Biology subjects

Choudhury, P.

Publications and source records attributed to Choudhury, P..

9 recordsLinked to original sources

bioRxiv2025

Coronavirus protein interaction mapping in bat and human cells identifies molecular and genetic switches for immune evasion and replication

Coronaviruses, including SARS-CoV-2, can cause severe disease in humans, whereas reservoir hosts like Rhinolophus bats remain asymptomatic. To investigate how host-specific protein-protein interactions (PPIs) influence infection, we generated comparative PPI maps for SARS-CoV-2 and its bat-origin relative RaTG13 using affinity purification-mass spectrometry (AP-MS) in human and Rhinolophus ferrumequinum (RFe) bat cells. This approach identified both conserved and virus- and host-specific interactions that regulate infection dynamics. Notably, SARS-CoV-2 required a non-synonymous mutation in nucleocapsid to replicate in bat cells expressing human ACE2 and TMPRSS2. Analysis of the viral protein Orf9b revealed differential interactions with mitochondrial proteins Tom70 and MTARC2. A single residue difference in Orf9b between SARS-CoV-2 and RaTG13 functions as a molecular switch, weakening Tom70 binding and immune evasion in human cells while enhancing interaction with the bat-specific restriction factor MTARC2. These findings demonstrate how a single-residue substitution can reshape virus-host interactions and contribute to immune evasion and host adaptation.

systems biology↗

bioRxiv2025

STARR-CRAAVT: A platform to design cell type-specific regulatory elements for next-generation gene therapy

Precise control of transgene expression through novel enhancer-promoter combinations is a promising strategy for advancing gene therapy mediated by adeno-associated virus (AAV). We present STARR-CRAAVT, a novel STARR-Seq-derived platform to screen for enhancers in the AAV context. Using HepG2 and HaCaT cells as screening models, we applied STARR-CRAAVT for the identification of cell type-specific enhancers. We integrated epigenetic datasets into an in-silico library of putative HepG2 enhancers and captured corresponding fragments from genomic DNA. The fragments were processed into AAV libraries and applied to HepG2 and HaCaT cells. STARR-CRAAVT analysis revealed a selective activity of the libraries confirming the HepG2-directed in-silico design and the specificity of single enhancer-promoter combinations could be validated using luciferase reporter assays. In addition, we scrutinized the impact of key experimental parameters on enhancer identification and found that the used promoter type had significant influence on the ability of candidates to act as enhancers. Furthermore, switching the location of identified enhancers in reporter assays revealed that the level of enhancer activity is highly dependent on the position in the AAV genome. Taken together, our study yields novel insights into enhancer function and demonstrates that STARR-CRAAVT can be employed to identify cell type-specific enhancers, highlighting promoter preference and enhancer positioning as key considerations for enhancer screening campaigns.

genomics↗

bioRxiv2025

In Silico Evaluation and Therapeutic Targeting of LVDD9B Protein for WSSV Inhibition: Molecular and Ecological Insights for Aquaculture Solutions

BackgroundThis study aimed to investigate structural dynamics, binding interactions, stability, pharmacokinetics, ecological risks, and bioactivity of shrimp receptor protein LVDD9B to identify potential therapeutic candidates against White Spot Syndrome Virus (WSSV). MethodsLVDD9B proteins 3D structure was predicted using SWISS-MODEL and validated with ProSA and Ramachandran plots. Protein-protein docking between LVDD9B and VP26 (WSSV protein) was performed using HADDOCK 2.4 server. Molecular docking, dynamics simulations, binding-free energy calculations, principal component analysis (PCA), electrostatic, and vibrational frequency analyses evaluated binding affinity, stability and polarity of complexes. Results128-amino-acids of LVDD9B protein was predicted as predominantly cytoplasmic with stable, and hydrophilic, with structural analysis identified key secondary structures and conserved chitin-binding site. Docking studies revealed strong interactions between LVDD9B and VP26, supported by hydrogen-bonds and salt bridges. Molecular dynamics simulations demonstrated stable complexes with fluctuating RMSD values, and MM/GBSA calculations indicated favorable binding free energies. Pharmacokinetic analysis highlighted promising bioavailability and drug-like properties for Luteolin and Quercetin from Cuscuta reflexa, while ecological assessment identified Cosmosiin as least hazardous, with Quercetin and Luteolin showing higher toxicity. PCA revealed stable protein-ligand complexes with flexibility in Apo form. Isorhoifolin exhibited the lowest internal energy (-2099.4722 Hartree) and highest dipole moment (8.1833 Debye). Frontier orbital analysis showed HOMO-LUMO gaps (4.05-4.34 eV) influencing reactivity, while MEP and vibrational frequency analyses supported compound stability and bioactivity. ConclusionsThis study explores LVDD9Bs structural and interaction dynamics for developing antiviral therapy against WSSV, highlighting therapeutic potential of Cosmosiin, Isorhoifolin, Quercetin and Luteolin based on their pharmacokinetic and ecological profiles.

bioinformatics↗

bioRxiv2024

Decoding polyubiquitin regulation of KV7. 1 functional expression with engineered linkage-selective deubiquitinases

Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the ubiquitin code that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP- targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.

