Biology subjects

Shehata, M.

Publications and source records attributed to Shehata, M..

8 recordsLinked to original sources

bioRxiv2025

N-Glycans Modulate HIV-1 Env Conformational Plasticity

Human immunodeficiency virus-1 (HIV-1) remains a global health crisis, with over 39 million people living with the virus and no effective vaccine available. Central to HIV infection and immune evasion is the envelope glycoprotein (Env), a heavily glycosylated class I fusion protein that mediates viral entry and is the sole immunogenic target. Despite the recent advancements provided by imaging techniques, the characterization of Envs structure and dynamics within its native membrane environment remains incomplete. Here, we present microsecond-long, all-atom molecular dynamics simulations of the full-length Env glycoprotein embedded in a biologically relevant lipid bilayer with a complete glycosylation profile. Our simulations reveal a pronounced tilting motion of Env relative to the membrane, with supporting evidence from cryo-electron tomography, which also captures Env tilting within the native membrane. Importantly, we identify a critical role for N-linked glycans at N88 and N611 in modulating the tilting transition. These findings highlight the dual role of Envs glycan shield as both a protective barrier against neutralizing antibodies and a structural modulator of conformational plasticity. While providing an atomically detailed view of Env in a native membrane environment and advancing the general understanding of its glycan shield and its vulnerabilities, this work also suggests a possible strategy to modulate Envs conformational plasticity.

biophysics↗

bioRxiv2024

FLIMPA: A versatile software for Fluorescence Lifetime Imaging Microscopy Phasor Analysis

Fluorescence lifetime imaging microscopy (FLIM) is an advanced microscopy technique capable of providing a deeper understanding of the molecular environment of a fluorophore. While FLIM data were traditionally analysed through the exponential fitting of the fluorophores emission decays, the use of phasor plots is increasingly becoming the preferred standard. This is due to their ability to visualise the distribution of fluorescent lifetimes within a sample, offering insights into molecular interactions in the sample without the need for model assumptions regarding the exponential decay behaviour of the fluorophores. However, so far most researchers have had to rely on commercial phasor plot software packages, which are closed-source and rely on proprietary data formats. In this paper, we introduce FLIMPA, an opensource, stand-alone software for phasor plot analysis that provides many of the features found in commercial software, and more. FLIMPA is fully developed in Python and offers advanced tools for data analysis and visualisation. It enhances FLIM data comparison by integrating phasor points from multiple trials and experimental conditions into a single plot, while also providing the possibility to explore detailed, localised insights within individual samples. We apply FLIMPA to introduce a cell-based assay for the quantification of microtubule depolymerisation, measured through fluorescence lifetime changes of SiR-tubulin, in response to various concentrations of Nocodazole, a microtubule depolymerising drug relevant to anti-cancer treatment.

biophysics↗

bioRxiv2023

Simulation-Driven Design of Stabilized SARS-CoV-2 Spike S2 Immunogens

The full-length prefusion-stabilized SARS-CoV-2 spike (S) is the principal antigen of COVID-19 vaccines. Vaccine efficacy has been impacted by emerging variants of concern that accumulate most of the sequence modifications in the immunodominant S1 subunit. S2, in contrast, is the most evolutionarily conserved region of the spike and can elicit broadly neutralizing and protective antibodies. Yet, S2s usage as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design S2-only immunogens stabilized in a closed prefusion conformation. Molecular simulations provide a mechanistic characterization of the S2 trimers opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Structural characterization via cryo-EM shows the molecular basis of S2 stabilization in the closed prefusion conformation. Informed by molecular simulations and corroborated by experiments, we report an engineered S2 immunogen that exhibits increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses.

