Biology subjects

Samanta, D.

Publications and source records attributed to Samanta, D..

9 recordsLinked to original sources

bioRxiv2026

Influenza virus Non Structural protein 1 (NS1) Chaperones Nucleoprotein (NP) Oligomerization to Coordinate Ribonucleoprotein Assembly and Genome Replication

Influenza virus nucleoprotein (NP) co-transcriptionally enwraps viral genomic/antigenomic RNAs to assemble viral ribonucleoprotein complexes (RNPs), while the non-structural protein-1 (NS1) is classically recognized as an antagonist of host antiviral responses. Although NP-NS1 interaction has long been reported, the molecular basis of this protein-protein interaction and its relevance in the virus life cycle remain elusive. Here, we define the NP-NS1 interaction interface and uncover a noncanonical role of NS1 in regulating RNP assembly and viral genome replication. Using integrated biochemical, biophysical, and structural analyses, we demonstrate that the RNA-binding domain (RBD) of NS1 directly engages the NP tail-loop, conferring high affinity for monomeric NP. Functional assays reveal that a cytoplasm-restricted NS1 variant acts as an NP-specific chaperone by stabilizing monomeric NP through sequestration of its intrinsically flexible tail-loop and by scaffolding NP-NP interactions. During influenza virus infection, phosphorylation at the homotypic interface restricts NP in a replication-competent monomeric state. Cytoplasmic NS1 recruits these phosphorylated monomeric NPs into assembling RNPs, thereby facilitating viral genome replication and virus propagation. Collectively, our findings establish NS1 as a selective cytoplasmic chaperone of NP and reveal a novel regulatory axis governing influenza virus RNP biogenesis and replication.

molecular biology↗

bioRxiv2025

Gene regulatory network transitions reveal the central transcription factors in lung adenocarcinoma progression

Transcription factors play a central role in cancer growth, progression, and metastasis, and contribute to intratumor phenotypic plasticity which enable drug tolerance and cancer relapse. Changes in the regulatory activities of transcription factors in cancer may not always be detected by investigating mutational signatures or differential expression of the transcription factor, as done in traditional analysis. In addition, past studies have focused on the activities of transcription factors in tumors as a whole and thus, have not fully captured the heterogeneity in gene regulation among different cell types within the tumor microenvironment. In this work, through an analysis of the transitions in regulatory network architecture and gene regulation dynamics, we identify the central transcription factors associated with lung adenocarcinoma progression. The gene NR2F1, associated with neurodevelopment and cancer dormancy, emerge as a key transcription factor in the progression of lung adenocarcinoma. We further identify transcription factors that are only active in cancer samples and uncover how changes in gene regulation dynamics influence intratumor heterogeneity. Taken together, our work elucidates the transitions in gene regulatory network during cancer progression, identifies central transcription factors in this process, and reveals the complex regulatory changes cooccurring in different cell types within the tumor microenvironment.

systems biology↗

bioRxiv2024

Coxiella burnetii-containing vacuoles interact with host recycling-endosomal proteins Rab11a and Rab35 for vacuolar expansion

Coxiella burnetii is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. Although acute Q fever manifests as atypical pneumonia, chronic infection may lead to life-threatening endocarditis. The lack of effective antibiotics and a licensed vaccine for Coxiella in the U.S. warrants further research into Coxiella pathogenesis. Within the host cells, Coxiella replicates in an acidic phagolysosome-like vacuole termed Coxiella-containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for Coxiella survival and that the Coxiella Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the immature endosomes in Coxiella-infected cells remained unclear. Our recent CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. A quantitation of Rab11a/Rab35-positive CCVs at 3- and 6-days post-infection (dpi) revealed that at 3 dpi, [~]52% of CCVs were positive for Rab11a, whereas only [~]39% CCVs were positive for Rab35. This pattern reversed at 6-dpi, when only [~]22% of CCVs were positive for Rab11a and [~]64% of CCVs were positive for Rab35. These data suggest that the CCV preferentially interacts with Rab11a and Rab35-positive recycling endosomes depending on the stage of maturation. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in Coxiella-infected cells compared to mock cells suggesting that Coxiella increases the recycling endosome content in infected cells. Finally, a siRNA-mediated knockdown of both Rab11a and Rab35 resulted in significantly smaller CCVs, suggesting that recycling endosomal Rab proteins are essential for CCV expansion. Overall, our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for vacuolar expansion and potentially uncovers novel host cell factors essential for Coxiella pathogenesis.

