Structure-Function of Falcipains: Malarial Cysteine Proteases

Evidence indicates that cysteine proteases play essential role in malaria parasites; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria. Studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis. The best characterized Plasmodium cysteine proteases are falcipains, which are papain family enzymes. Falcipain-2 and falcipain-3 are major hemoglobinases of P. falciparum. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. Overall, the complexes of falcipain-2 and falcipain-3 with small and macromolecular inhibitors provide structural insight to facilitate the design or modification of effective drug treatment against malaria. Drug development targeting falcipains should be aided by a strong foundation of biochemical and structural studies

Neuro-Olfactory Regulation and Salivary Actions: A Coordinated Event for Successful Blood-Feeding Behavior of Mosquitoes

The synergistic actions of the nongenetic and genetic factors are crucial to shape mosquitoes’ feeding behavior. Unlike males, adult female mosquitoes are evolved with unique ability to take blood meals from a vertebrate host for reproductive success which eventually makes them a potential vector. Processing and integration of chemical information in the neuro-olfactory system followed by salivary actions facilitate blood meal uptake process. Thus, deciphering the underlying molecular mechanism of odor sensing through the detection machinery (olfactory system), odor processing and decision-making by decision machinery (brain), and regulation of saliva secretion by the action machinery (salivary gland) is likely to reveal molecular pathways which can be targeted to disrupt mosquitoes’ feeding behavior. Here we summarize how smart actions of highly specialized neurosensory systems guide and manage feeding behavior associated complex events of (i) successful navigation to find a suitable host, (ii) making food choice decisions, and (iii) regulation of the salivary gland actions in mosquitoes

Protein-Protein Interactions in Malaria: Emerging Arena for Future Chemotherapeutics

Malaria is one of the most deadly diseases infecting humans. Advances in elimination and vector control have reduced the global malaria burden in the past decade; however, the emerging threat of drug resistance and suboptimal vaccine efficacies threaten global eradication efforts. Unlocking novel drug and vaccine targets while simultaneously mitigating spread of resistant strains seems to be the need of the hour. Protein-protein interactions (PPIs), an integral part of host-pathogen cross-talk and parasite survival, have only recently emerged as promising drug targets. Large PPI networks (interactome) are being developed to better our understanding of various parasite biochemical pathways. In this chapter, we throw light on several newly characterized protein-protein interactions between the host (humans) and parasite (plasmodium) in key processes such as hemoglobin degradation, enzyme regulation, protein export, egress, invasion, and drug resistance and further discuss their viability for development as novel chemotherapeutic targets

Recognition of fold- and function-specific sites in the ligand-binding domain of the thyroid hormone receptor-like family

Background: The thyroid hormone receptor-like (THR-like) family is the largest transcription factors family belonging to the nuclear receptor superfamily, which directly binds to DNA and regulates the gene expression and thereby controls various metabolic processes in a ligand-dependent manner. The THR-like family contains receptors THRs, RARs, VDR, PPARs, RORs, Rev-erbs, CAR, PXR, LXRs, and others. THR-like receptors are involved in many aspects of human health, including development, metabolism and homeostasis. Therefore, it is considered an important therapeutic target for various diseases such as osteoporosis, rickets, diabetes, etc. Methods: In this study, we have performed an extensive sequence and structure analysis of the ligand-binding domain (LBD) of the THR-like family spanning multiple taxa. We have use different computational tools (information-theoretic measures; relative entropy) to predict the key residues responsible for fold and functional specificity in the LBD of the THR-like family. The MSA of THR-like LBDs was further used as input in conservation studies and phylogenetic clustering studies. Results: Phylogenetic analysis of the LBD domain of THR-like proteins resulted in the clustering of eight subfamilies based on their sequence homology. The conservation analysis by relative entropy (RE) revealed that structurally important residues are conserved throughout the LBDs in the THR-like family. The multi-harmony conservation analysis further predicted specificity in determining residues in LBDs of THR-like subfamilies. Finally, fold and functional specificity determining residues (residues critical for ligand, DBD and coregulators binding) were mapped on the three-dimensional structure of thyroid hormone receptor protein. We then compiled a list of natural mutations in THR-like LBDs and mapped them along with fold and function-specific mutations. Some of the mutations were found to have a link with severe diseases like hypothyroidism, rickets, obesity, lipodystrophy, epilepsy, etc. Conclusion: Our study identifies fold and function-specific residues in THR-like LBDs. We believe that this study will be useful in exploring the role of these residues in the binding of different drugs, ligands, and protein-protein interaction among partner proteins. So this study might be helpful in the rational design of either ligands or receptors

Molecular Dynamics of Mosquito-<em>Plasmodium vivax</em> Interaction: A Smart Strategy of Parasitism

Parallel to Plasmodium falciparum, P. vivax is a fast emerging challenge to control malaria in South-East Asia regions. Owing to unique biological differences such as the preference for invading reticulocytes, early maturation of sexual stages during the infection, the formation of hypnozoites, unavailability of in-vitro culture, the molecular relation of P. vivax development inside the mosquito host is poorly known. In this chapter, we briefly provide a basic overview of Mosquito-Plasmodium interaction and update current knowledge of tissue-specific viz. midgut, hemocyte, and salivary glands- molecular dynamics of Plasmodium vivax interaction during its developmental transformation inside the mosquito host, in specific

