A Coronin 1-Derived Peptide Inhibits Membrane Fusion by Modulating Membrane Organization and Dynamics.

Membrane fusion is considered the first step in the entry of enveloped viruses into the host cell. Several targeted strategies have been implemented to block viral entry by limiting the fusion protein to form a six-helix bundle, which is a prerequisite for fusion. Nonetheless, the development of broad-spectrum fusion inhibitors is essential to combat emerging and re-emerging viral infections. TG-23, a coronin 1, a tryptophan-aspartate-rich phagosomal protein-derived peptide, demonstrated inhibition of fusion between small unilamellar vesicles (SUVs) by modulating the membrane's physical properties. However, its inhibitory efficacy reduces with an increasing concentration of membrane cholesterol. The present work aims to develop a fusion inhibitor whose efficacy would be unaltered in the presence of membrane cholesterol. A stretch of the tryptophan-aspartic acid-containing peptide with a similar secondary structure and hydrophobicity profile of TG-23 from coronin 1 was synthesized, and its ability to inhibit SUV-SUV fusion with varying concentrations of membrane cholesterol was evaluated. Our results demonstrate that the GG-21 peptide inhibits fusion irrespective of the cholesterol content of the membrane. We have further evaluated the peptide-induced change in the membrane organization and dynamics utilizing arrays of steady-state and time-resolved fluorescence measurements and correlated these results with their effect on fusion. Interestingly, GG-21 displays inhibitory efficacy in a wide variety of lipid compositions despite having a secondary structure and physical properties similar to those of TG-23. Overall, our results advocate that the secondary structure and physical properties of the peptide may not be sufficient to predict its inhibitory efficacy.

Phosphatidylglycerol Acts as a Switch for Cholesterol-Dependent Membrane Binding of ApoE Signal Peptide.

The apolipoprotein E (ApoE) signal peptide is a short stretch of N-terminal amino acids that direct the ApoE protein to the endoplasmic reticulum after synthesis. Previous studies have shown that this peptide can bind to lipid membranes in a cholesterol-dependent manner; however, the mechanism of this interaction is yet to be clarified. In this study, we aimed to investigate how the composition of neighboring lipids affects the membrane-binding of the ApoE signal peptide. We found that a negatively charged lipid, such as phosphatidylglycerol, can act as a switch that reduces the binding efficiency of the peptide to cholesterol-rich membranes. Interestingly, phosphatidylethanolamine does not activate the cholesterol-dependent binding of the ApoE signal peptide yet acts synergistically to enhance the cholesterol sensitivity in phosphatidylglycerol-containing membranes. To the best of our knowledge, this is the first report of modulation of the affinity of a peptide for a membrane by a neighboring lipid rather than by the lipid-binding domain of the peptide. Our findings revealed a novel role of lipid diversity in modulating the membrane binding of the ApoE signal peptide and its potential implications in the unidirectional trafficking of a newly synthesized protein from the ribosomes to the endoplasmic reticulum.

Peptide-Induced Fusion of Dynamic Membrane Nanodomains: Implications in a Viral Entry.

Enveloped viruses infect host cells via protein-mediated membrane fusion. However, insights into the microscopic rearrangement induced by the viral proteins and peptides have not yet emerged. Here, we report a new methodology to extract viral fusion peptide (FP)-mediated biomembrane dynamical nanodomain fusion parameter, λ, based on stimulated emission depletion microscopy coupled with fluorescence correlation spectroscopy. We also define another dynamical parameter membrane gradient, defined in terms of the ratio of average lipid diffusion coefficients across dynamic crossover length scales, ξ. Significantly, we observe that λ as well as these mobility gradients are larger in the stiffer liquid-ordered (Lo) phase compared to the liquid-disordered phase and are more effective at the smaller nanodomain interfaces, which are only present in the Lo phase. The results could possibly help to resolve a long-standing puzzle about the enhanced fusogenicity of FP in the Lo phase. Results obtained from the diffusion results have been correlated with the human immunodeficiency virus gp41 FP-induced membrane fusion.

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors that are directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified which are mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Phosphatidylethanolamine and Cholesterol Promote Hemifusion Formation: A Tug of War between Membrane Interfacial Order and Intrinsic Negative Curvature of Lipids.

