Understanding the complex formation of falstatin; an endogenous macromolecular inhibitor of falcipains.

Proteolytic activity constitutes a fundamental process essential for the survival of the malaria parasite and is thus highly regulated. Falstatin, a protease inhibitor of Plasmodium falciparum, tightly regulates the activity of cysteine hemoglobinases, falcipain-2 and 3 (FP2, FP3), by inhibiting FP2 through a single surface exposed loop. However, the multimeric nature of falstatin and its interaction with FP2 remained unexplored. Here we report that the N-terminal falstatin region is highly disordered, and needs chaperone activity (heat-shock protein 70, HSP70) for its folding. Protein-protein interaction assays showed a significant interaction between falstatin and HSP70. Further, characterization of the falstatin multimer through a series of biophysical techniques identified the formation of a falstatin decamer, which was extremely thermostable. Computational analysis of the falstatin decamer showed the presence of five falstatin dimers, with each dimer aligned in a head-to-tail orientation. Further, the falstatin C-terminal region was revealed to be primarily involved in the oligomerization process. Stoichiometric analysis of the FP2-falstatin multimer showed the formation of a heterooligomeric complex in a 1:1 ratio, with the participation of ten subunits of each protein. Taken together, our results report a novel protease-inhibitor complex and strengthens our understanding of the regulatory mechanisms of major plasmodium hemoglobinases.

Transcriptional regulation of suppressors of cytokine signaling during infection with Mycobacterium tuberculosis in human THP-1-derived macrophages and in mice.

Mycobacterium tuberculosis (Mtb) infection leads to upregulation of Suppressors of Cytokine signaling (SOCS) expression in host macrophages (Mϕ). SOCS proteins inhibit cytokine signaling by negatively regulating JAK/STAT. We investigated this host-pathogen dialectic at the level of transcription. We used phorbol-differentiated THP-1 Mϕ infected with Mtb to investigate preferential upregulation of some SOCS isoforms that are known to inhibit signaling by IFN-γ, IL-12, and IL-6. We examined time kinetics of likely transcription factors and signaling molecules upstream of SOCS transcription, and survival of intracellular Mtb following SOCS upregulation. Our results suggest a plausible mechanism that involves PGE2 secretion during infection to induce the PKA/CREB axis, culminating in nuclear translocation of C/EBPß to induce expression of SOCS1. Mtb-infected Mϕ secreted IL-10, suggesting a mechanism of induction of STAT3, which may subsequently induce SOCS3. We provide evidence of temporal variation in SOCS isoform exspression and decay. Small-interfering RNA-mediated knockdown of SOCS1 and SOCS3 restored the pro-inflammatory milieu and reduced Mtb viability. In mice infected with Mtb, SOCS isoforms persisted across Days 28-85 post infection. Our results suggest that differential temporal regulation of SOCS isoforms by Mtb drives the host immune response towards a phenotype that facilitates the pathogen's survival.

Signature transcriptome analysis of stage specific atherosclerotic plaques of patients.

BACKGROUND: Inflammation plays an important role in all the stages of atherosclerotic plaque development. The current study aimed at assessing the altered expression of genes functioning in inflammation within the early stage (ES) and advanced stage (AS) atherosclerotic plaques obtained from patients undergoing coronary artery bypass grafting (CABG) surgery and identifying biomarker panel/s that may detect the status of plaque stages using peripheral blood samples. METHODS: A section of ES and AS plaques and normal left internal mammary arteries (LIMA) were obtained from 8 patients undergoing the CABG surgery. Total RNA isolated was analyzed for mRNA and miRNA expression profile by Affymetrix arrays. A significant number of mRNAs was found to be differentially expressed in ES and AS plaque tissues relative to LIMA. The pathway analysis of differentially expressed mRNAs in the two plaque stages was also performed using DAVID Bioinformatics Database. RESULTS: The mRNAs were found to be involved in critical inflammatory processes such as the toll-like receptor signaling pathway and cytokine-cytokine receptor interaction. Few miRNAs targeting these mRNAs were also altered in the two plaque conditions. QRT-PCR results showed a similar expression pattern of a few of the mRNAs and miRNAs in peripheral blood of the same patients relative to healthy controls. CONCLUSION: Changes in mRNA and miRNA expression associated with various inflammatory processes occur in different atherosclerotic stage plaques as well as peripheral blood. Detection of such variations in patients' blood can be used as a possible prognostic tool to detect and/or predict the risk and stage of atherosclerosis.

The nucleotide specificity of succinyl-CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone.

Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl-CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild-type), generated by refolding of the individual P. falciparum SCSß and Blastocystis SCSα subunits, demonstrated ADP-forming activity (KmATP  = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSß subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required.

Ginkgo biloba attenuates aluminum lactate-induced neurotoxicity in reproductive senescent female rats: behavioral, biochemical, and histopathological study.

