Biology subjects

Deshmukh, A.

Publications and source records attributed to Deshmukh, A..

24 records · Page 2Linked to original sources

bioRxiv2023

Toxoplasma gondii aspartic protease 5 (TgASP5): Understanding structural details and inhibition mechanism.

Toxoplasma gondii, a worldwide prevalent parasite, is responsible for causing toxoplasmosis by infecting almost all warm-blooded animals, including humans. To establish a successful infection, the parasite exports a series of effector proteins which modulates the host immune system; Golgi-resident T. gondii aspartyl protease 5 (TgASP5) plays an essential role in the maturation and export of these effector proteins. This is the first report of the detailed structural investigation of the TgASP5 mature enzyme. Molecular modeling and all-atom simulation provided in-depth knowledge of the active site architecture of TgASP5. The analysis of the binding mode of TEXEL substrate highlighted the importance of the active site residues forming the pocket; the Ser505, Ala776 and Tyr689 in the S2 binding pocket provide the specificity towards Arg at the P2 position. Our study also provides insights into the binding mode of the known inhibitor RRLStatine. Screening the known aspartic protease inhibitors against TgASP5 active site and performing 100 ns all-atom molecular dynamic simulations, MM-PBSA binding energy calculations provided the best nine inhibitor protein complexes. Besides that, Principal Component Analysis (PCA) was employed to identify the change in protein dynamics with respect to the substrate and ligand binding. TgASP5 is essential for the fitness and virulence of the parasite; inhibiting this enzyme can be a possible therapeutic strategy against toxoplasmosis. Our study put forth the inhibitors which can act as initial scaffolds for developing potent mechanistic inhibitors against TgASP5.

bioinformatics↗

bioRxiv2022

Characterization of Plasmodium falciparum RAP domain proteins-RAP291 and RAP070, and their association with ribosomal RNAs

Plasmodium genomes encode multiple RAP (RNA-binding domain abundant in Apicomplexan) domain proteins that contain a conserved module of 56 to 73 amino acids. Here, we characterized two of the P. falciparum RAP domain proteins; PfRAP291 & PfRAP070, for their expression and role at asexual blood stages. RNA binding assays and high-throughput CLIP-seq analysis showed that these proteins mainly bind ribosome associated RNAs. Blue-native PAGE and protein-protein interaction studies suggested association of these proteins with MSP-1 complex. Anti-PfRAP291 and anti-PfRAP070 antibodies showed moderate inhibitions in in-vitro merozoite invasion assays. Together, these results suggest multiple roles of these proteins; PfRAP291 and PfRAP070, in merozoite invasion and in ribosome regulation during asexual stages of the parasite.

microbiology↗

bioRxiv2021

Molecular insights into the inhibition of plasmepsins by HIV-1 protease inhibitors: Implications for antimalarial drug discovery

1.Malaria is a deadly disease, and the worldwide emergence of drug resistance in the Plasmodium parasites demands the development of novel and potent antimalarials. HIV-1 protease inhibitors (HIV-1 PIs) alleviate the Plasmodium pathogenesis during malaria/HIV-1 co-infection plausibly by inhibiting vacuolar plasmepsins (PMs), the hemoglobin degrading proteases from P. falciparum. All five FDA-approved HIV-1 PIs tested against PMII exhibit the Ki values in the low micromolar range of which RTV and LPV display the highest inhibition activity. Both inhibitors impede in vitro growth of P. falciparum at low micromolar IC50 values. We report the first crystal structures of PMII complexed with RTV and LPV that reveal the binding mode and interactions of the inhibitors in the active site as well as elucidate the mechanism underlying their differential potency. The conformational flexibility of the P4 group in RTV allows it to explore multiple regions of the S4 pocket. The present study establishes vacuolar PMs as potential drug targets of HIV-1 PIs. The molecular details explaining the inhibitory mechanism of HIV-1 PIs might be crucial in designing novel and potent antimalarial analogs. This work strengthens the prospect of drug repurposing as an alternative strategy towards antimalarial treatments and provides an opportunity to tackle malaria and HIV-1 co-infection.

biochemistry↗

bioRxiv2021

Structures of plasmepsin X from P. falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme are involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

biophysics↗

bioRxiv2021

Artemisinin acts by inhibiting Plasmodium falciparum Ddi1, a retropepsin, resulting into the accumulation of ubiquitinated proteins

Reduced sensitivity of the human malaria parasite, Plasmodium falciparum, to Artemisinin and its derivatives (ARTs) threatens the global efforts towards eliminating malaria. ARTs have been shown to cause ubiquitous cellular and genetic insults, which results in the activation of the unfolded protein response (UPR) pathways. The UPR restores protein homeostasis, which otherwise would be toxic to cellular survival. Here, we interrogated the role of DNA-damage inducible protein 1 (PfDdi1), a unique proteasome-interacting retropepsin in mediating the actions of the ARTs. We demonstrate that PfDdi1 is an active A2 family protease that hydrolyzes ubiquitinated substrates. We further show that treatment with ARTs lead to the accumulation of ubiquitinated proteins in the parasites and blocks the destruction of the ubiquitinated substrates by PfDdi1. Besides, whereas the PfDdi1 is predominantly localised in the cytoplasm, exposure of the parasites to ARTs leads to DNA fragmentation and increased recruitment of the PfDdi1 into the nucleus. Furthermore, Ddi1 knock-out Saccharomyces cerevisiae cells are more suceptible to ARTs and the PfDdI1 protein robustly restores the corresponding functions in the knock-out cells. Together, these results show that ARTs act by inducing DNA and protein damage, and impairing the damage recovery by inhibiting the activity of PfDdi1, an essential ubiquitin-proteasome retropepsin.

cell biology↗

bioRxiv2019

A possible role for epigenetic feedback regulation in the dynamics of the Epithelial-Mesenchymal Transition (EMT)

The epithelial-mesenchymal transition (EMT) often plays a critical role in cancer metastasis and chemoresistance, and decoding its dynamics is crucial to design effective therapeutics. EMT is regulated at multiple levels transcriptional, translational, protein stability, and epigenetics; the mechanisms by which epigenetic regulation can alter the dynamics of EMT remain elusive. Here, to identify the possible effects of epigenetic regulation in EMT, we incorporate a feedback term in our previously proposed model of EMT regulation of the miR 200/ZEB/miR 34/SNAIL circuit. This epigenetic feedback that stabilizes long-term transcriptional activity can alter the relative stability and distribution of states in a given cell population, particularly when incorporated in the inhibitory effect on miR 200 from ZEB. This feedback can stabilize the mesenchymal state, thus making transitions out of that state difficult. Conversely, epigenetic regulation of the self activation of ZEB has only minor effects. Our model predicts that this effect could be seen in experiments, when epithelial cells are treated with an external EMT inducing signal for a sufficiently long period of time and then allowed to recover. Our preliminary experimental data indeed shows that a prolonged TGF beta; exposure gives rise to increasing difficult reversion back to the epithelial state. Thus, this integrated theoretical experimental approach yields insights into how an epigenetic feedback may alter the dynamics of EMT.

biophysics↗