Biology subjects

Thakur, B.

Publications and source records attributed to Thakur, B..

15 recordsLinked to original sources

bioRxiv2026

Peptide inhibitors recognize prefusion viral fusion proteins with heterogeneous stoichiometry and rapid kinetics

Peptide fusion inhibitors, an important class of antivirals, block viral entry by targeting fusion proteins required for membrane fusion. However, their interactions with intact trimeric fusion proteins remain elusive; direct observation of binding on virions or native-like trimers has been lacking. Here, we developed a single-molecule imaging platform to visualize peptide binding in real time. LP-98 bound HIV-1 Envelope (Env) trimers on virions and, unexpectedly, prefusion-stabilized soluble Env trimers, with higher affinity for virion-associated Env and, among soluble trimers, a mutant Env. RSV fusion-inhibiting T-118 and 4ca similarly engaged prefusion-stabilized fusion (F) trimers with rapid kinetics and high-nanomolar affinities. Binding to prefusion Env or F demonstrates that peptide inhibitors can act earlier than the canonical prehairpin-intermediate model suggests. Individual binding events revealed heterogeneous peptide-to-trimer stoichiometries, with single-peptide occupancy predominating, while stepwise and simultaneous events revealed multiple routes to higher occupancy. These findings expand the canonical model of peptide fusion inhibition and provide previously inaccessible mechanistic insights into how antiviral peptide fusion inhibitors act.

microbiology↗

bioRxiv2024

Effectiveness of FLASH vs conventional dose rate radiotherapy in a model of orthotopic, murine breast cancer

PurposeRadiotherapy is an effective breast cancer treatment that enhances local tumor control and prolongs overall survival yet is associated with undesirable side effects which can impair quality of life. Ultra-high dose rate radiotherapy (FLASH) has been shown to induce less normal tissue toxicity while producing comparable tumor growth delay in a variety of preclinical tumor models when compared with conventional dose rate radiotherapy (CONV). However, growth delay is not a surrogate for tumor eradication, which is a critical endpoint of cancer therapy, and studies using FLASH in breast cancer are limited. We sought to evaluate whether FLASH produced comparable tumor control to CONV in a breast cancer model with tumor eradication as the primary endpoint. Methods and Materials106 cells from the radiation sensitive mammary tumor cell line Py117 were used to create non-metastatic, syngeneic, orthotopic tumors in the left 4th mammary fat pad of C57BL/6J mice (n=67). Tumors were established for two distinct sequential irradiation studies (Rounds 1 and 2), utilizing either large (7.5 mm into the body) or small (5 mm) treatment tumor margins, respectively. For Round 1, mice were divided into groups with either small (20-40 mm3) or large (250-800 mm3) tumors, whereas only small tumors were included in Round 2. Tumors were irradiated with FLASH (93, 192 and 200 Gy/s) or CONV (0.08 Gy/s) using 16.6 MeV FLASH and 15.7 MeV CONV electron beams. Mice in the small tumor cohort were treated with single fractions of 20, 25, or 30 Gy. The larger tumors were treated with a single fraction of 30 Gy. Tumor eradication was determined by palpation and with histology as needed to clarify physical findings. ResultsSingle fractions of FLASH and CONV demonstrated comparable treatment responses within matched cohorts of small and large tumors. A portion of small tumors treated with single fractions of 20 or 25 Gy were eradicated though most regrew within 2 to 3 weeks. Eradication of small tumors was best seen treated with 30 Gy and a large treatment tumor margin. These mice had no tumor regrowth at 30 days with either FLASH or CONV: however, euthanasia criteria were met at the 30-day time point due to concerns over skin toxicity for both FLASH and CONV groups. Small tumors treated with 30 Gy and a smaller treatment tumor margin had less skin toxicity with 75% of mice remaining tumor free at 48 days. 30 Gy FLASH and CONV applied to larger tumors demonstrated growth delay equally with a partial reduction in size but without tumor eradication. ConclusionsFLASH and CONV produced comparable tumor control in this model of orthotopic, murine breast tumors. Single fractions of 30 Gy with both FLASH and CONV applied to small tumors achieved the highest rates of tumor eradication in particular when delivered with a wider treatment margin. Skin toxicity seen at this dose and in this location could be ameliorated with the use of multiple fractions or different tangents in future studies. Efforts at eradicating larger tumors would require testing higher single fraction doses, multiple fractions, and/or hypofractionated treatment regimens. The equivalent effectiveness between FLASH and CONV in this study of murine breast tumors supports ongoing evaluation of FLASH for use in treating human breast cancer. To this end future efforts at tumor eradication with single fraction FLASH doses with comprehensive evaluation of the toxicity of organs at risk as compared to CONV will be necessary. Additionally, studies of dose-response in a range of tumor volumes with additional breast cancer cell lines and tumors, including human xenografts, along with refined target margins, will guide future studies into the use of FLASH in the adjuvant therapy of primary human breast cancer.

