Biology subjects

Chakraborty, J.

Publications and source records attributed to Chakraborty, J..

11 recordsLinked to original sources

bioRxiv2026

Lateral interactions override nucleotide state in determining FtsZ filament curvature

The bacterial tubulin homolog FtsZ assembles into dynamic filaments that form the cytokinetic Z-ring and drives constriction during cell division. Whether a nucleotide-dependent FtsZ filament curvature plays a role in constriction is often debated. Here, we combine cryo-electron microscopy and molecular dynamics simulations to understand the structural basis of FtsZ filament curvature. Cryo-EM structures of GTP-bound Spiroplasma FtsZ filaments in two curved states emphasize that curvature is an intrinsic property of FtsZ filament, confirming recent models in which GTP hydrolysis does not dictate protofilament bending in the tubulin family. Consistently, molecular dynamics simulations demonstrate that GTP-bound filaments can adopt a range of curved conformations. The preferred intrinsic curvature appears to be such that the C-terminal end of the globular domain faces the convex surface. Structural analyses of the curved conformations identify dynamic and stationary zones at the longitudinal interfaces of the protofilament, suggesting that structural plasticity of the intermonomer interface contributes to filament bending. Furthermore, we demonstrate that lateral interactions between adjacent protofilaments straighten the filaments, overriding their relaxed curved states. Optimal orientations of lateral interactions in the Z-ring assembly could be brought about by other interacting proteins of the divisome machinery. The straighter filament conformation is likely to stimulate a higher GTPase activity. Together, our findings establish lateral association as a primary determinant for straight FtsZ filaments, analogous to the tubulin protofilaments in a microtubule lattice. The snapshots of structural states provide a mechanistic basis for how the intrinsic curvature facilitates association on the membrane and the physiological relevance of transitions between bent and straight conformations of the FtsZ filament during Z-ring assembly and constriction.

biochemistry↗

bioRxiv2026

Systematic functional dissection of germline noncoding risk variants impacting clonal hematopoiesis

Clonal hematopoiesis of indeterminate potential (CHIP) is a precursor condition characterized by the expansion of blood cell clones harboring somatic mutations originating in hematopoietic stem cells (HSCs). Since individuals with CHIP face a high risk of developing myeloid malignancies, targeting CHIP clones could provide a viable strategy for leukemia prevention. Despite its clinical significance, the mechanisms underlying CHIP predisposition and progression remain poorly understood. Recent genome wide association studies (GWAS) have identified several non-coding genetic loci that are strongly associated with CHIP; however, their underlying mechanisms still remain unknown. We hypothesize that risk variants in these non-coding loci modulate enhancer elements active in HSCs. To test this, we selected 1,374 non-coding variants from 51 loci associated for CHIP risk in the UK Biobank and screened them for regulatory activity using a Massively Parallel Reporter Assay (MPRA). We performed our lentiviral MPRA screen in MUTZ-3 cells, a human hematopoietic cell line relevant to HSCs, which express CD34 surface marker and are dependent on HSC-specific transcription factors. Using a MPRA library of [~]73,000 constructs in CD34+ fraction of MUTZ-3 cells, we identified 87 variants representing 32 GWAS loci. We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MPRA variants that affect the transcription of NKD2, FLT3, and MSI2. Our functional studies on MSI2 indicate that presence of higher levels of MSI2 mediated by CHIP risk allele enhances the clonal expansion of TET2 knockout hematopoietic stem and progenitor cells, providing a mechanistic link whereby non-coding genetic variants can influence the expansion of mutant CHIP clones.

genetics↗

bioRxiv2024

PP2C phosphatase Pic14 negatively regulates tomato Pto/Prf-triggered immunity by inhibiting MAPK activation

Type 2C protein phosphatases (PP2Cs) are emerging as important regulators of plant immune responses, although little is known about how they might impact nucleotide-binding, leucine-rich repeat (NLR)-triggered immunity (NTI). We discovered that expression of the PP2C-immunity associated candidate 14 gene (Pic14) is induced upon activation of the Pto/Prf-mediated NTI response in tomato. Pto/Prf recognize the effector AvrPto translocated into plant cells by the pathogen Pseudomonas syringae pv. tomato (Pst) and activate a MAPK cascade and other responses which together confer resistance to bacterial speck disease. Pic14 encodes a PP2C with an N-terminal kinase-interacting motif (KIM) and a C-terminal phosphatase domain. Upon inoculation with Pst-AvrPto, Pto/Prf-expressing tomato plants with loss-of-function mutations in Pic14 developed less speck disease, specifically in older leaves, compared to wild-type plants. Transient expression of Pic14 in leaves of Nicotiana benthamiana and tomato inhibited cell death typically induced by Pto/Prf and the MAPK cascade members M3K and Mkk2. The cell death-suppressing activity of Pic14 was dependent on the KIM and the catalytic phosphatase domain. Pic14 inhibited M3K- and Mkk2-mediated activation of immunity-associated MAPKs and Pic14 was shown to be an active phosphatase that physically interacts with and dephosphorylates Mkk2 in a KIM-dependent manner. Together, our results reveal Pic14 as an important negative regulator of Pto/Prf-triggered immunity by interacting with and dephosphorylating Mkk2. SIGNIFICANCE STATEMENTPlant intracellular immune receptors, typically nucleotide-binding, leucine-rich repeat proteins (NLRs) such as the tomato Prf protein activate NLR-triggered immunity (NTI) in response to specific pathogen virulence proteins. This paper reveals how a protein phosphatase interacts with and dephosphorylates a key signaling component acting downstream of Pto/Prf, likely to moderate negative effects of NTI on growth or other plant processes.

