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.

Characterization of Plasmodium falciparum prohibitins as novel targets to block infection in humans by impairing the growth and transmission of the parasite.

Prohibitins (PHBs) are highly conserved pleiotropic proteins as they have been shown to mediate key cellular functions. Here, we characterize PHBs encoding putative genes ofPlasmodium falciparum by exploiting different orthologous models. We demonstrated that PfPHB1 (PF3D7_0829200) and PfPHB2 (PF3D7_1014700) are expressed in asexual and sexual blood stages of the parasite. Immunostaining indicated hese proteins as mitochondrial residents as they were found to be localized as branched structures. We further validated PfPHBs as organellar proteins residing in Plasmodium mitochondria, where they interact with each other. Functional characterization was done in Saccharomyces cerevisiae orthologous model by expressing PfPHB1 and PfPHB2 in cells harboring respective mutants. The PfPHBs functionally complemented the yeast PHB1 and PHB2 mutants, where the proteins were found to be involved in stabilizing the mitochondrial DNA, retaining mitochondrial integrity and rescuing yeast cell growth. Further, Rocaglamide (Roc-A), a known inhibitor of PHBs and anti-cancerous agent, was tested against PfPHBs and as an antimalarial. Roc-A treatment retarded the growth of PHB1, PHB2, and ethidium bromide petite yeast mutants. Moreover, Roc-A inhibited growth of yeast PHBs mutants that were functionally complemented with PfPHBs, validating P. falciparum PHBs as one of the molecular targets for Roc-A. Roc-A treatment led to growth inhibition of artemisinin-sensitive (3D7), artemisinin-resistant (R539T) and chloroquine-resistant (RKL-9) parasites in nanomolar ranges. The compound was able to retard gametocyte and oocyst growth with significant morphological aberrations. Based on our findings, we propose the presence of functional mitochondrial PfPHB1 and PfPHB2 in P. falciparum and their druggability to block parasite growth.

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.