cancer biology↗

bioRxiv2024

Anti-HIV-1 B cell antigen receptor signaling and structure

The B cell antigen receptor (BCR) complex, comprised of antigen recognition and signaling components, functions in initiating B cell activation. While structural studies have described BCR domain organization, gaps remain in our understanding of its antigen binding domain (Fab, fragment antigen-binding) disposition, and how antigen binding is sensed to initiate signaling. Here, we report antigen affinity and signaling of the immunoglobulin (Ig) class IgM and IgG BCRs and define conformational states of full-length BCRs of two human broadly neutralizing antibodies, the glycan-specific, heavy chain domain-swapped, I-shaped 2G12, and a canonical Y-shaped antibody, CH31, that recognizes the CD4-binding site on the HIV-1 Envelope protein (Env). The BCRs adopted the shapes (I or Y) of their respective soluble antibodies, and both Ig class of BCRs of the same specificity bound Env trimers with similar affinities. We observed antigen-valenc y dependent differential signaling by the 2G12 IgM and IgG BCRs with trimeric Envs. Cryo-electron microscopy of the 2G12 IgG and CH31 IgM BCRs revealed varied Fab orientations. Structural comparisons revealed hinge points and regions of flexibility in the BCRs suggesting a highly dynamic structure of the BCR complex. Taken together, our results provide an integrated understanding of BCR structure, conformation, antigen recognition and signaling, and provide the basis for understanding antigen induced BCR signal transmission. One Sentence SummaryCryo-electron microscopy structures of full-length B cell antigen receptor complex provide a novel dynamic BCR model and a basis for understanding antigen binding induced signal transmission.

immunology↗

bioRxiv2024

Conformational trajectory of the HIV-1 fusion peptide during CD4-induced envelope opening

The hydrophobic fusion peptide (FP), a critical component of the HIV-1 entry machinery, is located at the N terminal stretch of the envelope (Env) gp41 subunit1-3. The receptor-binding gp120 subunit of Env forms a heterodimer with gp41 and assembles into a trimer, in which FP is accessible for antibody binding3. Env conformational changes or "opening" that follow receptor binding result in FP relocating to a newly formed interprotomer pocket at the gp41-gp120 interface where it is sterically inaccessible to antibody4. The mechanistic steps connecting the entry-related transition of antibody accessible-to-inaccessible FP configurations remain unresolved. Here, using SOSIP-stabilized Env ectodomains5, we visualized atomic-level details of a functional entry intermediate, where partially open Env was bound to receptor CD4, co-receptor mimetic antibody 17b, and FP-targeting antibody VRC34.01, demonstrating that FP remains antibody accessible despite substantial receptor-induced Env opening. We determined a series of structures delineating stepwise opening of Env from its closed state to a newly resolved intermediate and defining downstream re-organizations of the gp120-gp41 interface that ultimately resulted in FP burial in an antibody-inaccessible configuration. Our studies improve our understanding of HIV-1 entry and provide information on entry-related conformation reorganization of a key site of HIV vulnerability to neutralizing antibody.

microbiology↗

bioRxiv2024

SARS-CoV-2 Omicron lineage XBB spike structures, conformations, antigenicity, and receptor recognition

