Deciphering molecular mechanisms stabilizing the reovirus-binding complex.

Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.

Activation of neurotoxic A1-reactive astrocytes by SFTS virus infection accelerates fatal brain damage in IFNAR1-/- mice.

Severe fever with thrombocytopenia syndrome (SFTS) has a high mortality rate compared to other infectious diseases. SFTS is particularly associated with a high risk of mortality in immunocompromised individuals, while most patients who die of SFTS exhibit symptoms of severe encephalitis before death. However, the region of brain damage and mechanisms by which the SFTS virus (SFTSV) causes encephalitis remains unknown. Here, we revealed that SFTSV infects the brainstem and spinal cord, which are regions of the brain associated with respiratory function, and motor nerves in IFNAR1-/- mice. Further, we show that A1-reactive astrocytes are activated, causing nerve cell death, in infected mice. Primary astrocytes of SFTSV-infected IFNAR1-/- mice also induced neuronal cell death through the activation of A1-reactive astrocytes. Herein, we showed that SFTSV induces fatal neuroinflammation in the brain regions important for respiratory function and motor nerve, which may underlie mortality in SFTS patients. This study provides new insights for the treatment of SFTS, for which there is currently no therapeutic approach.

Host Subcellular Organelles: Targets of Viral Manipulation.

Viruses have evolved sophisticated mechanisms to manipulate host cell processes and utilize intracellular organelles to facilitate their replication. These complex interactions between viruses and cellular organelles allow them to hijack the cellular machinery and impair homeostasis. Moreover, viral infection alters the cell membrane's structure and composition and induces vesicle formation to facilitate intracellular trafficking of viral components. However, the research focus has predominantly been on the immune response elicited by viruses, often overlooking the significant alterations that viruses induce in cellular organelles. Gaining a deeper understanding of these virus-induced cellular changes is crucial for elucidating the full life cycle of viruses and developing potent antiviral therapies. Exploring virus-induced cellular changes could substantially improve our understanding of viral infection mechanisms.

Stepwise Enzymatic-Dependent Mechanism of Ebola Virus Binding to Cell Surface Receptors Monitored by AFM.

Ebola virus (EBOV) is responsible for several outbreaks of hemorrhagic fever with high mortality, raising great public concern. Several cell surface receptors have been identified to mediate EBOV binding and internalization, including phosphatidylserine (PS) receptors (TIM-1) and C-type lectin receptors (DC-SIGNR). However, the role of TIM-1 during early cell surface binding remains elusive and in particular whether TIM-1 acts as a specific receptor for EBOV. Here, we used force-distance curve-based atomic force microscopy (FD-based AFM) to quantify the binding between TIM-1/DC-SIGNR and EBOV glycoprotein (GP) and observed that both receptors specifically bind to GP with high-affinity. Since TIM-1 can also directly interact with PS at the single-molecule level, we also confirmed that TIM-1 acts as dual-function receptors of EBOV. These results highlight the direct involvement of multiple high-affinity receptors in the first steps of binding to cell surfaces, thus offering new perspectives for the development of anti-EBOV therapeutic molecules.

Viruses Binding to Host Receptors Interacts with Autophagy.

Viruses must cross the plasma membrane to infect cells, making them eager to overcome this barrier in order to replicate in hosts. They bind to cell surface receptors as the first step of initiating entry. Viruses can use several surface molecules that allow them to evade defense mechanisms. Various mechanisms are stimulated to defend against viruses upon their entry into cells. Autophagy, one of the defense systems, degrades cellular components to maintain homeostasis. The presence of viruses in the cytosol regulates autophagy; however, the mechanisms by which viral binding to receptors regulates autophagy have not yet been fully established. This review discusses recent findings on autophagy induced by interactions between viruses and receptors. It provides novel perspectives on the mechanism of autophagy as regulated by viruses.

Autophagy in Cell Survival and Death.

