Biology subjects

Raut, P.

Publications and source records attributed to Raut, P..

4 recordsLinked to original sources

bioRxiv2026

The Influenza Hemagglutinin Cytoplasmic Tail Domain Interacts with Phosphatidylinositol 4,5-bisphosphate

During the influenza viral life cycle, the viral glycoprotein hemagglutinin (HA) mediates binding, entry, and fusion. Densely packed clusters of HA trimers at the plasma membrane are required to produce infectious virions; however, the mechanism of HA clustering is still unknown. We have shown previously that HA co-clusters with and modulates phosphatidylinositol 4,5-bisphosphate (PIP2) in host cell plasma membranes (PM). Here, we further characterize the relationship between HA and PIP2 using molecular dynamics simulations (MD) and fluorescence photoactivation localization microscopy (FPALM) to elucidate a mechanism of HA-PIP2 interaction. We found that the interaction occurs largely between the PIP2 head group and the cytoplasmic tail domain (CTD) of HA. Mutations of the CTD were made to alter charge (HARE, HARREQ), palmitoylation sites (HAMAY), or a combination thereof (HAREMAY, RREQMAY). MD showed that HARREQ and RREQMAY had the strongest effect on HA-PIP2 interactions through a depletion in the radial distribution function of PIP2 around HA at distances [≤]2.5 nm. FPALM revealed that HA cluster density at the PM was significantly reduced by CTD mutations, with the largest reduction occurring in mutants where the CTD charge and acylation were both altered (HAREMAY). HAREMAY clusters were also found to have larger circularities and perimeters, implying a structural change to the clusters. Mutations in the HA transmembrane domain also caused modest changes to the cluster properties of HA and its co-clustering with PIP2. FPALM showed PIP2 co-clustering with HA was also affected by HA mutations with more free PIP2 localized under HAREMAY clusters. A chemical model of simultaneous HA-PIP2 and PH-PIP2 binding enables interpretation of HA-PIP2 interactions and reveals quantitative differences between PIP2 binding by HA CTD mutants. We conclude that the mechanism of HA-PIP2 interaction consists of at least electrostatic and hydrophobic components. Our insights into the mechanism of HA-PIP2 interaction, and the prevalence of putative PIP2-interacting domains in a number of viral spike proteins suggest it may be fruitful to identify methods of disrupting interactions between phosphoinositides and viral proteins.

biophysics↗

bioRxiv2024

Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APPSAA knock-in mouse model of Alzheimer disease

IntroductionReduced brain energy metabolism, mTOR dysregulation, and extracellular amyloid-{beta} oligomer (xcA{beta}O) buildup characterize AD; how they collectively promote neurodegeneration is poorly understood. We previously reported that xcA{beta}Os inhibit Nutrient-induced Mitochondrial Activity (NiMA) in cultured neurons. We now report NiMA disruption in vivo. MethodsBrain energy metabolism and oxygen consumption were recorded in APPSAA/+ mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. ResultsNiMA is inhibited in APPSAA/+ mice before other defects are detected in these amyloid-{beta}-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. GSK3{beta} signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3{beta} with lithium or TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APPSAA mice. ConclusionNiMA disruption in vivo occurs before histopathological changes and cognitive decline in APPSAA mice, and may represent an early stage in human AD.

neuroscience↗

bioRxiv2021

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 M, 1 hour) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 {micro}M) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

biophysics↗

bioRxiv2020

Mainly on the Plane: Deep Subsurface Bacterial Proteins Bind and Alter Clathrate Structure

Gas clathrates are both a resource and a hindrance. They store massive quantities of natural gas but also can clog natural gas pipelines, with disastrous consequences. Eco-friendly technologies for controlling and modulating gas clathrate growth are needed. Type I Antifreeze Proteins (AFPs) from cold-water fish have been shown to bind to gas clathrates via repeating motifs of threonine and alanine. We tested whether proteins encoded in the genomes of bacteria native to natural gas clathrates bind to and alter clathrate morphology. We identified putative clathrate-binding proteins (CBPs) with multiple threonine/alanine motifs in a putative operon (cbp) in metagenomes from natural clathrate deposits. We recombinantly expressed and purified five CbpA proteins, four of which were stable, and experimentally confirmed that CbpAs bound to tetrahydrofuran (THF) clathrate, a low-pressure analog for structure II gas clathrate. When grown in the presence of CbpAs, THF clathrate was polycrystalline and plate-like instead of forming single, octahedral crystals. Two CbpAs yielded branching clathrate crystals, similar to the effect of Type I AFP, while the other two produced hexagonal crystals parallel to the [1 1 1] plane, suggesting two distinct binding modes. Bacterial CBPs may find future utility in industry, such as maintaining a plate-like structure during gas clathrate transportation. Table of Contents Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