A recombinant lineage of the SARS-CoV-2 Omicron variant, named XBB, appeared in late 2022 and evolved descendants that successively swept local and global populations. XBB lineage members were noted for their improved immune evasion and transmissibility. Here, we determine cryo-EM structures of XBB.1.5, XBB.1.16, EG.5 and EG.5.1 spike (S) ectodomains to reveal reinforced 3-RBD-down receptor inaccessible closed states mediated by interprotomer receptor binding domain (RBD) interactions previously observed in BA.1 and BA.2. Improved XBB.1.5 and XBB.1.16 RBD stability compensated for stability loss caused by early Omicron mutations, while the F456L substitution reduced EG.5 RBD stability. S1 subunit mutations had long-range impacts on conformation and epitope presentation in the S2 subunit. Our results reveal continued S protein evolution via simultaneous optimization of multiple parameters including stability, receptor binding and immune evasion, and the dramatic effects of relatively few residue substitutions in altering the S protein conformational landscape.

biochemistry↗

bioRxiv2024

Context-dependent acetylation of the virulence regulator PhoP accounts for carbon-source specific mycobacterial growth inhibition under acidic conditions

Mycobacterium tuberculosis PhoP is essential for intracellular survival and virulence of the tubercle bacilli. Genetic evidences coupled with biochemical studies uncover that PhoP affects various aspects of M. tuberculosis pathophysiology including pH sensing during intracellular adaptation and carbon source utilization. Building on this observation, herein we report essentiality of the phoP locus in carbon-source specific mycobacterial growth. Further, our results on mycobacterial growth in the presence of different carbon sources suggest accumulation of acetyl CoA, a metabolic intermediate which acetylates major transcription factors. To explore the mechanism, we examined in vivo acetylation of PhoP, and our results suggest a link between acetylation of PhoP and mycobacterial carbon source utilization. Using a genetic screening, we identified PhoP-specific mycobacterial acetylases. Our two major findings that (a) acidic conditions of growth inhibit PhoP acetylation, which represses PhoP regulon by interfering with DNA binding activity of the regulator and (b) mycobacteria expressing acetylation-defective PhoP shows growth inhibition, together suggest a role of acetylation on mycobacterial growth via carbon-source utilization. These results have implications on intracellular survival and growth program of mycobacteria under varying environmental cues.

microbiology↗

bioRxiv2023

Vaccine induction of CD4-mimicking broadly neutralizing antibody precursors in macaques

The CD4 binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the CD4bs is occluded by glycans, immunogen expansion of appropriate naive B cells, and selection of functional antibody mutations. Here, we demonstrate immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAbs bound to HIV-1 Env demonstrated binding angles similar to human bnAbs and heavy chain second complementarity determining region-dependent binding characteristic of all known human CD4-mimicking bnAbs. Macaque nAbs were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAbs. This vaccine study initiated the B cells from which derive CD4bs bnAbs in primates, accomplishing the key first step in development of an effective HIV-1 vaccine.

immunology↗

bioRxiv2022

Structure-based stabilization of SOSIP Env enhances recombinant ectodomain durability and yield

The envelope glycoprotein (Env) is the main focus of HIV-1 vaccine development due to its critical role in viral entry. Despite advances in protein engineering, many Env proteins remain recalcitrant to recombinant expression due to their inherent metastability, making biochemical and immunological experiments impractical or impossible. Here we report a novel proline-stabilization strategy to facilitate the production of prefusion Env trimers. This approach, termed "2P", works synergistically with previously described SOSIP mutations and dramatically increases the yield of recombinantly expressed Env ectodomains without altering the antigenic or conformational properties of native Env. We determined that the 2P mutations function by enhancing the durability of the prefusion conformation and that this stabilization strategy is broadly applicable to evolutionarily and antigenically diverse Envs. These findings provide a new Env stabilization platform to facilitate biochemical research and expand the number of Env variants that can be developed as future HIV-1 vaccine candidates.

biochemistry↗

bioRxiv2022

UVC-based air disinfection system for rapid inactivation of SARS-CoV-2 present in the air

