Biology subjects

Chakraborti, S.

Publications and source records attributed to Chakraborti, S..

15 recordsLinked to original sources

bioRxiv2026

A Poly(rC)-Binding Protein Mediates Iron Delivery to Ferritin in Anopheles stephensi

Iron homeostasis is essential for both the Plasmodium parasite and its hosts, the mosquito and human; however, the molecular mechanisms governing intracellular iron trafficking remain poorly understood. In mammals, poly(rC)-binding proteins (PCBPs) function as major iron chaperones by delivering Fe2+ to ferritin and other iron-dependent proteins, yet their role in insects has not been investigated. Here, we provide the first structural and functional characterization of the PCBP from a major malaria vector, Anopheles stephensi. Comparative structural and evolutionary analyses showed that insect PCBPs are most closely related to human PCBP3. Coordinated expression and co-localization of AsPCBP with ferritin following blood feeding suggested a conserved role in mosquito iron homeostasis. Functional analyses demonstrated that KH2-KH3 linker and KH3 domain of PCBP, plays an important role in the AsPCBP-ferritin interaction and that deletion of these regions, unexpectedly increased ferritin binding affinity but impaired iron delivery, indicating that these regions are dispensable for complex formation but essential for efficient iron transfer. Collectively, our results identify PCBP as a key component of mosquito iron homeostasis that could be exploited for the development of novel antimalarial strategies.

biochemistry↗

bioRxiv2026

Pleiotropic antimalarial activities and immunomodulation exhibited by Himalayan Buransh (Rhododendron arboreum) in human and rodent malaria models

Emerging drug resistance against the malaria parasite is worrisome and necessitates the development of novel antimalarials. Himalayan Buransh (Rhododendron arboreum) is a well-known medicinal plant found in the northern states of India. In this study, we observed the pleiotropic antimalarial activities and immunomodulation exhibited by the aqueous extract of Buransh flower (AEBF). AEBF demonstrated significant IC50 values (16-29 {micro}g/ml) against the asexual stages of various P. falciparum strains (3D7, Dd2-chloroquine-resistant and C580Y-artemisinin resistant). The oral administration of AEBF (200 mg/kg) in mice, suppressed [~]80% P. berghei parasitemia, improved mean survival time (MST-23.5 days) and prevented splenomegaly. Notably, the combination of AEBF and artesunate not only cleared primary infection, but also conferred sustained immunity. This immunomodulatory effect, driven by protective IFN-{gamma} resulted in reduced parasitemia during a homologous challenge without the need for further treatment. It is important to highlight the malaria transmission blocking activity of AEBF, resulting in reduced sexual stage male gametocyte exflagellation. Furthermore, the virtual drug screening of selected bioactive constituents from Buransh flower demonstrated potent binding against multiple P. falciparum proteins, suggested a pleiotropic mode of action. Altogether, our results corroborated the first ever evidence of the multistage antimalarial potential of Buransh flower, supported by in vitro cell studies, in vivo rodent malaria model and in silico docking analyses. Based on our studys findings and the traditional use of Buransh juice as a medicinal beverage in Uttarakhand, India, we propose exploring it as an adjunct therapy for drug-resistant malaria, subject to further clinical validation.

pathology↗

bioRxiv2026

Mosquito-directed PROTACs to block malaria transmission

The mosquito stage of the Plasmodium falciparum life cycle is an attractive target for intervention since it is crucial for the sexual reproduction and transmission of parasites to human host. Mosquito determinants crucial for parasite infection and growth pose as lucrative targets for transmission blockers. Owing to the fact that p38 MAPK has role in immune response and vector competence, we have evaluated the potential of PROTAC molecule (NR-7h) to degrade Anopheles stephensi p38 MAPK (Asp38 MAPK), a conserved serine/threonine kinase involved in stress reactions, midgut homeostasis, and parasite survival. PROTAC-mediated degradation of Asp38 MAPK led to the disrupted development of the parasite, suggesting its crucial function in vector competence. Furthermore, NR-7h-treated mosquitoes showed higher expression of immune genes such Rel-2, TEP1, APL1, and NOS, suggesting that p38 MAPK regulates host immunity in a way that promotes parasite persistence. PROTAC-mediated degradation of target proteins, provides a more persistent and resistance-proof therapeutic effect than traditional kinase inhibitors. Our findings establish PROTACs as a novel vector-targeted strategy for the development of endectocides to limit malaria transmission.