microbiology↗

bioRxiv2023

In silico and in vitro characterization of the mycobacterial protein Ku to unravel its role in non-homologous end-joining DNA repair

Non-homologous end-joining DNA repair is essential for the survival and sustenance of M. tuberculosis (Mtb) in the dormant stage of its life cycle. The ability of Mtb to sustain itself in the inactive form has been reported to be the critical factor for its resilience over the years. To unravel one of the salient features of the Mtbs arsenal, we exploited in silico and in vitro tools to characterize the DNA binding properties of mycobacterial protein Ku (mKu) and its role in mycobacterial NHEJ. Here, we report the strong affinity of mKu for linear dsDNA exhibiting positive cooperativity for dsDNAs ({zeta}40bp). Molecular dynamics complemented with in vitro experiments showed that the DNA binding of mKu provides stability to both mKu homodimer and the DNA. Furthermore, mKu end-capping of DNA was seen to protect the DNA termini against nucleolytic degradation by exonuclease. The DNA-mKu association formed higher-order oligomers probably due to the lodgement of two DNA molecules at opposite ends of the mKu homodimer. The ability of mKu to form continuous filament-like structures with DNA indicated its potential role in mycobacterial NHEJ synapsis.

biochemistry↗

bioRxiv2023

Nanopore Sensors for Enhanced Detection of Nanoparticles

Nanopore sensing is a technique based on the Coulter principle to analyze and characterize nanoscale materials with single entity resolution. However, its use in nanoparticle characterization has been constrained by the need to tailor the nanopore aperture size to the size of the analyte, precluding the analysis of heterogenous samples. Additionally, nanopore sensors often require the use of high salt concentrations to improve the signal-to-noise ratio, which further limits their ability to study a wide range of nanoparticles that are unstable at high ionic strength. Here, we report the development of nanopore sensors enhanced by a polymer electrolyte system, enabling the analysis of heterogenous nanoparticle mixtures at low ionic strength. We present a finite element model to explain the anomalous conductive/resistive pulse signals observed and compare these results with experiments. Furthermore, we demonstrate the wide applicability of the method by characterizing metallic nanospheres of varied sizes, plasmonic nanostars with various degrees of branching, and protein-based spherical nucleic acids with different oligonucleotide loadings. Our system will complement the toolbox of nanomaterials characterization techniques and will enable real-time optimization workflow for engineering a wide range of nanomaterials.

biophysics↗

bioRxiv2022

Enrichment of breast cancer stem cells following cytotoxic chemotherapy is mediated by hypoxia-inducible factors

Breast cancers (BCs) that do not express the estrogen or progesterone receptor or human epidermal growth factor receptor 2 are known as triple negative breast cancers (TNBCs). Women with TNBC receive non-targeted chemotherapy with a durable response rate of less than 20%. BC stem cells (BCSCs) are a small subpopulation of BC cells that are characterized by the capacity for infinite self-renewal; are the only BC cells capable of forming a secondary (recurrent or metastatic) BC; and must be eliminated in order to eradicate BC. Hypoxia-inducible factors (HIFs) activate hundreds of genes in TNBCs and HIF-1 expression in the diagnostic tumor biopsy is associated with patient mortality. In this paper, we report that treatment of TNBC cells with cytotoxic chemotherapy increased HIF-1 and HIF-2 protein levels and HIF target gene expression. Chemotherapy also increased the percentage of BCSCs through pathways involving interleukin-6 (IL-6), IL-8, and multidrug resistance 1. HIF inhibitors blocked increased BCSC specification in response to cytotoxic chemotherapy and combination therapy led to tumor eradication. Increased HIF target gene expression in BC biopsies was correlated with increased mortality, especially in those patients treated with chemotherapy alone. Our results suggest that HIF-dependent BCSC enrichment provides a molecular and cellular basis for the high incidence of relapse in women with TNBC.