biophysics↗

bioRxiv2023

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD=SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties. Author summaryVirus infections often cause organ damage via excessive inflammation and oxidative stress. The identification of host-directed treatments that reduce inflammation, accumulation of reactive oxygen species, and viral infectivity is an important goal of antiviral drug development. One advantage of host-directed antivirals is that their targets are encoded by the stable host genome, making emergence of viral resistance less likely. The KEAP1/NRF2 signaling pathway is the most important pathway in humans that protects cells from oxidative stress, and it also induces antiviral and anti-inflammatory responses. NRF2 activators, therefore, are promising candidates for development of host-directed antivirals. We evaluated three NRF2-activating compounds as host-directed treatments for influenza A virus (IAV) infection. All three compounds reduced viral replication, cellular inflammation, and reactive oxygen species. Surprisingly, these effects were completely independent of NRF2 signaling. Instead, we found that these compounds (particularly 4-octyl itaconate) interfere with export of viral RNA/protein complexes from the nucleus, thereby reducing release of viral particles. The most plausible explanation is that the "natural" target of these compounds, KEAP1 (which limits NRF2 signaling), and the nuclear export factor XPO1 (which is required for egress of IAV from the nucleus) contain similar binding sites, thus allowing "NRF2 activators" to also inhibit XPO1.

microbiology↗

bioRxiv2022

A transcriptional response to replication stress selectively expands a subset of BRCA2-mutant mammary epithelial cells

BRCA2 mutation carriers preferentially develop luminal-like breast cancers, but it remains unclear how BRCA2 mutations affect mammary epithelial subpopulations. Here, we report that Brca2mut/WT mammary organoids subjected to replication stress activated a transcriptional response that selectively expands Brca2mut/WT luminal cells lacking hormone receptor expression (HR-). While CyTOF analyses revealed comparable epithelial compositions among wildtype and Brca2mut/WT mammary glands, Brca2mut/WT HR- luminal cells exhibited greater organoid formation and preferentially survived and expanded under replication stress. ScRNA-seq analysis corroborated the expansion of HR- luminal cells which express elevated levels of Tetraspanin-8 (Tspan8) and Thrsp mRNA, and pathways implicated in replication stress survival including Type I interferon responses. Notably, CRISPR/Cas9-mediated deletion of Tspan8 or Thrsp prevented Brca2mut/WT HR- luminal cell expansion. Our findings indicate that Brca2mut/WT cells have an activate a transcriptional response after replication stress that preferentially favours outgrowth of HR- luminal cells through the expression of interferon-responsive and mammary alveolar genes.

cancer biology↗

bioRxiv2021

Broad antiviral effects of Echinacea purpurea against SARS-CoV-2 variants of concern and potential mechanism of action

BackgroundSARS-CoV-2 variants of concern (VOC) represent an alarming threat as they show altered biological behavior and may escape vaccination effectiveness. Some exhibit increased pathogenicity and transmissibility compared to the original wild type WUHAN (Hu-1). Broad-spectrum antivirals could complement and further enhance preventive benefits achieved through SARS-CoV-2 vaccination campaigns MethodsThe anti-coronavirus activity of Echinacea purpurea (Echinaforce(R) extract, EF) against (i) VOCs B1.1.7 (alpha), B.1.351.1 (beta), P.1 (gamma), B1.617.2 (delta), AV.1 (Scottish) and B1.525 (eta), (ii) SARS-CoV-2 spike (S) protein-pseudotyped viral particles and reference strain OC43 as well as (iii) wild-type SARS-CoV-2 (Hu-1) were analyzed. Molecular dynamics (MD) were applied to study interaction of Echinaceas phytochemical markers with known pharmacological viral and host cell targets. ResultsEF extract broadly inhibited propagation of all investigated SARS-CoV-2 VOCs as well as entry of SARS-CoV-2 pseudoparticles at EC50s ranging from 3.62 to 12.03 {micro}g/ml. Preventive addition of 20 {micro}g/ml EF to epithelial cells significantly reduced sequential infection with SARS-CoV-2 (Hu-1) as well as with the common human strain OC43. MD analyses showed constant binding affinities to Hu-1, B1.1.7, B.1.351, P.1 and B1.617.2-typic S protein variants for alkylamides, caftaric acidand feruoyl-tartaric acid in EF extract. They further indicated that the EF extract could possibly interact with TMPRSS-2, a serine protease required for virus endocytosis. ConclusionsEF extract demonstrated stable antiviral activity across 6 tested VOCs, which is likely due to the constant affinity of the contained phytochemical marker substances to all spike variants. A possible interaction of EF with TMPRSS-2 partially would explain cell protective benefits of the extract by inhibition of endocytosis. EF may therefore offer a supportive addition to vaccination endeavors in the control of existing and future SARS-CoV-2 virus mutations.