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

An Epithelial Serine Protease, AgESP, Is Required for Plasmodium Invasion in the Mosquito Anopheles gambiae

Background: Plasmodium parasites need to cross the midgut and salivary gland epithelia to complete their life cycle in the mosquito. However, our understanding of the molecular mechanism and the mosquito genes that participate in this process is still very limited. Methodology/Principal Findings: We identified an Anopheles gambiae epithelial serine protease (AgESP) that is constitutively expressed in the submicrovillar region of mosquito midgut epithelial cells and in the basal side of the salivary glands that is critical for Plasmodium parasites to cross these two epithelial barriers. AgESP silencing greatly reduces Plasmodium berghei and Plasmodium falciparum midgut invasion and prevents the transcriptional activation of gelsolin, a key regulator of actin remodeling and a reported Plasmodium agonist. AgESP expression is highly induced in midgut cells invaded by Plasmodium, suggesting that this protease also participates in the apoptotic response to invasion. In salivary gland epithelial cells, AgESP is localized on the basal side–the surface with which sporozoites interact. AgESP expression in the salivary gland is also induced in response to P. berghei and P. falciparum sporozoite invasion, and AgESP silencing significantly reduces the number of sporozoites that invade this organ. Conclusion: Our findings indicate that AgESP is required for Plasmodium parasites to effectively traverse the midgut and salivary gland epithelial barriers. Plasmodium parasites need to modify the actin cytoskeleton of mosquito epithelial cells t

<span style="mso-bidi-language:HI">Identification and characterization of a new putative c-type lysozyme <span style="mso-bidi-language:HI">from malaria vector <i>Anopheles stephensi</i> </span></span>

15-19Lysozyme (E.C. 3.<span style="color:black; mso-bidi-language:HI">2.1<span style="color:#242424;mso-bidi-language: HI">.17) activity is reported from the malaria vector Anopheles stephensi. The activity was detected in the salivary gland and midgut using bacteriolytic radial diffusion assay. Spectrophotometric analysis indicated that higher level of lysozyme activity was maintained in both midgut and salivary gland tissues. The activity reached the highest level in 4-8 days old mosquitoes. Genomic PCR amplification revealed the presence of at least two putative lysozyme genes in the mosquito genome. Preliminary analysis of one of the 413 bp genomic fragments showed 56% identity to the lysozyme of mosquito A. gambiae. However, the nature and origin of the putative cloned lysozyme gene remains elusive. </span

Transcriptional responses of attractin gene in the mosquito Anopheles culicifacies: A synergistic neuro-olfactory regulation

Background & objectives: Attractin, is a large multi-domain protein which has regulatory functions in multiple physiological processes and thus have strong therapeutic potential. In invertebrates, it was first identified as a water-borne protein pheromone that plays important role in chemical communication and coordinates reproductive activities. But its role in mosquitoes/insects remains unknown. Our unexpected discovery of attractin homolog from the olfactory tissue of Anopheles culicifacies mosquito prompted us to investigate the possible role of Ac-attractin (Ac-atrn) in diverse behavioural responses, e.g. feeding, mating and other non-genetic stresses. Methods: A homology search analysis was performed to identify the full length attractin (Ac-atrn) gene of Anopheles culicifacies mosquito. To unravel its molecular function during external and internal stresses, extensive real-time PCR was performed in the neuro-olfactory tissues of the adult mosquitoes as well as in the larval stages. Further, a behavioural assay was conducted to elucidate its role in mosquitoes mating behaviour. Results: The results indicated that Ac-atrn is a 3942 bp long transcript which encodes a 1313 amino acid protein, having multiple domains including CUB, EGF, Keltch, etc, with 80–90% homology to other insect/mosquito homologs. Ac-atrn gene was dominantly expressed in the young larvae and its expression was elevated in response to the fresh food supply in the starved larvae. Cold stress temporarily arrested the expression of Ac-atrn gene. In case of adult mosquitoes, olfactory and brain tissue showed relatively higher expression of Ac-atrn than reproductive organs. Although, starvation did not yield significant changes in olfactory tissues, but aging and nutritional stress modulated Ac-atrn expression in the brain tissue. Furthermore, a circadian rhythm dependent change in the expression of Ac-atrn of virgin and mated mosquitoes (both sexes), indicates that Ac-atrn might also have a pheromone guided role during swarm formation and mating behaviour. Interpretation & conclusion: The relative expression profiling of Ac-atrn gene in the larvae during nutritional and cold stress suggested its possible role in mediating chemical communication towards the food source and in thermal regulation of young larvae. Similarly, it might have crucial regulatory role in the stress management and survival of adult mosquitoes. The results revealed that Ac-atrn gene is a global regulator of many physiological processes in mosquitoes including stress response and mating behaviour and thus might be a potential target to design novel intervention strategy against mosquitoes