestinal disease and liver abscess, and have expanded our understanding of the remarkable killing ability of this parasite. Here, I summarize recent work on the interactions between E. histolytica and human intestine, and between E. histolytica and hepatocytes, and discuss what these studies tell us about the role of inflammation and programmed cell death in the pathogenesis of amoebiasis. 相似文献
15.
Entamoeba histolytica is a protozoan parasite that can invade the intestinal mucosa. Infection induces production of secretory immunoglobulin A (SIgA) antibodies that can diminish the adhesion between E. histolytica trophozoites and epithelial cells in vitro and reduce the rate of new infections in children. SIgA antibodies produced by asymptomatic cyst carriers could play a protective role against the damage caused by E. histolytica. To identify membrane antigens capable of inducing SIgA response in E. histolytica cyst carriers, salivary SIgA antibodies were confronted with blotted plasma membrane proteins from amebae. A surface 115-kDa ameba protein was recognized by 62% of the human SIgA antibodies tested. The 115-kDa protein is not a mannose-containing glycoprotein and has no protease activity. Rabbit anti-115-kDa protein antibodies were capable of reducing erythrophagocytosis but were unable to protect culture cells from the cytopathic damage caused by E. histolytica. However, anti-115-kDa protein antibodies induced surface receptor redistribution. 相似文献
16.
Ali V Shigeta Y Tokumoto U Takahashi Y Nozaki T 《The Journal of biological chemistry》2004,279(16):16863-16874
We have characterized the iron-sulfur (Fe-S) cluster formation in an anaerobic amitochondrial protozoan parasite, Entamoeba histolytica, in which Fe-S proteins play an important role in energy metabolism and electron transfer. A genomewide search showed that E. histolytica apparently possesses a simplified and non-redundant NIF (nitrogen fixation)-like system for the Fe-S cluster formation, composed of only a catalytic component, NifS, and a scaffold component, NifU. Amino acid alignment and phylogenetic analyses revealed that both amebic NifS and NifU (EhNifS and EhNifU, respectively) showed a close kinship to orthologs from epsilon-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of epsilon-proteobacteria to E. histolytica. The EhNifS protein expressed in E. coli was present as a homodimer, showing cysteine desulfurase activity with a very basic optimum pH compared with NifS from other organisms. Eh-NifU protein existed as a tetramer and contained one stable [2Fe-2S]2+ cluster per monomer, revealed by spectroscopic and iron analyses. Fractionation of the whole parasite lysate by anion exchange chromatography revealed three major cysteine desulfurase activities, one of which corresponded to the EhNifS protein, verified by immunoblot analysis using the specific EhNifS antibody; the other two peaks corresponded to methionine gamma-lyase and cysteine synthase. Finally, ectopic expression of the EhNifS and EhNifU genes successfully complemented, under anaerobic but not aerobic conditions, the growth defect of an Escherichia coli strain, in which both the isc and suf operons were deleted, suggesting that EhNifS and EhNifU are necessary and sufficient for Fe-S clusters of non-nitrogenase Fe-S proteins to form under anaerobic conditions. This is the first demonstration of the presence and biological significance of the NIF-like system in eukaryotes. 相似文献
17.
The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G 相似文献
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