The novel coronavirus disease 2019 (COVID-19) infections have rapidly spread throughout the world, and the virus has acquired an ability to spread via aerosols even at long distances. Hand washing, face-masking, and social distancing are the primary preventive measures against infections. With mounting scientific evidence, World Health Organisation (WHO) declared COVID-19 an air-borne disease. This ensued the need to disinfect air to reduce the transmission. Ultraviolet C (UVC) comprising the light radiation of 200-280 nm range is a commonly used method for inactivation of pathogens. The heating, ventilation, and air conditioning (HVAC) systems are not beneficial in closed spaces due to poor or no ability to damage circulating viruses. Therefore, standard infection-prevention practices coupled with a strategy to reduce infectious viral load in air substantially might be helpful in reducing virus transmissibility. In this study, we implemented UV light-based strategies to combat COVID-19 and future pandemics. We tested various disinfection protocols by using UVC-based air purification systems and currently installed such a system in workspaces, rushed out places, hospitals and healthcare facilities for surface, air, and water disinfection. In this study, we designed a prototype device to test the dose of UVC required to inactivate SARS-CoV-2 in aerosols and demonstrate that the radiation rapidly destroys the virus in aerosols. The UVC treatment renders the virus non-infectious due to chemical modification of nucleic acid. We also demonstrate that UVC treatment alters the Spike protein conformation that may further affect the infectivity of the virus. We show by using a mathematical model based on the experimental data that UVC-based air disinfection strategy can substantially reduce the risk of virus transmission. The systematic treatment by UVC of air in the closed spaces via ventilation systems could be helpful in reducing the active viral load in the air.

microbiology↗

bioRxiv2022

Enhancing high-temperature degradation of polyethylene terephthalate through a synergistic division of enzyme labour between a solid-degrading thermostable cutinase and a reaction intermediate-degrading thermostable carboxylesterase

Cutinases degrade PET (polyethylene terephthalate) into various degradation intermediates (DIs) such as OET (oligoethylene terephthalate), BHET (bis-hydroxyethyl terephthalate), and MHET (mono-hydroxyethyl terephthalate), and eventually into TPA (terephthalic acid), which is the terminal product of degradation. Unlike PET, which is insoluble, TPA and the DIs are sparingly soluble in water. This causes both DIs and TPA to be partitioned into aqueous solution, where DIs accumulate without undergoing significant further degradation, despite being better substrates of cutinase than solid PET. This frustrates the creation of a circular economy involving PET and TPA (since TPA must be separated from DIs before re-condensation into PET). We argue that the non-degradation of DIs owes to cutinase becoming progressively depleted from solution, through binding to solid PET. This creates a conundrum, in that degradation of PET is anticipated to be inversely correlated with degradation of DIs (at least while solid PET remains available to deplete cutinase from solution), causing any improvement of the cutinases PET-binding efficiency to only further ensure non-degradation of released DIs. Here, we propose the deployment of a second DI-degrading enzyme; one that remains in solution, and acts as an assistant to the master PET-invading cutinase acting upon PETs surface. We demonstrate that one such dual-enzyme system, consisting of a thermostable Thermus thermophilus carboxylesterase (TTCE), characterized here for the first time, and the already-used thermostable leaf-branch compost cutinase (LCC), allows complete degradation of all products of PET hydrolysis into TPA in solution, at 60 {degrees}C, even in the presence of residual solid PET.

biochemistry↗

bioRxiv2021

N-terminal extensions strengthen hydrophobic inter-subunit interactions between HU's C-terminal domains to frustrate heterodimer formation

HU is a nucleoid-associated protein (NAP) that helps bacterial chromosomal DNA to remain compact. Escherichia coli contains two homologs of HU that are ~ 70 % identical: HU-A and HU-B. The early log phase, late log phase, and stationary phase of E. coli growth are reported to be dominated, respectively, by HU-AA homodimers, HU-BB homo-dimers, and HU-AB heterodimers. Here, we show that the formation of HU-AB heterodimers occurs to a much lower degree in HU chains that have a displaced N-terminus, whether through addition of an N-terminal affinity (polyhistidine) tag, or fusion of a fluorescent protein. A combination of mass spectrometry, spectroscopy, chromatography, and electrophoresis (exploring glutaraldehyde crosslinking of subunits) was used to study the mixing, co-expression, unfolding and refolding of HU-AA and HU-BB homodimers. The data suggests that, in HU polypeptides with N-terminal extension, whereas inter-subunit contacts between the alpha helical N-terminal domains (NTDs) undergo facile unfolding and dissociation, inter-subunit contacts between the beta sheet- and IDR-dominated C-terminal domains (CTDs) fail to do so, due to persistence of hydrophobic inter-subunit interactions between two beta sheets. This persistence causes HU to remain nominally dimeric even after substantive unfolding, and frustrates subunit exchange and heterodimer formation.

