Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.

Identification of a new quaternary association for legume lectins.

Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l-fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N-linked carbohydrate at Asn4 and the unusual LTA dimer-dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l-fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l-fucosyl carbohydrates.

Crystallization and preliminary X-ray diffraction analysis of an anti-H(O) lectin from Lotus tetragonolobus seeds.

The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K. A complete structural data set was collected at 2.00 angstroms resolution using a synchrotron-radiation source. LTA crystals were found to be monoclinic, belonging to space group P2(1), with unit-cell parameters a = 68.89, b = 65.83, c = 102.53 angstroms, alpha = gamma = 90, beta = 92 degrees. Molecular replacement yielded a solution with a correlation coefficient and R factor of 34.4 and 51.6%, respectively. Preliminary analysis of the molecular-replacement solution indicates a new quaternary association in the LTA structure. Crystallographic refinement is under way.