plant biology↗

bioRxiv2024

Related PP2C phosphatases Pic3 and Pic12 negatively regulate immunity in tomato to Pseudomonas syringae

Type 2C protein phosphatases (PP2Cs) constitute a large family in most plant species but relatively few of them have been implicated in immunity. To identify and characterize PP2C phosphatases that affect tomato immunity, we used CRISPR/Cas9 to generate loss-of-function mutations in 11 PP2C-encoding genes whose expression is altered in response to immune elicitors or pathogens. We report that two closely related PP2C phosphatases, Pic3 and Pic12, are involved in regulating resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Loss-of-function mutations in Pic3 lead to enhanced resistance to Pst in older but not younger leaves, whereas such mutations in Pic12 resulted in enhanced resistance in both older and younger leaves. Overexpression of Pic3 and Pic12 proteins in leaves of Nicotiana benthamiana inhibited resistance to Pst and this effect was dependent on Pic3/12 phosphatase activity and an N-terminal palmitoylation motif associated with localization to the cell periphery. Pic3 but not Pic12 had a slight negative effect on flagellin-associated reactive oxygen species generation, although their involvement in the response to Pst appeared independent of flagellin. RNA-sequencing analysis of Rio Grande (RG)-PtoR wild-type plants and two independent RG-pic3 mutants revealed that the enhanced disease resistance in RG-pic3 older leaves is associated with increased transcript abundance of multiple defense related genes. RG-pic3/RG-pic12 double mutant plants exhibited stronger disease resistance than RG-pic3 or RG-pic12 single mutants. Together, our results reveal that Pic3 and Pic12 negatively regulate tomato immunity in an additive manner through flagellin-independent pathways.

plant biology↗

bioRxiv2023

Structural Requirements for Reverse Transcription by a Diversity-generating Retroelement

Diversity-generating retroelements (DGRs) create massive protein sequence variation in ecologically diverse microbes. Variation occurs during reverse transcription of a protein-encoding RNA template coupled to misincorporation at adenosines. In the prototypical Bordetella bacteriophage DGR, the template must be surrounded by upstream and downstream RNA segments for cDNA synthesis by the reverse transcriptase bRT and associated protein Avd. The function of the surrounding RNA was unknown. Cryo-EM revealed that this RNA enveloped bRT and lay over barrel-shaped Avd, forming an intimate ribonucleoprotein (RNP). An abundance of essential interactions between RNA structural elements and bRT-Avd precisely positioned an RNA homoduplex for initiation of cDNA synthesis by cis-priming. Our results explain how the surrounding RNA primes cDNA synthesis, promotes processivity, terminates polymerization, and strictly limits mutagenesis to select proteins through mechanisms that are likely conserved in DGRs from distant taxa.

microbiology↗

bioRxiv2023

An amphipathic helix facilitates direct membrane binding of Mycoplasma FtsZ

Cell division in bacteria is initiated by constriction of the Z-ring comprising two essential proteins FtsZ and FtsA. Despite our knowledge about the crucial function of the Z-ring in bacterial division, the precise roles and mechanism of how FtsZ and FtsA drive cell constriction remain elusive. FtsZ/FtsA in wall-less bacteria like mycoplasmas is an ideal model system for obtaining mechanistic insights into Z-ring constriction in the absence of cell wall machinery. In this study, we have analyzed FtsZ and FtsA sequences of 113 mycoplasma species and compared with the corresponding protein sequences in cell-walled bacteria. We report a phylogenetically distinct group of 12 species that possess FtsZs without the canonical FtsA interacting conserved C-terminal peptide (CCTP) motif. Interestingly, these FtsZs contain a putative membrane-binding amphipathic helix as an N-terminal or C-terminal extension to the globular FtsZ domain. As a proof-of-concept, we experimentally show that the proposed C-terminal amphipathic helix in M. genitalium FtsZ binds liposomes in vitro as well as localizes to E. coli membrane in vivo. Additionally, we identify a putative cholesterol recognition motif within the C-terminal amphipathic helix region of M. genitalium FtsZ. Our study catalogues the functional variations of membrane attachment by the FtsZ and FtsA system in cell wall-less mycoplasmas and provides a new perspective to study novel functions of FtsZ/A system in cell division. ImportanceZ-ring and peptidoglycan synthesis machinery both play crucial roles in bacterial cell division. Currently, our knowledge about how FtsZ and FtsA, the two primary components of the Z-ring, function, is limited to cell-walled bacteria where ring constriction is coupled to peptidoglycan synthesis. Cell wall-less bacterial FtsZ/A system is an excellent model to study the mechanism of Z-ring constriction in the absence of cell wall synthesis machinery. Here, we analysed FtsZ protein sequences across mycoplasma species and identified their characteristic sequence features. Our study reveals a novel group of FtsZs from mycoplasma with an inherent membrane binding and probable cholesterol sensing amphipathic motif, which serves as a new paradigm to explore fundamental roles of FtsZ and FtsA in Z-ring constriction during bacterial division.

