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Outbreaks of viral diseases are on the rise, fueling the search for antiviral therapeutics that act on a broad range of viruses while remaining safe to human host cells. In this research, we leverage the finding that the plasma membranes of host cells and the lipid bilayers surrounding enveloped viruses differ in lipid composition. We feature Piscidin 1 (P1), a cationic host defense peptide (HDP) that has antimicrobial effects and membrane activity associated with its N-terminal region where a cluster of aromatic residues and copper-binding motif reside. While few HDPs have demonstrated antiviral activity, P1 acts in the micromolar range against several enveloped viruses that vary in envelope lipid composition. Notably, it inhibits HIV-1, a virus that has an envelope enriched in cholesterol, a lipid associated with higher membrane order and stability. Here, we first document through plaque assays that P1 boasts strong activity against SARS-CoV-2, which has an envelope low in cholesterol. Second, we extend previous studies done with homogeneous bilayers and devise cholesterol-containing zwitterionic membranes that contain the liquid disordered (Ld; low in cholesterol) and ordered (Lo, rich in cholesterol) phases. Using dye leakage assays and cryo-electron microscopy on vesicles, we show that P1 has dramatic permeabilizing capability on the Lo/Ld, an effect matched by a strong ability to aggregate, fuse, and thin the membranes. Differential scanning calorimetry and NMR experiments demonstrate that P1 mixes the lipid content of vesicles and alters the stability of the Lo. Structural studies by NMR indicate that P1 interacts with the Lo/Ld by folding into an α-helix that lies parallel to the membrane surface. Altogether, these results show that P1 is more disruptive to phase-separated than homogenous cholesterol-containing bilayers, suggesting an ability to target domain boundaries. Overall, this multi-faceted research highlights how a peptide that interacts strongly with membranes through an aromatic-rich N-terminal motif disrupt viral envelope mimics. This represents an important step towards the development of novel peptides with broad-spectrum antiviral activity.

RESUMEN

Developing new antimicrobials as alternatives to conventional antibiotics has become an urgent race to eradicate drug-resistant bacteria and to save human lives. Conventionally, antimicrobial molecules are studied independently even though they can be cosecreted in vivo. In this research, we investigate two classes of naturally derived antimicrobials: sophorolipid (SL) esters as modified yeast-derived glycolipid biosurfactants that feature high biocompatibility and low production cost; piscidins, which are host defense peptides (HDPs) from fish. While HDPs such as piscidins target the membrane of pathogens, and thus result in low incidence of resistance, SLs are not well understood on a mechanistic level. Here, we demonstrate that combining SL-hexyl ester (SL-HE) with subinhibitory concentration of piscidins 1 (P1) and 3 (P3) stimulates strong antimicrobial synergy, potentiating a promising therapeutic window. Permeabilization assays and biophysical studies employing circular dichroism, NMR, mass spectrometry, and X-ray diffraction are performed to investigate the mechanism underlying this powerful synergy. We reveal four key mechanistic features underlying the synergistic action: (1) P1/3 binds to SL-HE aggregates, becoming α-helical; (2) piscidin-glycolipid assemblies synergistically accumulate on membranes; (3) SL-HE used alone or bound to P1/3 associates with phospholipid bilayers where it induces defects; (4) piscidin-glycolipid complexes disrupt the bilayer structure more dramatically and differently than either compound alone, with phase separation occurring when both agents are present. Overall, dramatic enhancement in antimicrobial activity is associated with the use of two membrane-active agents, with the glycolipid playing the roles of prefolding the peptide, coordinating the delivery of both agents to bacterial surfaces, recruiting the peptide to the pathogenic membranes, and supporting membrane disruption by the peptide. Given that SLs are ubiquitously and safely used in consumer products, the SL/peptide formulation engineered and mechanistically characterized in this study could represent fertile ground to develop novel synergistic agents against drug-resistant bacteria.

