Applying flow cytometry to identify the modes of action of membrane-active peptides in a label-free and high-throughput fashion

Wichmann, Nanna; Lund, Philip M.; Hansen, Morten Borre; Hjørringgaard, Claudia U.; Larsen, Jørgen Hannover; Kristensen, Kasper; Andresen, Thomas Lars

doi:10.1016/j.bbamem.2021.183820

Cited by 7 publications

(6 citation statements)

References 67 publications

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“…They enclosed 1 mol% of DOPE jointed to Atto655 as membrane label, 20 mol% of the negatively charged POPS to set the negative membrane similarly to the biological membranes, and 79 mol%. Using the system developed created the possibility of differentiating the vesicle action mechanisms [ 97 ]. Different investigations focus on developing simple, accurate, and real-time methods to determine the MAP’s effects on targeted cells.…”

Section: Mechanisms Of Actionmentioning

confidence: 99%

Membrane-Active Peptides and Their Potential Biomedical Application

Gostaviceanu,

Gavrilaş,

Copolovici

et al. 2023

Pharmaceutics

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Membrane-active peptides (MAPs) possess unique properties that make them valuable tools for studying membrane structure and function and promising candidates for therapeutic applications. This review paper provides an overview of the fundamental aspects of MAPs, focusing on their membrane interaction mechanisms and potential applications. MAPs exhibit various structural features, including amphipathic structures and specific amino acid residues, enabling selective interaction with multiple membranes. Their mechanisms of action involve disrupting lipid bilayers through different pathways, depending on peptide properties and membrane composition. The therapeutic potential of MAPs is significant. They have demonstrated antimicrobial activity against bacteria and fungi, making them promising alternatives to conventional antibiotics. MAPs can selectively target cancer cells and induce apoptosis, opening new avenues in cancer therapeutics. Additionally, MAPs serve as drug delivery vectors, facilitating the transport of therapeutic cargoes across cell membranes. They represent a fascinating class of biomolecules with significant potential in basic research and clinical applications. Understanding their mechanisms of action and designing peptides with enhanced selectivity and efficacy will further expand their utility in diverse fields. Exploring MAPs holds promise for developing novel therapeutic strategies against infections, cancer, and drug delivery challenges.

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Section: Mechanisms Of Actionmentioning

confidence: 99%

Membrane-Active Peptides and Their Potential Biomedical Application

Gostaviceanu,

Gavrilaş,

Copolovici

et al. 2023

Pharmaceutics

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“…The liposome calcein release is an established technique to determine membrane permeation [16,30,44]. LUVs consisting of POPC were loaded with self-quenched calcein.…”

Section: Calcein Release Assaymentioning

confidence: 99%

“…Detailed studies on membrane permeation have traditionally focused on membrane active peptides [23][24][25][26][27]. The membrane permeation mechanism of these peptides are conventionally determined via bulk leakage experiments based on large unilamellar vesicles (LUVs), including classical calcein leakage setups [12,16,26,[28][29][30][31], ANTS/DPX requenching assays [26,[31][32][33], or more recently fluorescent lifetime measurements of calcein release [24,[34][35][36]. Based on the final integrity of the LUVs and the resulting leakage profile two mechanistic terms have been defined: All-or-None (AoN)…”

Section: Introductionmentioning

confidence: 99%

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism

Lund,

Kristensen,

Larsen

et al. 2023

Preprint

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A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.

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“…By using an experimental setup with vesicles of varying diameter, the curvature can be linked to the fluorescence intensity, thus quantifying curvature-induced sorting [ 78 , 80 ] ( Figure 5 B). Flow cytometry is a promising alternative to microscopy for high-throughput experiments (thousands of vesicles) [ 81 ]. By comparison with MD simulations of vesicles with the same lipid composition and protein content, researchers can investigate molecular details that are hard to probe experimentally.…”

Section: Comparison With Experimentsmentioning

confidence: 99%

Molecular Dynamics Simulations of Curved Lipid Membranes

Larsen

2022

IJMS

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Eukaryotic cells contain membranes with various curvatures, from the near-plane plasma membrane to the highly curved membranes of organelles, vesicles, and membrane protrusions. These curvatures are generated and sustained by curvature-inducing proteins, peptides, and lipids, and describing these mechanisms is an important scientific challenge. In addition to that, some molecules can sense membrane curvature and thereby be trafficked to specific locations. The description of curvature sensing is another fundamental challenge. Curved lipid membranes and their interplay with membrane-associated proteins can be investigated with molecular dynamics (MD) simulations. Various methods for simulating curved membranes with MD are discussed here, including tools for setting up simulation of vesicles and methods for sustaining membrane curvature. The latter are divided into methods that exploit scaffolding virtual beads, methods that use curvature-inducing molecules, and methods applying virtual forces. The variety of simulation tools allow researcher to closely match the conditions of experimental studies of membrane curvatures.

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Applying flow cytometry to identify the modes of action of membrane-active peptides in a label-free and high-throughput fashion

Cited by 7 publications

References 67 publications

Membrane-Active Peptides and Their Potential Biomedical Application

Membrane-Active Peptides and Their Potential Biomedical Application

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism

Molecular Dynamics Simulations of Curved Lipid Membranes

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