Extensive use of aluminum (Al) in industry, cooking utensils, and wrapping or freezing the food items, due to its cheapness and abundance in the environment, has become a major concern. Growing evidence supports that environmental pollutant Al promotes the aggregation of amyloid beta (Aß) in the brain, which is the main pathological marker of Alzheimer's disease (AD). Further, AD- and Al-induced neurotoxic effects are more common among women following reproductive senescence due to decline in estrogen. Though clinically Ginkgo biloba extract (GBE) has been exploited as a memory enhancer, its role in Al-induced neurotoxicity in reproductive senescent female rats needs to be evaluated. Animals were exposed to intraperitoneal dose (10 mg/kg b.wt) of Al and oral dose (100 mg/kg b.wt.) of GBE daily for 6 weeks. A significant decline in the Al-induced Aß aggregates was observed in hippocampal and cortical regions of the brain with GBE supplementation, as confirmed by thioflavin (ThT) and Congo red staining. GBE administration significantly decreased the reactive oxygen species, lipid peroxidation, nitric oxide, and citrulline levels in comparison to Al-treated rats. On the contrary, a significant increase in the reduced glutathione, GSH/GSSG ratio as well as in the activities of antioxidant enzymes was observed with GBE administration. Based on the above results, GBE prevented the neuronal loss in the hippocampus and cortex, hence caused significant improvement in the learning and memory of the animals in terms of AChE activity, serotonin levels, Morris water maze, and active and passive avoidance tests. In conclusion, GBE has alleviated the behavioral, biochemical, and histopathological alterations due to Al toxicity in rats. However, molecular studies are going on to better understand the mechanism of GBE protection against the environmental toxicant Al exposure. Graphical abstract .

Metacaspase-3 of Plasmodium falciparum: An atypical trypsin-like serine protease.

Metacaspases are clan CD cysteine peptidases found in plants, fungi and protozoa that possess a conserved Peptidase_C14 domain, homologous to the human caspases and a catalytic His/Cys dyad. Earlier reports have indicated the role of metacaspases in cell death; however, metacaspases of human malaria parasite remains poorly understood. In this study, we aimed to functionally characterize a novel malarial protease, P. falciparum metacaspase-3 (PfMCA3). Unlike other clan CD peptidases, PfMCA3 has an atypical active site serine (Ser1865) residue in place of canonical cysteine and it phylogenetically forms a distinct branch across the species. To investigate whether this domain retains catalytic activity, we expressed, purified and refolded the Peptidase_C14 domain of PfMCA3 which was found to express in all asexual stages. PfMCA3 exhibited trypsin-like serine protease activity with ser1865 acting as catalytic residue to cleave trypsin oligopeptide substrate. PfMCA3 is inhibited by trypsin-like serine protease inhibitors. Our study found that PfMCA3 enzymatic activity was abrogated when catalytic serine1865 (S1865A) was mutated. Moreover, PfMCA3 was found to be inactive against caspase substrate. Overall, our study characterizes a novel metacaspase of P. falciparum, different from human caspases and not responsible for the caspase-like activity, therefore, could be considered as a potential chemotherapeutic target.

Crucial residues in falcipains that mediate hemoglobin hydrolysis.

Falcipain-2 (FP2) and falcipain-3 (FP3) constitute the major hemoglobinases of Plasmodium falciparum. Previous biochemical and structural studies have explained the mechanism of inhibition of these enzymes by small molecules. However, a residue-level protein-protein interaction (PPI) with its natural macromolecular substrate, hemoglobin is not fully characterized. Earlier studies have identified a short motif in the C-terminal of FP2, an exosite protruding away from the active site, essential for hemoglobin degradation. Our structural and mutagenesis studies suggest that hemoglobin interacts with FP2 via specific interactions mediated by Glu185 and Val187 within the C-terminal motif, which are essential for hemoglobin binding. Since FP3 is also a major hemoglobinase and essential for parasite survival, we further demonstrate its interactions with hemoglobin. Our results suggest that Asp194 of FP3 is required for hemoglobin hydrolysis and residue-swap experiments confirmed that this position is functionally conserved between the two hemoglobinases. Residues involved in protein-protein interactions constitute important targets for drug-mediated inhibition. Targeting protein-protein interactions at exosites may likely be less susceptible to emergence of drug resistance and thus is a new field to explore in malaria.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets.

Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases.

Genome-wide endogenous DAF-16/FOXO recruitment dynamics during lowered insulin signalling in C. elegans.

Lowering insulin-IGF-1-like signalling (IIS) activates FOXO transcription factors (TF) to extend life span across species. To study the dynamics of FOXO chromatin occupancy under this condition in C. elegans, we report the first recruitment profile of endogenous DAF-16 and show that the response is conserved. DAF-16 predominantly acts as a transcriptional activator and binding within the 0.5 kb promoter-proximal region results in maximum induction of downstream targets that code for proteins involved in detoxification and longevity. Interestingly, genes that are activated under low IIS already have higher DAF-16 recruited to their promoters in WT. DAF-16 binds to variants of the FOXO consensus sequence in the promoter proximal regions of genes that are exclusively targeted during low IIS. We also define a set of 'core' direct targets, after comparing multiple studies, which tend to co-express and contribute robustly towards IIS-associated phenotypes. Additionally, we show that nuclear hormone receptor DAF-12 as well as zinc-finger TF EOR-1 may bind DNA in close proximity to DAF-16 and distinct TF classes that are direct targets of DAF-16 may be instrumental in regulating its indirect targets. Together, our study provides fundamental insights into the transcriptional biology of FOXO/DAF-16 and gene regulation downstream of the IIS pathway.