Membrane fusion is an important process for the survival of eukaryotes. The shape of lipids plays an important role in fusion by stabilizing nonlamellar fusion intermediates. Lipids with intrinsic positive curvature such as lysophosphatidylcholine and others inhibit hemifusion formation, whereas lipids having intrinsic negative curvature, e.g., phosphatidylethanolamine and cholesterol (CH), are known to promote hemifusion formation. In this work, we have measured the effect of dioleoylphosphatidylethanolamine (DOPE) and CH on the depth-dependent organization, dynamics, and fusion of dioleoylphosphatidylcholine membranes. Both DOPE and CH promote hemifusion formation despite their ability to order the interfacial and acyl chain region of the membrane and block water percolation at these regions. Generally, membrane ordering and inhibition of water percolation at the acyl chain region are detrimental to membrane fusion. This clearly emphasizes the importance of intrinsic negative curvature of lipids in membrane fusion. Interestingly, DOPE and CH show differential effects on the rate of hemifusion formation, which might be owing to their ability to induce order at the interfacial region and intrinsic negative curvature. Overall, our result is significant in understanding the role of lipidic shape in membrane fusion.

Microbiome analysis reveals potential for modulation of gut microbiota through polysaccharide-based prebiotic feeding in Oreochromis niloticus (Linnaeus, 1758).

Characterization and functional profiling of the gut microbiota are essential for guiding nutritional interventions in fish and achieving favorable host-microbe interactions. Thus, we conducted a 30 days study to explore and document the gut microbial community of O. niloticus, as well as to evaluate the effects of a polysaccharide-based prebiotics with 0.5% and 0.75% Aloe vera extract on the gut microbiome through genomic analysis. The V3-V4 region of 16S rRNA was amplified and sequenced using Illumina HiSeq 2500, resulting in 1,000,199 reads for operational taxonomic unit (OTU) identification. Out of 8,894 OTUs, 1,181 were selected for further analysis. Our results revealed that Planctomycetes, Firmicutes, Proteobacteria, Verrucomicrobia, Actinobacteria, and Fusobacteria were the dominant phyla in both control and treatment samples. Higher doses of prebiotics were found to improve Planctomycetes and Firmicutes while decreasing Proteobacteria and Verrucomicrobia. We observed increasing trends in the abundance of Bacilli, Bacillaceae, and Bacillus bacteria at the class, family, and genus levels, respectively, in a dose-dependent manner. These findings were consistent with the conventional colony count data, which showed a higher prevalence of Bacillus in prebiotic-supplemented groups. Moreover, predicted functional analysis using PICRUSt indicated a dose-dependent upregulation in glycolysis V, superpathway of glycol metabolism and degradation, glucose and xylose degradation, glycolysis II, and sulfoglycolysis pathways. Most of the energy, protein, and amino acid synthesis pathways were upregulated only at lower doses of prebiotic treatment. Our findings suggest that the gut microbiome of O. niloticus can be optimized through nutritional interventions with plant-based polysaccharides for improved growth performance in commercial fish.

Membrane cholesterol regulates the oligomerization and fusogenicity of SARS-CoV fusion peptide: implications in viral entry.

N-terminal residues (770-788) of the S2 glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) have been recognized as a potential fusion peptide that can be involved in the entry of the virus into the host cell. Membrane composition plays an important role in lipid-peptide interaction and the oligomeric status of the peptide. SARS-CoV fusion peptide (S2 fusion peptide) is known to undergo cholesterol-dependent oligomerization in the membrane; however, its significance in membrane fusion is still speculative. This study aimed to investigate the oligomerization of SARS-CoV fusion peptide in a membrane containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with varying concentrations of cholesterol, and to evaluate peptide-induced membrane fusion to correlate the importance of peptide oligomerization with membrane fusion. Peptide-induced modulation of membrane organization and dynamics was explored by steady-state and time-resolved fluorescence spectroscopic measurements using depth-dependent probes. The results clearly demonstrated the induction of S2 fusion peptide oligomerization by membrane cholesterol and the higher efficiency of the oligomer in promoting membrane fusion compared to its monomeric counterpart. Cholesterol-dependent peptide oligomerization and membrane fusion are important aspects of viral infection since the cholesterol level can change with age as well as with the onset of various pathophysiological conditions.