Autophagy is a degradative process to remove damaged or unnecessary cellular components, and it has been implicated in many biological processes during cell survival and death [...].

Probing PIEZO1 Localization upon Activation Using High-Resolution Atomic Force and Confocal Microscopy.

PIEZO1 ion channels are activated by mechanical stimuli, triggering intracellular chemical signals. Recent structural studies suggest that plasma membrane tension or local curvature changes modulate PIEZO1 channel gating and activation. However, whether PIEZO1 localization is governed by tension gradients or long-range mechanical perturbations across the cells is still unclear. Here, we probe the nanoscale localization of PIEZO1 on red blood cells (RBCs) at high resolution (∼30 nm), and we report for the first time the existence of submicrometric PIEZO1 clusters in native conditions. Upon interaction with Yoda1, an allosteric modulator, PIEZO1 clusters increase in abundance in regions of higher membrane tension and lower curvature. We further show that PIEZO1 ion channels interact with the spectrin cytoskeleton in both resting and activated states. Our results point toward a strong interplay between plasma membrane tension gradients, curvature, and cytoskeleton association of PIEZO1.

Isocitrate protects DJ-1 null dopaminergic cells from oxidative stress through NADP+-dependent isocitrate dehydrogenase (IDH).

DJ-1 is one of the causative genes for early onset familiar Parkinson's disease (PD) and is also considered to influence the pathogenesis of sporadic PD. DJ-1 has various physiological functions which converge on controlling intracellular reactive oxygen species (ROS) levels. In RNA-sequencing analyses searching for novel anti-oxidant genes downstream of DJ-1, a gene encoding NADP+-dependent isocitrate dehydrogenase (IDH), which converts isocitrate into α-ketoglutarate, was detected. Loss of IDH induced hyper-sensitivity to oxidative stress accompanying age-dependent mitochondrial defects and dopaminergic (DA) neuron degeneration in Drosophila, indicating its critical roles in maintaining mitochondrial integrity and DA neuron survival. Further genetic analysis suggested that DJ-1 controls IDH gene expression through nuclear factor-E2-related factor2 (Nrf2). Using Drosophila and mammalian DA models, we found that IDH suppresses intracellular and mitochondrial ROS level and subsequent DA neuron loss downstream of DJ-1. Consistently, trimethyl isocitrate (TIC), a cell permeable isocitrate, protected mammalian DJ-1 null DA cells from oxidative stress in an IDH-dependent manner. These results suggest that isocitrate and its derivatives are novel treatments for PD associated with DJ-1 dysfunction.

Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1) Mutation and TRAP1 Inhibitor Gamitrinib-triphenylphosphonium (G-TPP) Induce a Forkhead Box O (FOXO)-dependent Cell Protective Signal from Mitochondria.

TRAP1 (tumor necrosis factor receptor-associated protein 1), a mitochondrial Hsp90 family chaperone, has been identified as a critical regulator of cell survival and bioenergetics in tumor cells. To discover novel signaling networks regulated by TRAP1, we generated Drosophila TRAP1 mutants. The mutants successfully developed into adults and produced fertile progeny, showing that TRAP1 is dispensable in development and reproduction. Surprisingly, mutation or knockdown of TRAP1 markedly enhanced Drosophila survival under oxidative stress. Moreover, TRAP1 mutation ameliorated mitochondrial dysfunction and dopaminergic (DA) neuron loss induced by deletion of a familial Parkinson disease gene PINK1 (Pten-induced kinase 1) in Drosophila. Gamitrinib-triphenylphosphonium, a mitochondria-targeted Hsp90 inhibitor that increases cell death in HeLa and MCF7 cells, consistently inhibited cell death induced by oxidative stress and mitochondrial dysfunction induced by PINK1 mutation in mouse embryonic fibroblast cells and DA cell models such as SH-SY5Y and SN4741 cells. Additionally, gamitrinib-triphenylphosphonium also suppressed the defective locomotive activity and DA neuron loss in Drosophila PINK1 null mutants. In further genetic analyses, we showed enhanced expression of Thor, a downstream target gene of transcription factor FOXO, in TRAP1 mutants. Furthermore, deletion of FOXO almost nullified the protective roles of TRAP1 mutation against oxidative stress and PINK1 mutation. These results strongly suggest that inhibition of the mitochondrial chaperone TRAP1 generates a retrograde cell protective signal from mitochondria to the nucleus in a FOXO-dependent manner.

Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling.

In their environment, cells must cope with mechanical stresses constantly. Among these, nanoscale deformations of plasma membrane induced by substrate nanotopography are now largely accepted as a biophysical stimulus influencing cell behavior and function. However, the mechanotransduction cascades involved and their precise molecular effects on cellular physiology are still poorly understood. Here, using homemade fluorescent nanostructured cell culture surfaces, we explored the role of Bin/Amphiphysin/Rvs (BAR) domain proteins as mechanosensors of plasma membrane geometry. Our data reveal that distinct subsets of BAR proteins bind to plasma membrane deformations in a membrane curvature radius-dependent manner. Furthermore, we show that membrane curvature promotes the formation of dynamic actin structures mediated by the Rho GTPase CDC42, the F-BAR protein CIP4, and the presence of PI(4,5)P2. In addition, these actin-enriched nanodomains can serve as platforms to regulate receptor signaling as they appear to contain interferon-γ receptor (IFNγ-R) and to lead to the partial inhibition of IFNγ-induced JAK/STAT signaling.

Manipulating RKIP reverses the metastatic potential of breast cancer cells.

Breast cancer is a common tumor type among women, with a high fatality due to metastasis. Metastasis suppressors encode proteins that inhibit the metastatic cascade independent of the primary tumor growth. Raf kinase inhibitory protein (RKIP) is one of the promising metastasis suppressor candidates. RKIP is reduced or lost in aggressive variants of different types of cancer. A few pre-clinical or clinical studies have capitalized on this protein as a possible therapeutic target. In this article, we employed two breast cancer cells to highlight the role of RKIP as an antimetastatic gene. One is the low metastatic MCF-7 with high RKIP expression, and the other is MDA-MB-231 highly metastatic cell with low RKIP expression. We used high-throughput data to explore how RKIP is lost in human tissues and its effect on cell mobility. Based on our previous work recapitulating the links between RKIP and SNAI, we experimentally manipulated RKIP in the cell models through its novel upstream NME1 and investigated the subsequent genotypic and phenotypic changes. We also demonstrated that RKIP explained the uneven migration abilities of the two cell types. Furthermore, we identified the regulatory circuit that might carry the effect of an existing drug, Epirubicin, on activating gene transcription. In conclusion, we propose and test a potential strategy to reverse the metastatic capability of breast cancer cells by chemically manipulating RKIP expression.

Fermented Soybean Paste Attenuates Biogenic Amine-Induced Liver Damage in Obese Mice.

Biogenic amines are cellular components produced by the decarboxylation of amino acids; however, excessive biogenic amine production causes adverse health problems. The relationship between hepatic damage and biogenic amine levels in nonalcoholic fatty liver disease (NAFLD) remains unclear. In this study, mice were fed a high-fat diet (HFD) for 10 weeks to induce obesity, presenting early-stage of NAFLD. We administered histamine (20 mg/kg) + tyramine (100 mg/kg) via oral gavage for 6 days to mice with HFD-induced early-stage NAFLD. The results showed that combined histamine and tyramine administration increased cleaved PARP-1 and IL-1ß in the liver, as well as MAO-A, total MAO, CRP, and AST/ALT levels. In contrast, the survival rate decreased in HFD-induced NAFLD mice. Treatment with manufactured or traditional fermented soybean paste decreased biogenically elevated hepatic cleaved PARP-1 and IL-1ß expression and blood plasma MAO-A, CRP, and AST/ALT levels in HFD-induced NAFLD mice. Additionally, the biogenic amine-induced reduction in survival rate was alleviated by fermented soybean paste in HFD-induced NAFLD mice. These results show that biogenic amine-induced liver damage can be exacerbated by obesity and may adversely affect life conservation. However, fermented soybean paste can reduce biogenic amine-induced liver damage in NAFLD mice. These results suggest a beneficial effect of fermented soybean paste on biogenic amine-induced liver damage and provide a new research perspective on the relationship between biogenic amines and obesity.

