Refolding of metacaspase 5 from Trypanosoma cruzi, structural characterization and the influence of c-terminal in protein recombinant production.

Metacaspases are known to have a fundamental role in apoptosis-like, a programmed cellular death (PCD) in plants, fungi, and protozoans. The last includes several parasites that cause diseases of great interest to public health, mostly without adequate treatment and included in the neglected tropical diseases category. One of them is Trypanosoma cruzi which causes Chagas disease and has two metacaspases involved in its PCD: TcMCA3 and TcMCA5. Their roles seemed different in PCD, TcMCA5 appears as a proapoptotic protein negatively regulated by its C-terminal sequence, while TcMCA3 is described as a cell cycle regulator. Despite this, the precise role of TcMCA3 and TcMCA5 and their atomic structures remain elusive. Therefore, developing methodologies to allow investigations of those metacaspases is relevant. Herein, we produced full-length and truncated versions of TcMCA5 and applied different strategies for their folded recombinant production from E. coli inclusion bodies. Biophysical assays probed the efficacy of the production method in providing a high yield of folded recombinant TcMCA5. Moreover, we modeled the TcMCA5 protein structure using experimental restraints obtained by XLMS. The experimental design for novel methods and the final protocol provided here can guide studies with other metacaspases. The production of TcMCA5 allows further investigations as protein crystallography, HTS drug discovery to create potential therapeutic in the treatment of Chagas' disease and in the way to clarify how the PCD works in the parasite.

Cross-linking mass spectrometry reveals structural insights of the glutamine synthetase from Leishmania braziliensis.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania braziliensis, commonly found in Brazil and associated with cutaneous and visceral forms of this disease. Like other organisms, L. braziliensis has an enzyme called glutamine synthetase (LbGS) that acts on the synthesis of glutamine from glutamate. This enzyme plays an essential role in the metabolism of these parasites and can be a potential therapeutic target for treating this disease. OBJECTIVES: Investigate LbGS structure and generate structural models of the protein. METHODS: We use the method of crosslinking mass spectrometry (XLMS) and generate structural models in silico using I-TASSER. FINDINGS: 42 XLs peptides were identified, of which 37 are explained in a monomeric model with the other five indicating LbGS dimerization and pentamers interaction region. The comparison of 3D models generated in the presence and absence of XLMS restrictions probed the benefits of modeling with XLMS highlighting the inappropriate folding due to the absence of spatial restrictions. MAIN CONCLUSIONS: In conclusion, we disclose the conservation of the active site and interface regions, but also unique features of LbGS showing the potential of XLMS to probe structural information and explore new drugs.

Dissecting dual specificity: Identifying key residues in L-asparaginase for enhanced acute lymphoid leukemia therapy and reduced adverse effects.

L-asparaginase from Escherichia coli (EcA) has been used for the treatment of acute lymphoid leukemia (ALL) since the 1970s. Nevertheless, the enzyme has a second specificity that results in glutaminase breakdown, resulting in depletion from the patient's body, causing severe adverse effects. Despite the huge interest in the use of this enzyme, the exact process of glutamine depletion is still unknown and there is no consensus regarding L-asparagine hydrolysis. Here, we investigate the role of T12, Y25, and T89 in asparaginase and glutaminase activities. We obtained individual clones containing mutations in the T12, Y25 or T89 residues. After the recombinant production of wild-type and mutated EcA, The purified samples were subjected to structural analysis using Nano Differential Scanning Fluorimetry, which revealed that all samples contained thermostable molecules in their active structural conformation, the homotetramer conformation. The quaternary conformation was confirmed by DLS and SEC. The activity enzymatic assay combined with molecular dynamics simulation identified the contribution of T12, Y25, and T89 residues in EcA glutaminase and asparaginase activities. Our results mapped the enzymatic behavior paving the way for the designing of improved EcA enzymes, which is important in the treatment of ALL.

Cross-linking mass spectrometry reveals structural insights of the glutamine synthetase from Leishmania braziliensis

BACKGROUND Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania braziliensis, commonly found in Brazil and associated with cutaneous and visceral forms of this disease. Like other organisms, L. braziliensis has an enzyme called glutamine synthetase (LbGS) that acts on the synthesis of glutamine from glutamate. This enzyme plays an essential role in the metabolism of these parasites and can be a potential therapeutic target for treating this disease. OBJECTIVES Investigate LbGS structure and generate structural models of the protein. METHODS We use the method of crosslinking mass spectrometry (XLMS) and generate structural models in silico using I-TASSER. FINDINGS 42 XLs peptides were identified, of which 37 are explained in a monomeric model with the other five indicating LbGS dimerization and pentamers interaction region. The comparison of 3D models generated in the presence and absence of XLMS restrictions probed the benefits of modeling with XLMS highlighting the inappropriate folding due to the absence of spatial restrictions. MAIN CONCLUSIONS In conclusion, we disclose the conservation of the active site and interface regions, but also unique features of LbGS showing the potential of XLMS to probe structural information and explore new drugs.