cancer biology↗

bioRxiv2021

Architecture of the linker-scaffold in the nuclear pore

The nuclear pore complex (NPC) is the sole bidirectional gateway for nucleocytoplasmic transport. Despite recent progress in elucidating the arrangement of the structured scaffold building blocks in the NPC symmetric core, their cohesion by multivalent unstructured linker proteins remained elusive. Combining biochemical reconstitution, high resolution structure determination, docking into cryo-electron tomographic reconstructions, and physiological validation, we elucidated the architecture of the entire linker-scaffold, yielding a near-atomic composite structure of the symmetric core accounting for [~]77 MDa of the human NPC. Whereas linkers generally play a rigidifying role, the linker-scaffold of the NPC provides the plasticity and robustness necessary for the reversible constriction and dilation of its central transport channel. Our results complete the structural characterization of the NPC symmetric core, providing a rich foundation for future functional studies. One sentence summaryAn interdisciplinary analysis established the near-atomic molecular architecture and evolutionary conservation of the linker-scaffold of the human nuclear pore complex.

biochemistry↗

bioRxiv2019

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Upon host cell infection, the obligate intracellular bacterium C. burnetii resides and multiplies within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring lysosomal markers as well as acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and subsequent bacterial replication in the mature CCV. Initial CCV acidification is required to activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we recently found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates CCV pH. Because heterotypic fusion with host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant ({Delta}dotA) C. burnetii. We observed significantly fewer LAMP1-positive lysosomes, along with less acidic \"mature\" endosomes (pH~5.8), in WT-infected cells, compared to mock or {Delta}dotA-infected cells. Further, while endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and {Delta}dotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells, indicating that the C. burnetii T4BSS inhibits endosomal maturation. Finally, increasing the number of acidic lysosomes by overexpressing the transcription factor EB inhibited C. burnetii growth, indicating lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii regulates CCV pH, possibly by reducing the number of host lysosomes available for heterotypic fusion.\n\nAuthor summaryThe obligate intracellular bacterium Coxiella burnetii causes human Q fever, which manifests as a flu-like illness but can develop into a life-threatening and difficult to treat endocarditis. C. burnetii, in contrast to many other intracellular bacteria, thrives within a lysosome-like vacuole in host cells. However, we previously found that the C. burnetii vacuole is not as acidic as lysosomes and increased acidification kills the bacteria, suggesting that C. burnetii regulates the pH of its vacuole. Here, we discovered that C. burnetii blocks endosomal maturation and acidification during host cell infection, resulting in fewer lysosomes in the host cell. Moreover, increasing lysosomes in the host cells blocked C. burnetii growth. Together, our study suggests that C. burnetii regulates vacuole acidity and blocks endosomal acidification in order to produce a permissive intracellular niche.

microbiology↗

bioRxiv2018

nPIST: A Novel Actin Binding Protein of trans-Golgi Network

npist is the neuronal isoform of PIST, a trans-golgi associated protein involved in major modulation of vesicular trafficking. nPIST interacts with glutamate delta2 receptor (GluR{delta}2) in Purkinje cells. Our study shows nPIST as a novel actin binding protein. Our structure based sequence analysis shows nPIST contains one WH2-like domain. Further our experimental analysis illustrates that fragment of nPIST consisting of WH2-like domain binds to actin. Moreover it was found that nPIST contains several regions involved in interaction with actin. The binding of nPIST to actin through multiple actin binding regions facilitated actin filament stabilization in vitro. In vivo, nPIST localized actin in perinuclear region as a blotch when ectopically expressed.

cell biology↗