Multivalent 9-O-Acetylated-sialic acid glycoclusters as potent inhibitors for SARS-CoV-2 infection.

The recent emergence of highly transmissible SARS-CoV-2 variants illustrates the urgent need to better understand the molecular details of the virus binding to its host cell and to develop anti-viral strategies. While many studies focused on the role of the angiotensin-converting enzyme 2 receptor in the infection, others suggest the important role of cell attachment factors such as glycans. Here, we use atomic force microscopy to study these early binding events with the focus on the role of sialic acids (SA). We show that SARS-CoV-2 binds specifically to 9-O-acetylated-SA with a moderate affinity, supporting its role as an attachment factor during virus landing to cell host surfaces. For therapeutic purposes and based on this finding, we have designed novel blocking molecules with various topologies and carrying a controlled number of SA residues, enhancing affinity through a multivalent effect. Inhibition assays show that the AcSA-derived glycoclusters are potent inhibitors of cell binding and infectivity, offering new perspectives in the treatment of SARS-CoV-2 infection.

Targeting Autophagy for Developing New Therapeutic Strategy in Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is a prevalent cause of low back pain. IVDD is characterized by abnormal expression of extracellular matrix components such as collagen and aggrecan. In addition, it results in dysfunctional growth, senescence, and death of intervertebral cells. The biological pathways involved in the development and progression of IVDD are not fully understood. Therefore, a better understanding of the molecular mechanisms underlying IVDD could aid in the development of strategies for prevention and treatment. Autophagy is a cellular process that removes damaged proteins and dysfunctional organelles, and its dysfunction is linked to a variety of diseases, including IVDD and osteoarthritis. In this review, we describe recent research findings on the role of autophagy in IVDD pathogenesis and highlight autophagy-targeting molecules which can be exploited to treat IVDD. Many studies exhibit that autophagy protects against and postpones disc degeneration. Further research is needed to determine whether autophagy is required for cell integrity in intervertebral discs and to establish autophagy as a viable therapeutic target for IVDD.

Nanophysical Mapping of Inflammasome Activation by Nanoparticles via Specific Cell Surface Recognition Events.

Silica nanoparticles (SiNP) trigger a range of innate immune responses in relevant essential organs, such as the liver and the lungs. Inflammatory reactions, including NLRP3 inflammasome activation, have been linked to particulate materials; however, the molecular mechanisms and key actors remain elusive. Although many receptors, including several scavenger receptors, were suggested to participate in SiNP cellular uptake, mechanistic evidence of their role on innate immunity is lacking. Here we present an atomic force microscopy-based approach to physico-mechanically map the specific interaction occurring between nanoparticles and scavenger receptor A1 (SRA1) in vitro on living lung epithelial cells. We find that SiNP recognition by SRA1 on human macrophages plays a key role in mediating NLRP3 inflammasome activation, and we identify cellular mechanical changes as clear indicators of inflammasome activation in human macrophages, greatly advancing our knowledge on the interplay among nanomaterials and innate immunity.

Reovirus directly engages integrin to recruit clathrin for entry into host cells.

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, ß1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct ß1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and ß1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and ß1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for ß1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and ß1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor.