molecular biology↗

bioRxiv2026

Adaptive Landscapes of Plasmodium Falciparum Dihydrofolate Reductase Reveal Pathways to Antifolate Resistance

Antifolate resistance in Plasmodium falciparum dihydrofolate reductase (pfDHFR) remains a major challenge for malaria control. To understand how this enzyme maintains function under antifolate selection, we developed PfPATH, a computational framework that integrates mutational fitness measurements, evolutionary interactions, and structural information to map adaptive trajectories. PfPATH adaptive walks reveal that evolution is constrained to a narrow ridge in sequence space defined by a small subset of residues. Most resistance mutations are individually deleterious and become viable only when supported by stabilizing changes, restricting adaptation to a few high-resistance pathways. Structure network analyses show that these mutations do not disrupt the global enzyme architecture but instead reorganize internal communication to preserve catalytic function. Consistent with this, molecular dynamics and free-energy analyses indicate that resistant variants remain stable while sampling a broader ensemble of low-energy conformational substates. Together, these results reveal a narrow and predictable route to antifolate escape.

biophysics↗

bioRxiv2025

Tuning the open-close equilibrium of Cereblon with small molecules influences protein degradation

Most PROTACs and molecular glue degraders currently approved or in clinical trials recruit Cereblon (CRBN) as the ubiquitin E3 ligase. Upon binding ligands and molecular glues, CRBN undergoes a significant structural rearrangement from an open to closed state, defined by the positioning of the thalidomide-binding domain (TBD) with respect to the Lon domain. However, the exact molecular basis for this ligand-induced conformational change and its implication to neo-substrate degradation remain elusive. During our campaign to discover novel CRBN binders, we found hits exhibiting distinct biophysical behaviour from classical thalidomide-based ligands. By combining orthogonal biophysical methods of differential scanning fluorimetry, isothermal titration calorimetry, and small-angle X-ray scattering, supported by X-ray crystallography and cryo-EM structures of ligand-bound complexes, we classify CRBN binders between those that can induce CRBN closure, and those that cannot. Mutational studies identify key residues in the CRBN ligand binding pocket and N-terminal belt that are essential for the Lon and TBD domains to come together in the closed state. Finally, we show that the probability to yield active degrader molecules is greatly influenced by whether binders can or cannot induce CRBN closure. Together, our study reveals new molecular insights into the structural basis for how CRBN open-closed equilibrium is directly modulated by compound binding and impact target degradability by CRBN, with important implications to the design of PROTACs and molecular glue degraders.

biochemistry↗

bioRxiv2025

Mosquito Ferritin: A Haem-Binding Iron Store Required for Egg Development and Targetable for Malaria Control

Iron metabolism is essential for both mosquito reproductive fitness and Plasmodium development within the vector, yet this axis remains insufficiently explored as a potential target for malaria control. Ferritin, the major iron-storage protein and a central regulator of iron homeostasis, has not previously been biochemically or functionally characterized in any hematophagous insect. Our computational and phylogenetic analyses reveal that mosquito ferritin represents a unique case, combining conserved iron-binding motifs with distinctive haem-coordinating residues that confer an exceptionally high haem-binding affinity exceeding that of bacterioferritin. In addition, through a screen of multiple iron chelators in Anopheles larvae, we identified potent candidates with potential utility for vector control. Together, these findings establish mosquito ferritin as an evolutionarily distinct and physiologically essential protein, and they highlight iron homeostasis as a promising dual-target axis for next-generation vector control strategies.