microbiology↗

bioRxiv2023

Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy

Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin decorated mitochondria are selectively recruiting autophagy receptors,which are required to terminate the organelle via autophagy. In this work we show a previously uncharacterized molecular pathway that correlates the activation of the Ca2+-dependent phosphatase Calcineurin to Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria, and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurin in vivo boosts basal mitophagy in neurons, and corrects locomotor dysfunction and mitochondrial respiratory defects of a Drosophila model of impaired mitochondrial functions. Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy.

cell biology↗

bioRxiv2023

Minuteman - A versatile cloud computational platform for collaborative research

Secure platforms for bio-computation are critical to foster increasingly complex and data-intensive collaborations involving biomedical data. Here, we present Minuteman - an open-source cloud computing platform that can be securely used across organizations. Minuteman can be used for hosting data sources and running computational pipelines in an organized way. The platform consists of three fundamental features, 1) data operations including collaborative data processing and analytics, 2) customizable user access management for secure dissemination of the data, and 3) interactive exploration of the data through 3rd party (e.g. shiny, dashboard etc.) applications that can be scaled using docker containers. Strict data access rules and user-specific roles are applied across the whole platform to maintain data security. Minuteman is ideal for scenarios where data security, and privileged access are critical, such as industry-academia collaborations, and multi-institution consortiums. Using single-cell transcriptomics preprocessing, analyses and visualization pipelines across labs, we showcase the utility of the Minuteman platform for biomedical data analyses. Minuteman code is available at (https://github.com/hayatlab/minuteman)

bioinformatics↗

bioRxiv2022

Structures of FtsZ from a cell-wall less bacterium Spiroplasma provide a mechanism for kinetic polarity

FtsZ, the tubulin homolog essential for bacterial cell division, assembles as Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we characterized the FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). SmFtsZ was found to be a slower GTPase and has higher critical concentration (CC) for polymerization compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R (close)- to T (open)- state favors polymerization. In FtsZs, a conformational switch from R (close)- to T (open)- state favors polymerization. We identified a residue, Phe224, located at the cleft between N-terminal domain (NTD) and C-terminal domain (CTD) of SmFtsZ, which is crucial for R- to T-state transition. The mutation F224M in SmFtsZ cleft resulted in higher GTPase activity and lower CC, whereas the corresponding M225F in EcFtsZ resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. Our structural analysis of interdomain interactions suggests that R- to T-state transition likely follows addition of a GTP-bound monomer to the filament through interaction of the preformed NTD. Hence, the addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain end, thus supporting the phenomenon of kinetic polarity in a single protofilament assembly.

biochemistry↗

bioRxiv2022

A salt bridge mediated resistance mechanism to FtsZ inhibitor PC190723 revealed by a single step cell-based screen

Bacterial cell division proteins, especially the tubulin homolog FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. Based on sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt-bridge interactions between the central H7 helix and beta-strands S9 and S10 mediate resistance to PC190723 in FtsZ. The single-step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.

cell biology↗

bioRxiv2020

Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission

The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has a capability of gaining rapid mutations as the virus spreads. Whole genome sequencing data offers a wide range of opportunities to study the mutation dynamics. The advantage of increasing amount of whole genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity and to investigate the implications of those mutations in protein stability and viral transmission. Four proteins, surface glycoprotein, nucleocapsid, ORF1ab and ORF8 showed frequent mutations, while envelop, membrane, ORF6 and ORF7a proteins showed conservation in terms of amino acid substitutions. Some of the mutations across different proteins showed co-occurrence, suggesting their functional cooperation in stability, transmission and adaptability. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Analysis of protein structure stability of surface glycoprotein mutants indicated viability of specific variants and are more prone to be temporally and spatially distributed across the globe. Similar empirical analysis of other proteins indicated existence of important functional implications of several variants. Analysis of co-occurred mutants indicated their structural and/or functional interaction among different SARS-COV-2 proteins. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing and containment of the disease.

genomics↗