Study of the interactions established between the viral glycoproteins and their host receptors is of critical importance for a better understanding of virus entry into cells. The novel coronavirus SARS-CoV-2 entry into host cells is mediated by its spike glycoprotein (S-glycoprotein), and the angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor. Here, we use atomic force microscopy to investigate the mechanisms by which the S-glycoprotein binds to the ACE2 receptor. We demonstrate, both on model surfaces and on living cells, that the receptor binding domain (RBD) serves as the binding interface within the S-glycoprotein with the ACE2 receptor and extract the kinetic and thermodynamic properties of this binding pocket. Altogether, these results provide a picture of the established interaction on living cells. Finally, we test several binding inhibitor peptides targeting the virus early attachment stages, offering new perspectives in the treatment of the SARS-CoV-2 infection.

Label-Free Imaging of Cholesterol Assemblies Reveals Hidden Nanomechanics of Breast Cancer Cells.

Tumor cells present profound alterations in their composition, structural organization, and functional properties. A landmark of cancer cells is an overall altered mechanical phenotype, which so far are linked to changes in their cytoskeletal regulation and organization. Evidence exists that the plasma membrane (PM) of cancer cells also shows drastic changes in its composition and organization. However, biomechanical characterization of PM remains limited mainly due to the difficulties encountered to investigate it in a quantitative and label-free manner. Here, the biomechanical properties of PM of a series of MCF10 cell lines, used as a model of breast cancer progression, are investigated. Notably, a strong correlation between the cell PM elasticity and oncogenesis is observed. The altered membrane composition under cancer progression, as emphasized by the PM-associated cholesterol levels, leads to a stiffening of the PM that is uncoupled from the elastic cytoskeletal properties. Conversely, cholesterol depletion of metastatic cells leads to a softening of their PM, restoring biomechanical properties similar to benign cells. As novel therapies based on targeting membrane lipids in cancer cells represent a promising approach in the field of anticancer drug development, this method contributes to deciphering the functional link between PM lipid content and disease.

Glycan-mediated enhancement of reovirus receptor binding.

Viral infection is an intricate process that requires the concerted action of both viral and host cell components. Entry of viruses into cells is initiated by interactions between viral proteins and their cell surface receptors. Despite recent progress, the molecular mechanisms underlying the multistep reovirus entry process are poorly understood. Using atomic force microscopy, we investigated how the reovirus σ1 attachment protein binds to both α-linked sialic acid (α-SA) and JAM-A cell-surface receptors. We discovered that initial σ1 binding to α-SA favors a strong multivalent anchorage to JAM-A. The enhanced JAM-A binding by virions following α-SA engagement is comparable to JAM-A binding by infectious subvirion particles (ISVPs) in the absence of α-SA. Since ISVPs have an extended σ1 conformer, this finding suggests that α-SA binding triggers a conformational change in σ1. These results provide new insights into the function of viral attachment proteins in the initiation of infection and open new avenues for the use of reoviruses as oncolytic agents.

Spatial Activation of TORC1 Is Regulated by Hedgehog and E2F1 Signaling in the Drosophila Eye.

Target of rapamycin complex 1 (TORC1) regulates cell growth in response to nutrients and growth factors. Although TORC1 signaling has been thoroughly studied at the cellular level, the regulation of TORC1 in multicellular tissues and organs has remained elusive. Here we found that TORC1 is selectively activated in the second mitotic wave (SMW), the terminal synchronous cell division, of the developing Drosophila eye. We demonstrated that Hedgehog (Hh) signaling regulates TORC1 through E2F1 and the cyclin D/Cdk4 complex in the SMW, and this regulation is independent from insulin and amino acid signaling pathways. TORC1 is necessary for the proper G1/S transition of the cells, and the activation of TORC1 rescues the cell-cycle defect of Hh signaling-deficient cells in the SMW. Based on this evolutionarily conserved regulation of TORC1 by Hh signaling, we propose that Hh-dependent developmental signaling pathways spatially regulate TORC1 activity in multicellular organisms.