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Spider silk is biocompatible, biodegradable, and rivals some of the best synthetic materials in terms of strength and toughness. Despite extensive research, comprehensive experimental evidence of the formation and morphology of its internal structure is still limited and controversially discussed. Here, we report the complete mechanical decomposition of natural silk fibers from the golden silk orb-weaver Trichonephila clavipes into ≈10 nm-diameter nanofibrils, the material's apparent fundamental building blocks. Furthermore, we produced nanofibrils of virtually identical morphology by triggering an intrinsic self-assembly mechanism of the silk proteins. Independent physico-chemical fibrillation triggers were revealed, enabling fiber assembly from stored precursors "at-will". This knowledge furthers the understanding of this exceptional material's fundamentals, and ultimately, leads toward the realization of silk-based high-performance materials. STATEMENT OF SIGNIFICANCE: Spider silk is one of the strongest and toughest biomaterials, rivaling the best man-made materials. The origins of these traits are still under debate but are mostly attributed to the material's intriguing hierarchical structure. Here we fully disassembled spider silk into 10 nm-diameter nanofibrils for the first time and showed that nanofibrils of the same appearance can be produced via molecular self-assembly of spider silk proteins under certain conditions. This shows that nanofibrils are the key structural elements in silk and leads toward the production of high-performance future materials inspired by spider silk.

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This research reports on the membrane interactions of orexin A (OXA), an α-helical and amphipathic neuropeptide that contains 33 residues and two disulfide bonds in the N-terminal region. OXA, which activates the orexins 1 and 2 receptors in neural and immune cell membranes, has essential pleiotropic physiological effects, including at the levels of arousal, sleep/wakefulness, energy balance, neuroprotection, lipid signaling, the inflammatory response, and pain. As a result, the orexin system has become a prominent target to treat diseases such as sleep disorders, drug addiction, and inflammation. While the high-resolution structure of OXA has been investigated in water and bound to micelles, there is a lack of information about its conformation bound to phospholipid membranes and its receptors. NMR is a powerful method to investigate peptide structures in a membrane environment. To facilitate the NMR structural studies of OXA exposed to membranes, we present a novel synthetic scheme, leading to the production of isotopically-labeled material at high purity. A receptor activation assay shows that the 15N-labeled peptide is biologically active. Biophysical studies are performed using surface plasmon resonance, circular dichroism, and NMR to investigate the interactions of OXA with phospholipid bilayers. The results demonstrate a strong interaction between the peptide and phospholipids, an increase in α-helical content upon membrane binding, and an in-plane orientation of the C-terminal region critical to function. This new knowledge about structure-activity relationships in OXA could inspire the design of novel therapeutics that leverage the anti-inflammatory and neuro-protective functions of OXA, and therefore could help address neuroinflammation, a major issue associated with neurological disorders such as Alzheimer's disease.

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In mammals, IFN regulatory factor (IRF)1, IRF3, and IRF7 are three critical transcription factors that are pivotal for cooperative regulation of the type I IFN response. In this study, we explored the relative contribution of zebrafish (Danio rerio) IRF1 (DrIRF1), IRF3 (DrIRF3), and IRF7 (DrIRF7) (DrIRF1/3/7) to zebrafish IFNΦ1 (DrIFNΦ1) and IFNΦ3 (DrIFNΦ3) (DrIFNΦ1/3) activation. Following spring viremia of carp virus infection, DrIFNΦ1/3 and DrIRF1/3/7 transcripts are significantly induced in zebrafish tissues, which correlates with the replication of spring viremia of carp virus. DrIRF1/3/7 selectively bind to the IRF-binding element/IFN-stimulated regulatory element sites of DrIFNΦ1/3 promoters, with the exception that DrIRF3 has no preference for two IRF-binding element/IFN-stimulated regulatory element motifs within the DrIFNΦ3 promoter. Consistently, DrIRF3 alone activates DrIFNΦ1, but not DrIFNΦ3; DrIRF7 predominantly stimulates DrIFNΦ3; and DrIRF1 has similar potential to DrIFNΦ1 and DrIFNΦ3. Strikingly, DrIRF3 facilitates the binding of DrIRF1 and DrIRF7 to both zebrafish IFN promoters, and so does DrIRF7 for the binding of DrIRF1, particularly to the DrIFNΦ3 promoter. These binding properties correlate with differential responses of DrIFNΦ1 and DrIFNΦ3 to the combinatory stimulation of DrIRF1/3/7, depending on their relative amounts. Similar to the dual roles of human IRF3 in regulating IRF7-activated IFNα genes, DrIRF3 exerts dual effects on DrIRF1-mediated DrIFNΦ3 gene expression: an inhibitory effect at lower concentrations and a synergistic effect at higher concentrations. These data provide evidence that fish and mammals have evolved a similar IRF-dependent regulatory mechanism fine-tuning IFN gene activation.

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