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.

PatternLab V Handles Multiplex Spectra in Shotgun Proteomic Searches and Increases Identification.

Complex protein mixtures typically generate many tandem mass spectra produced by different peptides coisolated in the gas phase. Widely adopted proteomic data analysis environments usually fail to identify most of these spectra, succeeding at best in identifying only one of the multiple cofragmenting peptides. We present PatternLab V (PLV), an updated version of PatternLab that integrates the YADA 3 deconvolution algorithm to handle such cases efficiently. In general, we expect an increase of 10% in spectral identifications when dealing with complex proteomic samples. PLV is freely available at http://patternlabforproteomics.org.

In-solution behavior and protective potential of asparagine synthetase A from Trypanosoma cruzi.

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.

Procaspase-activating compound-1 induces apoptosis in Trypanosoma cruzi.

Some therapeutics for parasitic, cardiac and neurological diseases activate apoptosis. Therefore, the study of apoptotic proteins in pathogenic organisms is relevant. However, the molecular mechanism of apoptosis in unicellular organisms remain elusive, despite morphological evidence of its occurrence. In Trypanosoma cruzi, the causative agent of Chagas disease, metacaspase 3 (TcMCA3), seems to have a key role in parasite apoptosis. Accordingly, this work provides data concerning TcMCA3 regulation through its interaction with procaspase-activating compound 1 (PAC-1), a procaspase 3 activator. Indeed, PAC-1 reduced T. cruzi epimastigote viability with an IC50 of 14.12 µM and induced loss of mitochondrial potential and exposure of phosphatidylserine, features of the apoptotic process. Notwithstanding, those PAC-1-inducible effects were not conserved in metacyclic trypomastigotes. Moreover, PAC-1 reduced the viability of mammalian cells with a greater IC50 (25.70 µM) compared to T. cruzi epimastigotes, indicating distinct modes of binding between caspases and metacaspases. To shed light on the selectivity of metacaspases and caspases, we determined the structural features related to the PAC-1 binding sites in both types of proteins. These data are important for improving the understanding of the apoptosis pathway in T. cruzi so that TcMCA3 could be better targeted with future pharmaceuticals.

Dissecting biochemical peculiarities of the ATPase activity of TcSub2, a component of the mRNA export pathway in Trypanosoma cruzi.

The RNA helicase DEAD-box protein Sub2 (yeast)/UAP56 (mammals) is conserved across eukaryotes and is essential for mRNA export in trypanosomes. Despite the high conservation of Sub2 in lower eukaryotes such as Trypanosoma cruzi, the low conservation of other mRNA export factors raises questions regarding whether the mode of action of TcSub2 is similar to that of orthologs from other eukaryotes. Mutation of the conserved K87 residue of TcSub2 abolishes ATPase activity, showing that its ATPase domain is functional. However, the Vmax of TcSub2 was much higher than the Vmax described for the human protein UAP56, which suggests that the TcSub2 enzyme hydrolyzes ATP faster than its human homolog. Furthermore, we demonstrate that RNA association is less important to the activity of TcSub2 compared to UAP56. Our results show differences in activity of this protein, even though the structure of TcSub2 is very similar to UAP56. Functional complementation assays indicate that these differences may be common to other trypanosomatids. Distinct features of RNA influence and ATPase efficiency between UAP56 and TcSub2 may reflect distinct structures for functional sites of TcSub2. For this reason, ligand-based or structure-based methodologies can be applied to investigate the potential of TcSub2 as a target for new drugs.

Identification of potential targets for an anticoagulant pectin.

Heparin is a sulfated polysaccharide of animal origin showing excellent anticoagulant properties. Although it strongly inhibits the coagulation cascade, its interaction with multiple sites results in several side effects. An ideal alternative compound should not only possess anticoagulant and antithrombotic activities, but also provide specific binding to components of the coagulation cascade to decrease side effects and facilitate the control of pharmacologic actions in patient's body. In this work, we performed a scan of potential targets for chemically sulfated pectin from Citrus sinensis (SCP) that shows an efficient anticoagulant activity by combining proteomics and molecular docking techniques. Defining the interaction partners of SCP is fundamental to evaluate if its pharmacological side effects can be as harmful as those from heparin. SCP interacts directly with heparin cofactor II, probably favoring its interaction with thrombin. SCP interaction with antithrombin depends likely on its association with thrombin or factor Xa. In addition to the interaction with factors related to homeostasis, SCP may also act on the renin-angiotensin and on the complement systems. BIOLOGICAL SIGNIFICANCE: The knowledge of potential molecular targets of SCP provides clues to understand its mechanism of action in order to guide molecular changes in this compound to increase its specificity.

Biophysical Characterization of Alanine Aminotransferase from Trypanosoma cruzi.

Aminotransferases are an important group of enzymes that catalyze the transfer of an amino group of an amino acid into a keto acid. Alanine aminotransferase from Trypanosoma cruzi (TcALAT) was cloned, overexpressed and purified. Far-UV Circular Dichroism (CD), Dynamic Light Scattering (DLS), Analytical Size Exclusion Chromatography (aSEC) and Small Angle X-ray Scattering (SAXS) provide data concerning TcALAT biophysical behavior. CD analysis displayed a typical spectrum of α-ß proteins analogously as observed for other alanine aminotransferases. The protein is stable until 40oC and above that temperature starts to denatured. Its temperature of melting is equal to 50oC. DLS, aSEC and SAXS data show that protein is monomeric in solution. All these gather initial information on secondary and quaternary structures of TcALAT.