microbiology↗

bioRxiv2020

Team flow is a unique brain state associated with enhanced information integration and neural synchrony

Team flow occurs when a group of people reaches high task engagement while sharing a common goal as in sports teams and music bands. While team flow is a superior enjoyable experience to individuals experiencing flow or regular socialization, the neural basis for such superiority is still unclear. Here, we addressed this question utilizing a music rhythm task and electroencephalogram hyper-scanning. Experimental manipulations held the motor task constant while disrupted the hedonic musical correspondence to blocking flow or occluded the partners body and task feedback to block social interaction. The manipulations effectiveness was confirmed using psychometric ratings and an objective measure for the depth of flow experience through the inhibition of the auditory-evoked potential to a task-irrelevant stimulus. Spectral power analysis revealed higher beta/gamma power specific to team flow at the left temporal cortex. Causal interaction analysis revealed that the left temporal cortex receives information from areas encoding individual flow or socialization. The left temporal cortex was also significantly involved in integrated information at both the intra- and inter-brains levels. Moreover, team flow resulted in enhanced global inter-brain integrated information and neural synchrony. Thus, our report presents neural evidence that team flow results in a distinct brain state and suggests a neurocognitive mechanism by which the brain creates this unique experience. Data AvailabilityAll data and analysis codes used in the preparation of this article are available at https://osf.io/3b4hp.

neuroscience↗

bioRxiv2017

Autophagy enhances memory erasure through synaptic destabilization

There is substantial interest in memory reconsolidation as a target for the treatment of anxiety disorders such as post-traumatic stress disorder (PTSD). However, its applicability is restricted by reconsolidation-resistant conditions that constrain the initial memory destabilization. In this study, we investigated whether the induction of synaptic protein degradation through autophagy modulation, a major protein degradation pathway, can enhance memory destabilization upon retrieval and whether it can be utilized to overcome these conditions. Here, using male mice in an auditory fear reconsolidation model, we showed that autophagy contributes to memory destabilization and its induction can be utilized to enhance erasure of a reconsolidation-resistant auditory fear memory that depended on -amino-3-hydroxy-5-methyl4-isoxazolepropionic acid receptor (AMPAR) endocytosis. Using male mice in a contextual fear reconsolidation model, autophagy induction in the amygdala or in the hippocampus enhanced fear or contextual memory destabilization, respectively. The latter correlated with AMPAR degradation in the spines of the contextual memory-ensemble cells. Using male rats in an in vivo long-term potentiation reconsolidation model, autophagy induction enhanced synaptic destabilization in an N-methyl-D-aspartate receptor-dependent manner. These data indicate that induction of synaptic protein degradation can enhance both synaptic and memory destabilization upon reactivation and that autophagy inducers have the potential to be used as a therapeutic tool in the treatment of anxiety disorders.\n\nSignificance StatementIt has been reported that inhibiting synaptic protein degradation prevents memory destabilization. However, whether the reverse relation is true and whether it can be utilized to enhance memory destabilization is still unknown. Here we addressed this question on the behavioral, molecular and synaptic levels, and showed that induction of autophagy, a major protein degradation pathway, can enhance memory and synaptic destabilization upon reactivation. We also show that autophagy induction can be utilized to overcome a reconsolidation-resistant memory, suggesting autophagy inducers as a potential therapeutic tool in the treatment of anxiety disorders.

neuroscience↗