biochemistry↗

bioRxiv2025

Comparative study of manganese catalase monomers, interfaces and cage architecture within the ferritin superfamily

Manganese catalase protein is an example of a protein cage within the ferritin superfamily that focuses on enzymatic catalysis rather than on storage which other ferritin proteins are known for. Formed of 6 homomeric chains, it shows large subunit-subunit sidewise contacts and special interfacial interactions ensuring cage generation with only 6 subunits. We aim to explore manganese catalase at the monomer, subunit-subunit and cage level. As compared to other ferritin subtypes, we found manganese catalase monomers to have greater fraction of {beta}-turns at the secondary structure level and thermophilic manganese catalase monomers to have greater non-polar to polar residue ratio. At the interface level, the placement of subunits in manganese catalase cage with sidewise and angular interface was found to be distinctly different from the parallel and perpendicular interfaces present in other ferritin subtypes. Our study highlights the contribution of sidewise interfaces and terminal extensions in making the cage architecture possible in 6-mer manganese catalases. We also studied the quaternary structure of manganese catalase cage with respect to Classical ferritin (C-ferritin) and found manganese catalase to have smaller cavity volume and cavity surface area than C-ferritin but higher cavity surface to volume ratio. This observation along with the smaller distance between substrate entry point and active site as compared to C-ferritin highlights the structural distinction between catalytic enzymes like manganese catalase and storage proteins like ferritins. Thus, the study gives structural insight into manganese catalase protein cage with focus on its ability to form a cage architecture and show efficient catalytic activity.

bioinformatics↗

bioRxiv2025

Mycolic acid like lipids act as substrates for Mycobacterium tuberculosis melH

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has developed its own ways of evading defense mechanisms to counteract the lethal effects of reactive oxygen species (ROS) generated within the host macrophages during infection. The melH gene present in Mtb and Mycobacterium marinum (Mm) plays an important role to reduce ROS generated during infection. The melH gene encodes for an epoxide hydrolase. Bioinformatics data suggests that encoded enzyme utilizes lipid substrates for its function. Initially, we used a lipid fractionation approach coupled with liquid chromatography mass spectrometry (LC-MS) and treatment with active MelH enzyme to identify potential substrates for MelH. We found classes of mycolic acids, predominantly epoxy mycolic acids accumulate in the melH mutant and upon treatment with MelH are reduced in the lipid fraction. These results provide insight into how MelH encoded in the mel2 operon contributes to Mtb virulence and persistence and present further evidence for potential mechanisms of action if MelH is targeted for antitubercular drug discovery.

biochemistry↗

bioRxiv2024

Navigating the Fitness Landscapes of Plasmodium falciparum Dihydrofolate Reductase: Evolutionary Insights into Antifolate Resistance

The rapid emergence of drug resistance in malaria parasites poses a significant challenge to the efficacy of antifolate treatments. Traditional drug development approaches, which often rely on empirical screening with limited mechanistic insights, tend to overlook the complex evolutionary mechanisms that enable Plasmodium falciparum to evade drug inhibition while preserving enzyme functionality. In this study, we employed computational techniques to investigate the mutational landscape of dihydrofolate reductase (DHFR), focusing on regions essential for enzyme stability and resistance. Our analysis uncovered conserved residues essential for stability, mutation hotspots that enhance adaptability under drug pressure and co-evolving clusters revealing critical functional interdependencies. Through integrated approaches including mutational scanning, epistatic interaction modeling, and fitness trajectory mapping, we elucidated distinct evolutionary pathways that drive resistance. We were able to track the adaptive paths taken by wild-type residues upon mutation, revealing the steps required to reach high-fitness peaks within the rugged fitness landscape. These findings provide valuable insights into the molecular mechanisms of antifolate resistance. We suggest that future drug design should target co-evolving networks and conserved regions to support the development of next-generation therapies to overcome resistance.

