Analysis of the Paracoccidioides brasiliensis triosephosphate isomerase suggests the potential for adhesin function

Paracoccidioides brasiliensis is an important fungal pathogen. The disease it causes, paracoccidioidomycosis (PCM), ranges from localized pulmonary infection to systemic processes that endanger the life of the patient. Paracoccidioides brasiliensis adhesion to host tissues contributes to its virulence, but we know relatively little about molecules and the molecular mechanisms governing fungal adhesion to mammalian cells. Triosephosphate isomerase (TPI: EC 5.3.1.1) of P. brasiliensis (PbTPI) is a fungal antigen characterized by microsequencing of peptides. The protein, which is predominantly expressed in the yeast parasitic phase, localizes at the cell wall and in the cytoplasmic compartment. TPI and the respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis to in vitro cultured epithelial cells. TPI binds preferentially to laminin, as determined by peptide inhibition assays. Collectively, these results suggest that TPI is required for interactions between P. brasiliensis and extracellular matrix molecules such as laminin and that this interaction may play an important role in the fungal adherence and invasion of host cells.

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear (1)H NMR spectroscopy at 900 MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities. (c) 2005 Elsevier Ltd. All rights reserved.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA(2) homolog from Bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-1), a Lys49 phospholipase A(2) homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-1 disulfide bonds follow the normal pattern of group II PLA(2)s.

Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of tetramethylthiuram disulfide (thiram) for residual analysis in food using spectrophotometry coupled with a solid-phase reactor (SPR) in a flow system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys(P)) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the alpha-helix that contains Cys(P). Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8 angstrom resolution of Tsa1(C47S) in the decameric form [(alpha(2))(5)] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1(C47S) structure indicated that G1u50 and Arg146 participate in the stabilization of the Cys(P) alpha-helix. As a consequence, we raised the hypothesis that G1u50 and Arg146 might be relevant to the Cys(P) reactivity. Therefore, Tsa1(E50A) and Tsa1(R146Q) mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10(6) M-1 S-1. Remarkably, although Tsa1(E50A) and Tsa1(R146Q) were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that G1u50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx. (C) 2012 Elsevier Ltd. All rights reserved.

Electron Interactions with Disulfide Bridges

Because of its electronic properties, sulfur plays a major role in a variety of metabolic processes and, more in general, in the chemistry of life. In particular, S-S bridges between cysteines are present in the amino acid backbone of proteins. Protein disulfur radical anions may decay following different paths through competing intra and intermolecular routes, including bond cleavage, disproportionation, protein-protein cross linking, and electron transfer. Indeed, mass spectrometry ECD (electron capture dissociation massspectroscopy) studies have shown that capture of low-energy (<0.2 eV) electrons by multiply protonated proteins is followed by dissociation of S-S bonds holding two peptide chains together. In view of the importance of organic sulfur chemistry, we report on electron interactions with disulphide bridges. To study these interactions we used as prototypes the molecules dimethyl sulfide [(CH3)2S] and dimethyl disulfide [(H3C)S2(CH3)]. We seek to better understand the electron-induced cleavage of the disulfide bond. To explore dissociative processes we performed electron scattering calculations with the Schwinger Multichannel Method with pseudopotentials (SMCPP), recently parallelized with OpenMP directives and optimized with subroutines for linear algebra (BLAS) and LAPACK routines. Elastic cross sections obtained for different S-S bond lengths indicate stabilization of the anion formed by electron attachment to a σ*SS antibonding orbital, such that dissociation would be expected.

Thiosaccharine disulfide: synthesis, crystal structure, spectroscopic characterization and theoretical study

The title compound, (thiosaccharine disulfide), bis[1,10dioxide-2,3-dihidro-1,2-benzoisothiazol]disulfide, (tsac)2 has been synthesized and fully characterized by UV–Visible, IR, Raman, 1H and 13C NMR spectroscopy elemental analysis and structural X-ray crystallography. A DFT theoretical study has been performed and good agreement between experimental and theoretical values of structural parameters and vibration frequencies have been achieved.

Application of an in vitro drug screening assay based on the release of phosphoglucose isomerase to determine the structure-activity relationship of thiazolides against Echinococcus multilocularis metacestodes

OBJECTIVES: The disease alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is fatal if treatment is unsuccessful. Current treatment options are, at best, parasitostatic, and involve taking benzimidazoles (albendazole, mebendazole) for the whole of a patient's life. In conjunction with the recent development of optimized procedures for E. multilocularis metacestode cultivation, we aimed to develop a rapid and reliable drug screening test, which enables efficient screening of a large number of compounds in a relatively short time frame. METHODS: Metacestodes were treated in vitro with albendazole, the nitro-thiazole nitazoxanide and 29 nitazoxanide derivatives. The resulting leakage of phosphoglucose isomerase (PGI) activity into the medium supernatant was measured and provided an indication of compound efficacy. RESULTS: We show that upon in vitro culture of E. multilocularis metacestodes in the presence of active drugs such as albendazole, the nitro-thiazole nitazoxanide and 30 different nitazoxanide derivatives, the activity of PGI in culture supernatants increased. The increase in PGI activity correlated with the progressive degeneration and destruction of metacestode tissue in a time- and concentration-dependent manner, which allowed us to perform a structure-activity relationship analysis on the thiazolide compounds used in this study. CONCLUSIONS: The assay presented here is inexpensive, rapid, can be used in 24- and 96-well formats and will serve as an ideal tool for first-round in vitro tests on the efficacy of large numbers of antiparasitic compounds.

Echinococcus multilocularis phosphoglucose isomerase (EmPGI): a glycolytic enzyme involved in metacestode growth and parasite-host cell interactions

In Echinococcus multilocularis metacestodes, the surface-associated and highly glycosylated laminated layer, and molecules associated with this structure, is believed to be involved in modulating the host-parasite interface. We report on the molecular and functional characterisation of E. multilocularis phosphoglucose isomerase (EmPGI), which is a component of this laminated layer. The EmPGI amino acid sequence is virtually identical to that of its homologue in Echinococcus granulosus, and shares 64% identity and 86% similarity with human PGI. Mammalian PGI is a multi-functional protein which, besides its glycolytic function, can also act as a cytokine, growth factor and inducer of angiogenesis, and plays a role in tumour growth, development and metastasis formation. Recombinant EmPGI (recEmPGI) is also functionally active as a glycolytic enzyme and was found to be present, besides the laminated layer, in vesicle fluid and in germinal layer cell extracts. EmPGI is released from metacestodes and induces a humoral immune response in experimentally infected mice, and vaccination of mice with recEmPGI renders these mice more resistant towards secondary challenge infection, indicating that EmPGI plays an important role in parasite development and/or in modulating the host-parasite relationship. We show that recEmPGI stimulates the growth of isolated E. multilocularis germinal layer cells in vitro and selectively stimulates the proliferation of bovine adrenal cortex endothelial cells but not of human fibroblasts and rat hepatocytes. Thus, besides its role in glycolysis, EmPGI could also act as a factor that stimulates parasite growth and potentially induces the formation of novel blood vessels around the developing metacestode in vivo.

An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, nonreceptor binding conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.