Differential Behavior of Eicosapentaenoic and Docosahexaenoic Acids on the Organization, Dynamics, and Fusion of Homogeneous and Heterogeneous Membranes.

Membrane fusion is a common course in innumerable biological processes that helps in the survival of eukaryotes. Enveloped viruses utilize this process to enter the host cells. Generally, the membrane lipid compositions play an important role in membrane fusion by modulating the membrane's physical properties and the behavior of membrane proteins in the cellular milieu. In this work, we have demonstrated the role of polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, on the organization, dynamics, and fusion of homogeneous and heterogeneous membranes. We have exploited arrays of steady-state and time-resolved fluorescence spectroscopic methods and polyethylene glycol-induced membrane fusion assay to elucidate the behavior of EPA and DHA on dioleoyl phosphatidylcholine (DOPC)/cholesterol (CH) homogeneous and DOPC/sphingomyelin/CH heterogeneous membranes. Our results suggest that EPA and DHA display differential effects on two different membranes. The effects of PUFAs in homogeneous membranes are majorly attributed to their flexible chain dynamics, whereas the ability of PUFA-induced cholesterol transfer from the lo to the ld phase rules their behavior in heterogeneous membranes. Overall, our results provide detailed information on the effect of PUFAs on homogeneous and heterogeneous membranes.

Effect of polyunsaturated free fatty acids on the membrane fusion mechanism.

Membrane fusion is one of the important processes for the survival of eukaryotic cells and the entry of enveloped viruses into the host cells. Lipid composition plays a crucial role by modulating the organization and dynamics of the membrane, as well as the structure and conformation of membrane proteins. The diversity of the lipid acyl chain in its length and degree of unsaturation originates from the variation in free fatty acids (FFAs). We have studied the effect of linoleic (LA) and alpha-linolenic (ALA) acids on the depth-dependent organization, dynamics, and fusion of DOPC/DOPE (70/30 mol%) membranes utilizing steady-state and time-resolved fluorescence spectroscopic methods. Our results suggest that membranes with 5 mol% LA stabilize the stalk-intermediate and promote lipid mixing at the early stage of the process, i.e., the fusion follows the classical stalk model. Conversely, the extents of lipid and content mixing at the stalk intermediate are similar in the presence of 5 mol% of ALA, indicating the fusion mechanism as a nonclassical one like in the DOPC/DOPE (70/30 mol%) membranes. Our results provide an in-depth insight into the effect of the increasing degree of fatty acid tail unsaturation on membrane organization and dynamics and their impact on the membrane fusion mechanism.

Bacteriophages diversity in India's major river Ganga: a repository to regulate pathogenic bacteria in the aquatic environment.

Bacteriophages are key viruses that can kill thousands of harmful microbes generally present at polluted sites. Such bacteriophages are abundantly present in the river Ganga, where millions of people in India and abroad drink its water and take baths every day for spiritual reasons. Besides bacteriophages, several pathogenic and zoonotic microbes are present in the river Ganga. It is interesting to study the diversity and abundance of bacteria and their respective phages present in polluted or non-polluted sites. Thus, the metagenomics study was carried out at the most polluted sites of river Ganga near Kanpur and non-polluted sites at Farakka, which harbors several harmful bacteria and their phages. The results revealed a significantly higher percentage of Microviridae phage family, ssDNA viruses, and Mimiviridae virus family near Kanpur than Farakka. In addition, compared to Kanpur, Farakka has a more significant percentage of Myoviridae, an unidentified phage family, and Retroviridae viral families. Despite heavy drainage of untreated and contaminated effluents from the leather industry, pesticide industry, paper mills, metropolitan cities, and other sources, the vast number of said phages kills several harmful pathogenic microbes in polluted sites to maintain the Ganga water's healing power or natural sterility. In a polluted aquatic environment, the varieties of bacteriophages were identified in the Ganga and their interaction with the microbial host. The taxonomic diversity of several bacteriophages found in pathogenic host systems was investigated to get exceptional knowledge of these small viruses in the aquatic environment.