biophysics↗

bioRxiv2024

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degraders ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics. One sentence summaryA novel Cereblon construct (CRBNmidi) allows structural and biophysical enablement of ligand and degrader design

biochemistry↗

bioRxiv2023

Elucidation of the essentiality of lumazine synthase (RibH) for Mycobacterium tuberculosis survival and discovery of potent inhibitors for enhanced antimycobacterial therapy

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate the indispensable role of lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, in the survival of Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We show that genetic and functional ablation of RibH in M. tb cannot be compensated by exogenous supply of Riboflavin, or co-factors Flavin Adenine Dinucleotide (FAD) or Flavin Mononucleotide (FMN). Capitalizing on the essentiality of RibH, we employ a high-throughput molecular docking approach to screen [~]600,000 compounds and identify inhibitors of RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 inhibitors that exhibit potent antimycobacterial activity. These compounds effectively also eradicate intracellular M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these 3 compounds synergistically enhance the bactericidal effect of first-line anti-TB drugs, Isoniazid and Rifampicin. Corroborating with the in silico predicted high docking scores along with favorable ADME and toxicity profiles, all 3 compounds demonstrate exceptional binding affinity towards purified lumazine synthase enzyme in vitro, and display an acceptable safety profile in mammalian cells, with a high selective index. By providing mechanistic evidence for the essentiality of RibH in M. tb survival, and discovering potent RibH inhibitors with outstanding antimycobacterial activity, our study contributes to the development of superior TB treatment strategies and advances the global fight against this devastating disease.

microbiology↗

bioRxiv2022

Simultaneous visualization of cells and marker genes from scRNA-seq studies

The complexity of scRNA-sequencing datasets highlights the urgent need for enhanced clustering and visualization methods. Here, we propose Stardust, an iterative, force-directed graph layouting algorithm that enables the simultaneous embedding of cells and marker genes. Stardust, for the first time, allows a single-stop visualization of cells and marker genes on a single 2D map. While Stardust provides its own visualization pipeline, it can be plugged in with state-of-the-art methods such as Uniform Manifold Approximation and Projection (UMAP) and t-Distributed Stochastic Neighbor Embedding (tSNE). We benchmarked Stardust against popular visualization and clustering tools on both scRNA-seq and spatial transcriptomics datasets. In all cases, Stardust performs competitively in identifying and visualizing cell types in an accurate and spatially coherent manner.

bioinformatics↗

bioRxiv2021

Functional disruption of Transferrin expression alters reproductive physiologyin Anopheles culicifacies

