Search

Triosephosphate isomerase (TIM) is an enzyme of the glycolysis pathway which exists in almost all types of cells. Its structure is the prototype of a motif called TIM-barrel or (α/β) 8 barrel, which is the most common fold of all known enzyme structures. The simplest form in which TIM is catalytically active is a homodimer, in many species of bacteria and eukaryotes, or a homotetramer in some archaea. Here we show that the purified homodimeric TIMs from nine different species of eukaryotes and one of an extremophile bacterium spontaneously form higher order aggregates that can range from 3 to 21 dimers per macromolecular complex. We analysed these aggregates with clear native electrophoresis with normal and inverse polarity, blue native polyacrylamide gel electrophoresis, liquid chromatography, dynamic light scattering, thermal shift assay and transmission electron and fluorescence microscopies, we also performed bioinformatic analysis of the sequences of all enzymes to identify and predict regions that are prone to aggregation. Additionally, the capacity of TIM from Trypanosoma brucei to form fibrillar aggregates was characterized. Our results indicate that all the TIMs we studied are capable of forming oligomers of different sizes. This is significant because aggregation of TIM may be important in some of its non-catalytic moonlighting functions, like being a potent food allergen, or in its role associated with Alzheimer's disease.

Protein structure determination by X-ray crystallography guides structure-function and rational drug design studies. Helminths cause devastating diseases, including schistosomiasis that affects over one-third of the human population. Trematodes from the genus Schistosoma heavily depend on glycolysis; thus enzymes involved in this metabolic pathway are potential drug targets. Here we present a protocol to obtain crystal structures of recombinantly expressed triosephosphate isomerase from S. mansoni (SmTPI) that diffracted in house to a resolution of 2 Å.

Triosephosphate isomerase (TPI), the glycolytic enzyme that catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), has been frequently identified as a target of S-nitrosylation by proteomic studies. However, the effect of S-nitrosylation on its activity has only been explored in plants and algae. Here, we describe the in vitro S-nitrosylation of human TPI (hTPI), and the effect of the modification on its enzymatic parameters. NO-incorporation into the enzyme cysteine residues occurred by a time-dependent S-transnitrosylation from both, S-nitrosocysteine (CySNO) and S-nitrosoglutathione (GSNO), with CySNO being the more efficient NO-donor. Both X-ray crystal structure and mass spectrometry analyses showed that only Cys217 was S-nitrosylated. hTPI S-nitrosylation produced a 30% inhibition of the Vmax of the DHAP conversion to G3P, without affecting the Km for DHAP. This is the first study describing features of human TPI S-nitrosylation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Triose phosphate isomerase (TIM) is a cytoplasmic enzyme of prime importance in the mammalian glycolytic pathway. It has a major role in the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We have successfully purified a stable complex of TIM with β-globin subunit from the sheep kidney using a simple two-step chromatography procedure. It is seen for the first time that TIM is forming a stable complex with β-globin. The purified protein-protein complex was crystallized and preliminary diffraction data were collected at 2.1Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-β-globin complex by monitoring changes in the mean residue ellipticity at 222nm ([θ] 222 ) and difference absorption coefficient at 406nm (Δε 406 ) at pH 7.5 and 25°C. We have observed that GdmCl-induced denaturation is reversible. Coincidence of normalized transition curves of both physical properties ([θ] 222 and Δε 406 ) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ] 222 and Δε 406 were used to estimate the stability parameters of the protein-protein complex. This is the first report on the isolation, purification, crystallization and biophysical characterization of the naturally occurring complex of TIM with the β-globin subunit., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background & Objectives: Diarrhoea is the main clinical manifestation caused by intestinal parasitic infections in patients, with special reference to transplant recipients who require careful consideration to reduce morbidity and mortality. Further, molecular characterization of some important parasites is necessary to delineate the different modes of transmission to consider appropriate management strategies. We undertook this study to investigate the intestinal parasitic infections in transplant recipients with or without diarrhoea, and the genotypes of the isolated parasites were also determined., Methods: Stool samples from 38 transplant recipients comprising 29 post-renal, two liver and seven bone marrow transplant (BMT) recipients presenting with diarrhoea and 50 transplant recipients (42 post-renal transplant, eight BMT) without diarrhoea were examined for the presence of intestinal parasites by light microscopy using wet mount, modified Ziehl-Neelsen staining for intestinal coccidia and modified trichrome staining for microsporidia. Genotypes of Cryptosporidium species were determined by multilocus genotyping using small subunit ribosomal (SSUrRNA), Cryptosporidium oocyst wall protein (COWP) and dihydrofolate reductase (DHFR) as the target genes. Assemblage study for Giardia lamblia was performed using triose phosphate isomerase (TPI) as the target gene. Samples were also screened for bacterial, fungal and viral pathogens., Results: The parasites that were detected included Cryptosporidium species (21%, 8/38), Cystoisospora (Isospora) belli (8%, 3), Cyclospora cayetanensis (5%, 2), G. lamblia (11%, 4), Hymenolepis nana (11%, 4), Strongyloides stercoralis (3%, 1) and Blastocystis hominis (3%, 1). Multilocus genotyping of Cryptosporidium species at SSUrRNA, COWP and DHFR loci could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. All the C. hominis isolates were detected in adult post-renal transplant (PRT) recipients, whereas the C. parvum isolates included a child with BMT and an adult with PRT. Clostridium difficle, cytomegalovirus and Candida albicans were found in 2, 3 and 2 patients, respectively., Interpretation & Conclusions: In the present study, C. hominis was observed as an important parasite causing intestinal infections in transplant recipients. Multilocus genotyping of Cryptosporidium species could detect four isolates of C. hominis; two of C. parvum, one of mixed genotype and one could not be genotyped. Genotyping of G. lamblia revealed that assemblage B was most common., Competing Interests: None.

Triosephosphate isomerase (Tpi) is a glycolytic enzyme that is essential for efficient energy production in many pathogens. However, its function in Mycoplasma gallisepticum has not been fully elucidated. In this study, the mga0357 gene of M. gallisepticum, which encodes TpiA (MGTpiA), was amplified and expressed in Escherichia coli by IPTG induction. The purified recombinant MGTpiA protein exhibited catalytic activity that was similar to TPI from rabbit muscle, reducing NAD(+) to NADH. The MGTpiA was also found to be a surface-exposed protein by western blotting and immunofluorescence assays. In addition, cytadherence inhibition assays confirmed that the cytadherence of M. gallisepticum to the DF-1 cells was significantly inhibited by the anti-MGTpiA serum. The results of the study suggested that MGTpiA plays an important role in the metabolism and closely related to the M. gallisepticum pathogenicity., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)