Lipid composition dependent binding of apolipoprotein E signal peptide: Importance of membrane cholesterol in protein trafficking.

Soluble secretory and membrane proteins contain a short stretch of signal peptide (SP) at their N-terminal end, which gets cleaved after reaching the destination organelle. However, the importance of SP in protein trafficking is not fully understood. The lipid compositions of cellular organelles are highly heterogeneous, and the preference of SP toward a particular lipid composition might play a key role in unidirectional trafficking of protein. In order to understand the preference of Apolipoprotein E (ApoE) toward endoplasmic reticulum (ER), we have studied the interaction of its SP with membranes of varying lipid compositions. The importance of cholesterol is paramount as subcellular organelles contain differential amount of cholesterol; endoplasmic reticulum (ER) contains the least amount of cholesterol. We have utilized batteries of steady-state and time-resolved fluorescence techniques to understand the affinity of ApoE signal peptide toward membranes of varying lipid compositions. We observed that the ApoE signal peptide binds tightly with membranes devoid of cholesterol, and binding affinity reduces with increasing concentration of membrane cholesterol. Our results clearly suggest the importance of membrane composition in the unidirectional movement of ApoE toward ER. This property of SP can further be utilized for the development of organelle specific cargo delivery.

Identification of novel salt tolerance-associated proteins from the secretome of Enterococcus faecalis.

The ability of bacteria to adapt to the external environment is fundamental for their survival. A halotolerant microorganism Enterococcus faecalis able to grow under high salt stress conditions was isolated in the present study. The SDS-PAGE analysis of the secretome showed a protein band with a molecular weight of 28 kDa, gradually increased with an increase in salt concentration, and the highest intensity was observed at 15% salt stress condition. LC-MS/MS analysis of this particular band identified fourteen different proteins, out of which nine proteins were uncharacterized. Further, the function of uncharacterized proteins was predicted based on structure-function relationship using a reverse template search approach deciphering uncharacterized protein into type III polyketide synthases, stress-induced protein-1, Eed-h3k79me3, ba42 protein, 3-methyladenine DNA glycosylase, Atxa protein, membrane-bound respiratory hydrogenase, type-i restriction-modification system methylation subunit and ManxA. STRING network analysis further a showed strong association among the proteins. The processes predicted involvement of these proteins in signal transduction, ions transport, synthesis of the protective layer, cellular homeostasis and regulation of gene expression and different metabolic pathways. Thus, the fourteen proteins identified in the secretome play an essential role in maintaining cellular homeostasis in E. faecalis under high-salinity stress. This may represent a novel and previously unreported strategy by E. faecalis to maintain their normal growth and physiology under high salinity conditions.

Combination of Oleic Acid and the gp41 Fusion Peptide Switches the Phosphatidylethanolamine-Induced Membrane Fusion Mechanism from a Nonclassical to a Classical Stalk Model.

Membrane fusion is considered to be one of the crucial processes for the existence of eukaryotes and the entry of enveloped viruses into host cells. The fusion mechanism depends on the lipid composition of the membrane as well as the properties of fusion proteins or peptides. The gp41 fusion peptide from the human immunodeficiency virus (HIV) is known to catalyze membrane fusion by altering the physical properties of the membrane. Earlier, we demonstrated that a membrane containing 30 mol % phosphatidylethanolamine (PE) circumvents the classical stalk model because of its intrinsic negative curvature. In this work, we demonstrated how the gp41 fusion peptide influences the fusion mechanism of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phos-pho¬ethanolamine (DOPE) (70/30 mol %) membranes. We further evaluated the effect of the same peptide on the mechanism of fusion for membranes containing 30 mol % PE and a fatty acid with an intrinsic positive curvature (oleic acid (OA)). Our results show that gp41 switches the fusion mechanism from a nonclassical to a classical stalk model when membranes contain OA, but fails to do so for DOPC/DOPE membranes. This could be due to the extreme influence of the intrinsic negative curvature of PE, which is partially downregulated in the presence of OA.