biochemistry↗

bioRxiv2020

Mechanism of bacterial adhesion and embedment in a DNA biofilm matrix:Evidence that binding of outer membrane lipopolysaccharide (LPS) to HU is key

In biofilms, bacteria are embedded within a matrix of extracellular DNA (e-DNA). Since bacterial cells and e-DNA are both negatively-charged, a positively-charged substance must act like a glue to allow bacteria to be embedded within the DNA matrix. Here we show that HU (a highly-abundant, histone-like, nucleoid-associated, DNA-binding protein) facilitates bacterium-bacterium and bacterium-DNA interactions by binding to lipopolysaccharide (LPS), a bacterial outer membrane component. We demonstrate that LPS binds to both the canonical and non-canonical DNA-binding sites on HU. We propose that the hexose sugar-terminal phosphate moieties present in the lipid A head-group of LPS bind to the same lysine/arginine residues that are involved in binding of the pentose sugar-phosphate groups in DNA. Alternate binding of LPS and DNA by HUs DNA-binding sites could allow HU to bind to bacterial cells surfaces and thus elicit bacterium-bacterium and bacterium-DNA interactions in biofilms.

microbiology↗

bioRxiv2020

Dynamics of replication origin over-activation

We determined replication patterns in cancer cells in which the controls that normally prevent excess replication were disrupted ("re-replicating cells"). Single-fiber analyses suggested that replication origins were activated at a higher frequency in re-replicating cells. However, nascent strand sequencing demonstrated that re-replicating cells utilized the same pool of potential replication origins as normally replicating cells. Surprisingly, re-replicating cells exhibited a skewed initiation frequency correlating with replication timing. These patterns differed from the replication profiles observed in non-re-replicating cells exposed to replication stress, which activated a novel group of dormant origins not typically activated during normal mitotic growth. Hence, disruption of the molecular interactions that regulates origin initiation can activate two distinct pools of potential replication origins: re-replicating cells over-activate flexible origins while replication stress in normal mitotic growth activates dormant origins.

genomics↗

bioRxiv2019

BAMscale: quantification of DNA sequencing peaks and generation of scaled coverage tracks

BAMscale is a one-step tool that processes DNA sequencing datasets from chromatin binding (ChIP-seq) and chromatin state changes (ATAC-seq, END-seq) experiments to DNA replication data (OK-seq, NS-seq and replication timing). The outputs include normalized peak scores in text format and scaled coverage tracks (BigWig) which are directly accessible to data visualization programs. BAMscale (available at https://github.com/ncbi/BAMscale) effectively processes large sequencing datasets (~100Gb size) in minutes, outperforming currently available tools.

bioinformatics↗

bioRxiv2017

Soft drug-resistant ovarian cancer cells invade via two distinct mechanisms utilizing myosin IIB

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the invasion mechanisms of drug-resistant cancer cells remains incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are softer and more contractile than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR and dual cells, suggesting protease-dependent mode of invasion in CisR cells and protease-independent mode in PacR and dual cells. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin IIB signaling pathway regulates both modes of invasion. Myosin IIB modulates matrix metalloproteinase-9 (MMP-9) secretion in CisR cells and nuclear squeezing in PacR and dual cells, thereby highlighting its importance as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.\n\nFinancial SupportAuthors acknowledge financial support from IIT Bombay Healthcare Initiative, CSIR andDepartment of Biotechnology (Govt. of India) (Grant # BT/PR14658/MED/31/107/2010).AK and BTwere supported by fellowships from UGC and CSIR respectively (Govt. of India).\n\nAuthors declare no competing financial interests\n\nAuthor contributions: AK, PR and SS designed the experiments. AK performed most of the experiments and analyzed the data.BT and SG developed drug resistant cell lines and did RT-PCR. MM performed western blot. AD performed AFM experiments. SD performed gelatin zymography. ABB, PM, AM and AD helped with live cell imaging experiments. AK and SS wrote the manuscript. All authors read and approved the final manuscript.\n\nSummary statement: This study identifies drug-specific differences in the modes of invasion utilized by ovarian cancer cells, and demonstrates the role of myosin IIB in regulating both modes of invasion.

biophysics↗