Iron metabolism is crucial to maintain optimal physiological homeostasis of every organism and any alteration of the iron concentration (i.e. deficit or excess) can have adverse consequences. Transferrins are glycoproteins that play important role in iron transportation and have been widely characterized in vertebrates, and insects, but poorly studied in blood-feeding mosquitoes. Here, we characterized a 2102 bp long transcript AcTrf1a encoding putative transferrin homolog protein from mosquito An. culicifacies. A detailed in silico analysis predicts AcTrf1a (ACUA023913-RA) encodes 624 amino acid (aa) long polypeptide that carries transferrin domain. AcTrf1a also showed a putative N-linked glycosylation site, a characteristic feature of most of the mammalian transferrins and certain non-blood feeding insects. Structure modelling prediction confers the presence of an iron binding site at the N-terminal lobe of the transferrin. Our spatial and temporal expression analysis under altered pathophysiological conditions showed that AcTrf1a abundantly express in the fat-body, ovary, and its response is significantly altered (enhanced) after blood meal uptake, and exogenous bacterial challenge. Additionally, a non-heme iron supplementation of FeCl3 at 1 mM concentration not only augmented the AcTrf1a transcript expression in fat-body, also enhanced the reproductive fecundity of gravid adult female mosquitoes. RNAi mediated knockdown of AcTrf1a causes a significant reduction in the egg laying/fecundity, confirmed important role of transferrin in oocyte maturation. Further detailed characterization may help to select this transcript as a unique target to impair the mosquito reproductive outcome. HighlightsO_LIInsect transferrins are mostly glycoprotein of about 60-80 kDa molecular weight, involved in myriad physiological events and serve as a major iron transport protein. C_LIO_LIHere, we identified and characterized a 2102 bp long transcript encoding putative transferrin homolog of 624 aa long peptide, carrying only one fully functional transferrin domain at N-terminal from An. culicifacies. C_LIO_LISpatial and temporal expression analysis of AcTrf1a highlights an enriched expression in fat-body and ovary during vitellogenesis. C_LIO_LIIron supplementation and dsRNA mediated knockdown experiments together confer that AcTrf1a may have key role in the iron homeostasis regulation during oogenesis, and egg maturation in the gravid female mosquitoes. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/448311v1_fig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@a87ec0org.highwire.dtl.DTLVardef@197cecorg.highwire.dtl.DTLVardef@1588092org.highwire.dtl.DTLVardef@16110b4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig 1:C_FLOATNO Schematic presentation of iron transport from midgut to ovary by transferrin1 and oocyte reduction after AcTrf1a knockdown. Mosquito acquires iron either from blood meal or iron supplementation in sugar meal. Fat-body derived transferrin proceed towards the gut surface, load iron in its N-terminal iron-binding pocket and deliver iron to ovary. This blood meal iron is required by adult female for completion of gonotrophic cycle. (a) limited iron availability in sugar meal does not support the ovary development and hence no oogenesis; (b) when sugar meal is replaced by blood meal upregulation of transferrin protein results in rapid iron transport to various organs including ovary results in healthy ovarian growth; (c) RNAi mediated knockdown of this transporter protein transferrin in fat-body followed by blood meal, may cause reduced iron transport to ovary and consequently declines in oocyte load. C_FIG

molecular biology↗

bioRxiv2018

A novel sialylation site on Neisseria gonorrhoeae lipooligosaccharide links heptose II lactose expression with pathogenicity

Sialylation of lacto-W-neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for mAb 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho-W-acetylneuraminic acid (CMP-Neu5Ac)-containing media enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOS of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and a MS11 mutant with only lactose from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by 2-3-specific neuraminidase suggested an 2-6-linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with mAb 2C7; mAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. mAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose in vivo. Our findings reiterate the candidacy of the 2C7 epitope as a vaccine antigen and mAb 2C7 as an immunotherapeutic antibody.

microbiology↗

bioRxiv2016

DNA damage response decreases during induced pluripotent and embryonic stem cell differentiation

Genomic integrity is critical for preservation of stem cell function and is, maintained through a robust DNA damage response (DDR) with systemized DNA DSB repair by either the non-homologous end joining (NHEJ) pathway or the homologous recombination (HR) pathway. To examine DDR during stem cell differentiation, human embryonic (hES) and induced pluripotent stem (IPS) cells were exposed to DNA damaging agents and DNA damage signaling/repair measured. Differentiated cells displayed a higher frequency of residual DNA damage, chromosomal aberrations, cells with delayed {gamma}-H2AX foci disappearance and a reduced number of RAD51 foci. Factors impacting DNA DSB repair by HR formed reduced foci in differentiated cells. The reduction in repairosome foci formation after DNA damage was not due to changes in HR protein levels, which were unchanged by differentiation. Differentiated cells also displayed a higher frequency of stalled DNA replication forks and decreased firing of new replication origins from transient inhibition of DNA synthesis by hydroxyurea treatment. In addition, we observed that differentiated cells exhibit a higher frequency of R-loops. A similar decline in DDR was observed as early stage mouse astrocytes differentiated into later stage astrocytes. Our studies thus suggest that DSB repair by homologous recombination is increasingly impaired during stem cell differentiation while the NHEJ pathway is minimally altered.

cell biology↗