Mechanism of Membrane Fusion: Interplay of Lipid and Peptide.

Membrane fusion is an essential process for the survival of eukaryotes and the entry of enveloped viruses into host cells. A proper understanding of the mechanism of membrane fusion would provide us a handle to manipulate several biological pathways, and design efficient vaccines against emerging and re-emerging viral infections. Although fusion proteins take the central stage in catalyzing the process, role of lipid composition is also of paramount importance. Lipid composition modulates membrane organization and dynamics and impacts the lipid-protein (peptide) interaction. Moreover, the intrinsic curvature of lipids has strong impact on the formation of stalk and hemifusion diaphragm. Detection of transiently stable intermediates remains the bottleneck in the understanding of fusion mechanism. In order to circumvent this challenge, analytical methods can be employed to determine the kinetic parameters from ensemble average measurements of observables, such as lipid mixing, content mixing, and content leakage. The current review aims to present an analytical method that would aid our understanding of the fusion mechanism, provides a better insight into the role of lipid shape, and discusses the interplay of lipid and peptide in membrane fusion.

Trh positive strain of Vibrio parahaemolyticus induce immunity by modulating MAPK pathway: A molecular pathogenic insight in immune-related gene regulation.

Vibrio parahaemolyticus is a zoonotic bacterium that causes infections in shellfish, fish and higher vertebrates as well as in humans. The Tdh and Trh positive strains of V. parahaemolyticus are generally considered as major virulent strains. The pathogenic mechanisms of Trh positive strain of V. parahaemolyticus are poorly understood. Therefore, in the present study Indian Major Carp, Labeo rohita was intraperitoneally challenged with a Trh positive strain of V. parahaemolyticus below lethal dose 50 (LD50) to understand the innate immune response. A significant upregulation in the respiratory burst activity, myeloperoxidase activity and lysozyme activity of serum was observed in the challenged fishes. However, the serum alpha (α) 2-macro globulin activity and antiprotease activity remained unaltered in the infected fish. The relative expression study of some immune-related genes showed that after the experimental challenge the expression of immune-related genes viz., Toll-like receptor (TLR), Nucleotide-binding oligomerization domain (NOD), Interleukin-1ß (IL-ß), Interleukin-6 (IL-6), Tumor necrosis factor α (TNFα), Inducible nitric oxide synthase (iNOS), Complement factor 3a (C3a) and Heat shock proteins 70 (Hsp70) was upregulated during infection. Furthermore, overexpression of nuclear factor kappa light chain enhancer of activated B cells (NF-κß), Myeloid differentiation primary response 88 (MyD88), Mitogen-activated protein kinases (MAPK) and cysteine-aspartic proteases (Casp 1) was also observed after post-infection which clearly indicated that Trh positive V. parahaemolyticus activates MAPK pathway. The present study strengthens the understanding of molecular pathogenesis and provides insights on gene regulation during infection with Trh positive V. parahaemolyticus.

Effect of Phosphatidylethanolamine and Oleic Acid on Membrane Fusion: Phosphatidylethanolamine Circumvents the Classical Stalk Model.

Membrane fusion is one of the most important processes for the survival of eukaryotic cells and entry of enveloped viruses to the host cells. Lipid composition plays a crucial role in the process by modulating the organization and dynamics of the membrane, as well as the structure and conformation of membrane proteins. Phosphatidylethanolamine (PE), a lipid molecule with intrinsic negative curvature, promotes membrane fusion by stabilizing the non-lamellar intermediate structures in the fusion process. Conversely, oleic acid (OA), with intrinsic positive curvature, inhibits membrane fusion. The current study aimed to investigate polyethylene glycol-mediated lipid mixing, content mixing, content leakage, and depth-dependent membrane organization and dynamics, using arrays of steady-state and time-resolved fluorescence techniques, to determine the causative role of PE and OA in membrane fusion. The results demonstrated that the presence of 30 mol % PE in the membrane promotes membrane fusion through a mechanism that circumvents the classical stalk model. On the contrary, membranes containing OA showed reduced rate and extent of fusion, despite following the same mechanism. Collectively, our findings in terms of membrane organization and dynamics indicated a plausible role of PE and OA in membrane fusion.