molecular biology↗

bioRxiv2024

Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins-- ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis-- the E2 binding site, the catalytic cysteine, and the ubiquitin exosite-- as revealed by a 3.1 [A] cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and CaV2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.

biophysics↗

bioRxiv2023

Disease progression modelling reveals heterogeneity in trajectories of Lewy-type alpha-synuclein pathology

Lewy body (LB) disorders, characterized by the aggregation of misfolded -synuclein proteins, exhibit notable clinical heterogeneity. This may be due to variations in accumulation patterns of LB neuropathology. By applying data-driven disease progression modelling to regional neuropathological LB density scores from 814 brain donors, we describe three inferred trajectories of LB pathology that were characterized by differing clinicopathological presentation and longitudinal antemortem clinical progression. Most donors (81.9%) showed earliest pathology in the olfactory bulb, followed by accumulation in either limbic (60.8%) or brainstem (21.1%) regions. The remaining donors (18.1%) exhibited the first abnormalities in brainstem regions. Early limbic pathology was associated with Alzheimers disease-associated characteristics. Meanwhile, brainstem-first pathology was associated with progressive motor impairment and substantial LB pathology outside of the brain. Our data provides evidence for heterogeneity in the temporal spread of LB pathology, possibly explaining some of the clinical disparities observed in LBDs.

pathology↗

bioRxiv2023

Regulation of RNA methylation by therapy treatment, promotes tumor survival

Our data previously revealed that chemosurviving cancer cells translate specific genes. Here, we find that the m6A-RNA-methyltransferase, METTL3, increases transiently in chemotherapy-treated breast cancer and leukemic cells in vitro and in vivo. Consistently, m6A increases on RNA from chemo-treated cells, and is needed for chemosurvival. This is regulated by eIF2 phosphorylation and mTOR inhibition upon therapy treatment. METTL3 mRNA purification reveals that eIF3 promotes METTL3 translation that is reduced by mutating a 5'UTR m6A-motif or depleting METTL3. METTL3 increase is transient after therapy treatment, as metabolic enzymes that control methylation and thus m6A levels on METTL3 RNA, are altered over time after therapy. Increased METTL3 reduces proliferation and anti-viral immune response genes, and enhances invasion genes, which promote tumor survival. Consistently, overriding phospho-eIF2 prevents METTL3 elevation, and reduces chemosurvival and immune-cell migration. These data reveal that therapy-induced stress signals transiently upregulate METTL3 translation, to alter gene expression for tumor survival. One sentence summarym6A enzyme translation upon therapy stress, promotes tumor survival

molecular biology↗

bioRxiv2017

Characterization of a replicating mammalian orthoreovirus with tetracysteine tagged μNS for live cell visualization of viral factories

Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs) which are sites of viral transcription, translation, assembly, and replication. MRV non-structural protein, NS, comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced NS. We set out to develop a method to overcome this obstacle by utilizing the 6 amino-acid (CCPGCC) tetracysteine (TC)-tag and FlAsH-EDT2 reagent. The TC-tag was introduced into eight sites throughout NS, and the capacity of the TC-NS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC-tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-NS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore the rTC-NS viruses were utilized to infect cells and examine VF dynamics using live cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs, and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in depth studies of VF dynamics and host cell interactions.\n\nImportanceMRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection, but remain to be fully characterized. To study VFs, researchers have focused on visualizing the non-structural protein NS which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC-tag into the NS open reading frame. Characterization of each recombinant reovirus sheds light on NS interactions with viral proteins. Moreover, utilizing the TC labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

microbiology↗

bioRxiv2017

Mammalian Orthoreovirus Factories Modulate Stress Granule Protein Localization by Interaction with G3BP1

Mammalian orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2 which promotes formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early, then interferes with SG formation as infection proceeds. In this work, we found that SG associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of SG effector protein, G3BP1, with MRV non-structural protein {sigma}NS, which localizes to VFs via association with VF nucleating protein, NS. Deletion analysis of the {sigma}NS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that the association and VF localization of G3BP1 is not occurring solely through RNA or ribosomal binding, but requires both RNA and ribosomal binding domains of G3BP1 for maximal VFL localization and {sigma}NS association. Co-expression of {sigma}NS and NS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that {sigma}NS association with and relocalization of G3BP1 to the VF periphery plays a role in SG disruption to facilitate MRV replication in the host translational shutoff environment.\n\nIMPORTANCESGs and SG effector proteins have emerged as important, yet poorly understood, players in the hosts innate immune response to virus infection. MRV infection induces SGs early during infection that are dispersed and/or prevented from forming during late stages of infection despite continued activation of the eIF2 signaling pathway. Cellular and viral components involved in disruption of SGs during late stages of MRV infection remain to be elucidated. This work provides evidence that MRV disruption of SGs may be facilitated by association of MRV non-structural protein {sigma}NS with major SG effector protein G3BP1 and subsequent localization of G3BP1 and other SG associated proteins around the periphery of virus encoded factories, interrupting the normal formation of SGs. Our findings also reveal the importance of G3BP1 as an inhibitor of MRV replication during infection for the first time.

microbiology↗