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.

Artemisinin resistance in P. falciparum: probing the interacting partners of Kelch13 protein in parasite.

OBJECTIVES: Artemisinin (ART) resistance in Plasmodium is threatening the artemisinin combination therapies-the first line of defence against malaria. ART resistance has been established to be mediated by the Plasmodium Kelch13 (PfK13) protein. For the crucial role of PfK13 in multiple pathways of the Plasmodium life cycle and ART resistance, it is imperative that we investigate its interacting partners. METHODS: We recombinantly expressed PfK13-p (Bric a brac/Poxvirus and zinc finger and propeller domains), generating anti-PfK13-p antibodies to perform co-immunoprecipitation assays and probed PfK13 interacting partners. Surface plasmon resonance and pull-down assays were performed to establish physical interactions of representative proteins with PfK13-p. RESULTS: The co-immunoprecipitation assays identified 17 proteins with distinct functions in the parasite life cycle- protein folding, cellular metabolism, and protein binding and invasion. In addition to the overlap with previously identified proteins, our study identified 10 unique proteins. Fructose-biphosphate aldolase and heat shock protein 70 demonstrated strong biophysical interaction with PfK13-p, with KD values of 6.6 µM and 7.6 µM, respectively. Additionally, Plasmodium merozoite surface protein 1 formed a complex with PfK13-p, which is evident from the pull-down assay. CONCLUSION: This study adds to our knowledge of the PfK13 protein in mediating ART resistance by identifying new PfK13 interacting partners. Three representative proteins-fructose-biphosphate aldolase, heat shock protein 70, and merozoite surface protein 1-demonstrated clear evidence of biophysical interactions with PfK13-p. However, elucidation of the functional relevance of these physical interactions are crucial in context of PfK13 role in ART resistance.

Exploring the Immunodominant Epitopes of SARS-CoV-2 Nucleocapsid Protein as Exposure Biomarker.

Background The nucleocapsid protein (N protein) of SARS-CoV-2 is undeniably a potent target for the development of diagnostic tools due to its abundant expression and lower immune evasion pressure compared to spike (S) protein. Methods Blood samples of active COVID-19 infections (n=71) and post-COVID-19 (n=11) were collected from a tertiary care hospital in India; pre-COVID-19 (n=12) sera samples served as controls. Real-time reverse transcriptase-PCR (rRT-PCR) confirmed pooled sera samples (n=5) were used with PEPperCHIP® SARS-CoV-2 Proteome Microarray (PEPperPRINT GmbH, Germany) to screen immunodominant epitopes of SARS-CoV-2. Highly immunodominant epitopes were then commercially synthesized and further validated for their immunoreactivity by dot-blot and ELISA. Results The lowest detectable concentration (LDC) of the N1 peptide in the dot-blot assay was 12.5 µg demonstrating it to be fairly immunoreactive compared to control sera. IgG titers against the contiguous peptide (N2: 156AIVLQLPQGTTLPKGFYAEGS176) was found to be significantly higher (p=0.018) in post-COVID-19 compared to pre-COVID-19 control sera. These results suggested that N2-specific IgG titers buildup over time as expected in post-COVID-19 sera samples, while a non-significant immunoreactivity of the N2 peptide was also observed in active-COVID-19 sera samples. However, there were no significant differences in the total IgG titers between active COVID-19 infections, post-COVID-19 and pre-COVID-19 controls. Conclusion The N2-specific IgG titers in post-COVID-19 samples demonstrated the potential of N protein as an exposure biomarker, particularly in sero-surveillance studies.

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.

Current scenario and future strategies to fight artemisinin resistance.

Despite several setbacks in the fight against malaria such as insecticide and drug resistance as well as low efficacy of available vaccines, considerable success in reducing malaria burden has been achieved in the past decade. Artemisinins (ARTs and their combination therapies, ACTs), the current frontline drugs against uncomplicated malaria, rapidly kill plasmodial parasites and are non-toxic at short exposures. Though the exact mode of action remains unclear, the endoperoxide bridge, indispensable for ART activity, is thought to react with heme released from hemoglobin hydrolysis and generate free radicals that alkylate multiple protein targets, thereby disrupting proteostasis pathways. However, rapid development of ART resistance in recent years with no potential alternatives on the horizon threaten the elimination efforts. The Greater Mekong Subregion in South-East Asia continues to churn out mutants resistant to multiple ACTs and detected in increasingly expanding geographies. Extensive research on ART-resistant strains have identified a potential candidate Kelch13, crucial for mediating ART resistance. Parasites with mutations in the propeller domains of Plasmodium falciparum Kelch13 protein were shown to have enhanced phosphatidylinositol 3-kinase levels that were concomitant with delayed parasite clearance. Current research focused on understanding the mechanism of Kelch13-mediated ART resistance could provide better insights into Plasmodium resistome. This review covers the current proposed mechanisms of ART activity, resistance strategies adopted by the parasite in response to ACTs and possible future approaches to mitigate the spread of resistance from South-East Asia.

Correction to: Current scenario and future strategies to fight artemisinin resistance.

Author Atul Yadav would like to present his name as Atul only to be the same with his previous publications. The original article has been corrected.