Cholesterol: A key player in membrane fusion that modulates the efficacy of fusion inhibitor peptides.

The interaction of cholesterol with the neighboring lipids modulates several physical properties of the membrane. Mostly, it affects membrane fluidity, membrane permeability, lateral diffusion of lipids, bilayer thickness, and water penetration into the lipid bilayer. Due to the smaller head group to hydrophobic cross-sectional area of the tail, cholesterol induces intrinsic negative curvature to the membrane. The interaction of cholesterol with sphingolipids forms lipid rafts; generates phase separation in the membrane. The cholesterol-dependent modifications of membrane physical properties modulate viral infections by affecting the fusion between viral and host cell membranes. Cholesterol demonstrates a strong impact on the structure, depth of penetration, conformation, and organization of fusion peptides in membrane milieu. Further, cholesterol has been implicated to modify the fusion inhibitory efficiency of peptide-based membrane fusion inhibitors.

Lipid Headgroup Charge Controls Melittin Oligomerization in Membranes: Implications in Membrane Lysis.

Melittin, a hemolytic peptide present in bee venom, represents one of the most well-studied amphipathic antimicrobial peptides, particularly in terms of its membrane interaction and activity. Nevertheless, no consensus exists on the oligomeric state of membrane-bound melittin. We previously reported on the differential microenvironments experienced by melittin in zwitterionic and negatively charged phospholipid membranes. In this work, we explore the role of negatively charged lipids in the oligomerization of membrane-bound melittin (labeled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)) utilizing a quantitative photobleaching homo-FRET assay. Our results show that the presence of negatively charged lipids decreases melittin oligomeric size to ∼50% of that observed in zwitterionic membranes. This is possibly due to differential energetics of binding of the peptide monomer to membranes of different compositions and could explain the reduced lytic activity yet tighter binding of melittin in negatively charged membranes. These results constitute one of the first experimental observations on the role of phospholipid headgroup charge in the oligomerization of melittin in membranes and is relevant in light of previous apparently contradictory reports on oligomerization of membrane-bound melittin. Our results highlight the synergistic interplay of peptide-membrane binding events and peptide oligomerization in modulating the organization, dynamics, and function of amphipathic α-helical peptides.

Fusogenic Effect of Cholesterol Prevails over the Inhibitory Effect of a Peptide-Based Membrane Fusion Inhibitor.

Membrane fusion is the primary step in the entry of enveloped viruses into the host cell. Membrane composition modulates the membrane fusion by changing the organization dynamics of the fusion proteins, peptides, and membranes. The asymmetric lipid compositions of the viral envelope and the host cell influence the membrane fusion. Cholesterol is an important constituent of mammalian cells and plays a vital role in the entry of several viruses. In our pursuit of developing peptide-based general fusion inhibitors, we have previously shown that a coronin 1-derived peptide, TG-23, inhibited polyethylene glycol-induced fusion between symmetric membranes without cholesterol. In this work, we have studied the effect of TG-23 on the polyethylene glycol-mediated fusion between 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) (60/30/10 mol %) and DOPC/DOPE/DOPG/CH (50/30/10/10 mol %) membranes and between DOPC/DOPE/DOPG (60/30/10 mol %) and DOPC/DOPE/DOPG/CH (40/30/10/20 mol %) membranes. Our results demonstrate that the TG-23 peptide inhibited the fusion between membranes containing 0 and 10 mol % cholesterol though the efficacy is less than that of symmetric fusion between membranes devoid of cholesterol, and the inhibitory efficacy becomes negligible in the fusion between membranes containing 0 and 20 mol % cholesterol. Several steady-state and time-resolved fluorescence spectroscopic techniques have been successfully utilized to evaluate the organization, dynamics, and membrane penetration of the TG-23 peptide. Taken together, our results demonstrate that the reduction of the inhibitory effect of TG-23 in asymmetric membrane fusion containing cholesterol of varying concentrations is not due to the altered peptide structure, organization, and dynamics, rather owing to the intrinsic negative curvature-inducing property of cholesterol. Therefore, the membrane composition is an added complexity in the journey of developing peptide-based membrane fusion inhibitors.