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1.
Mass Spectrom Rev ; 2024 Apr 05.
Article in English | MEDLINE | ID: mdl-38576378

ABSTRACT

Affinity photolabeling is a smart method to study noncovalent and transient interactions and provide a submolecular picture of the contacts between interacting partners. In this review, we will focus on the identification of peptide partners using photoaffinity labeling coupled to mass spectrometry in different contexts such as in vitro with a purified potential partner, in model systems such as model membranes, and with live cells using both targeted and nontargeted proteomics studies. Different biological partners will be described, among which glycoconjugates, oligonucleotides, peptides, proteins, and lipids, with the photoreactive label inserted either on the peptide of interest or on the potential partner. Particular attention will be paid to the observation and characterization of specific rearrangements following the photolabeling reaction, which can help characterize photoadducts and provide a better understanding of the interacting systems and environment.

2.
Nat Commun ; 14(1): 1998, 2023 04 10.
Article in English | MEDLINE | ID: mdl-37032404

ABSTRACT

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells.


Subject(s)
Glycosaminoglycans , Heparitin Sulfate , Heparitin Sulfate/metabolism , Glycosaminoglycans/metabolism , Transcription Factors , Homeodomain Proteins/genetics , Sulfates , Chondroitin Sulfates/metabolism
3.
Q Rev Biophys ; 55: e10, 2022 08 18.
Article in English | MEDLINE | ID: mdl-35979810

ABSTRACT

Trp is unique among the amino acids since it is involved in many different types of noncovalent interactions such as electrostatic and hydrophobic ones, but also in π-π, π-cation, π-anion and π-ion pair interactions. In membranotropic peptides and proteins, Trp locates preferentially at the water-membrane interface. In antimicrobial or cell-penetrating peptides (AMPs and CPPs respectively), Trp is well-known for its strong role in the capacity of these peptides to interact and affect the membrane organisation of both bacteria and animal cells at the level of the lipid bilayer. This essential amino acid can however be involved in other types of interactions, not only with lipids, but also with other membrane partners, that are crucial to understand the functional roles of membranotropic peptides. This review is focused on this latter less known role of Trp and describes in details, both in qualitative and quantitative ways: (i) the physico-chemical properties of Trp; (ii) its effect in CPP internalisation; (iii) its importance in AMP activity; (iv) its role in the interaction of AMPs with glycoconjugates or lipids in bacteria membranes and the consequences on the activity of the peptides; (v) its role in the interaction of CPPs with negatively charged polysaccharides or lipids of animal membranes and the consequences on the activity of the peptides. We intend to bring highlights of the physico-chemical properties of Trp and describe its extensive possibilities of interactions, not only at the well-known level of the lipid bilayer, but with other less considered cell membrane components, such as carbohydrates and the extracellular matrix. The focus on these interactions will allow the reader to reevaluate reported studies. Altogether, our review gathers dedicated studies to show how unique are Trp properties, which should be taken into account to design future membranotropic peptides with expected antimicrobial or cell-penetrating activity.


Subject(s)
Anti-Infective Agents , Cell-Penetrating Peptides , Animals , Amino Acids , Anti-Bacterial Agents/chemistry , Anti-Infective Agents/chemistry , Antimicrobial Cationic Peptides/metabolism , Antimicrobial Peptides , Carbohydrates , Cations , Lipid Bilayers , Tryptophan/chemistry , Tryptophan/metabolism , Water
4.
ACS Chem Biol ; 17(6): 1427-1439, 2022 06 17.
Article in English | MEDLINE | ID: mdl-35608167

ABSTRACT

Cell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,5)P2 and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photo-crosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photo-crosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,5)P2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization.


Subject(s)
Cell-Penetrating Peptides , Carrier Proteins/metabolism , Cell-Penetrating Peptides/metabolism , Phosphatidylinositols , Protein Binding
5.
Methods Mol Biol ; 2383: 143-152, 2022.
Article in English | MEDLINE | ID: mdl-34766287

ABSTRACT

Fluorescence-based methods are widely used to detect crossing of peptides across model or biological membranes. For membrane-active peptides, i.e., peptides that have strong membrane tropism, fluorescence experiments must be accompanied by relevant controls, otherwise they can lead to inconsistent interpretation and underestimation of their limitations. Here we describe how to prepare samples to study fluorescent peptide crossing droplet interface bilayer (model membrane) or cell membrane (biological membrane) and the pitfalls that can affect observational qualitative and quantitative data.


Subject(s)
Cell-Penetrating Peptides , Cell Membrane/metabolism , Cell Membrane Permeability , Cell-Penetrating Peptides/metabolism , Fluorescence , Lipid Bilayers/metabolism
6.
Bioconjug Chem ; 32(8): 1503-1514, 2021 08 18.
Article in English | MEDLINE | ID: mdl-34160213

ABSTRACT

Membranotropic peptides is a class of peptides that exert their biological action at the level of cell membranes. Understanding how they interact with their different membrane binding partners (lipids, proteins, and/or glycoconjugates) is important to decipher their mechanism of action. Affinity photolabeling is a powerful method to study noncovalent interactions and provide a submolecular picture of the contacts between two interacting partners. In this review, we give a panorama of photolabeling-based studies of the interactions between membranotropic peptides and membranes using either photoreactive lipids or peptides.


Subject(s)
Cell Membrane/metabolism , Membrane Lipids/metabolism , Membrane Proteins/metabolism , Peptides/metabolism , Animals , Cell Membrane/chemistry , Humans , Light , Membrane Lipids/analysis , Membrane Proteins/analysis , Models, Molecular , Peptides/analysis , Staining and Labeling/methods
8.
J Cell Biol ; 220(4)2021 04 05.
Article in English | MEDLINE | ID: mdl-33740033

ABSTRACT

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.


Subject(s)
Drosophila Proteins/metabolism , Embryo, Nonmammalian/metabolism , Fatty Acid-Binding Proteins/metabolism , Pseudopodia/metabolism , Xenopus Proteins/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster , Fatty Acid-Binding Proteins/genetics , Pseudopodia/genetics , Xenopus , Xenopus Proteins/genetics
9.
Arch Biochem Biophys ; 699: 108751, 2021 03 15.
Article in English | MEDLINE | ID: mdl-33421380

ABSTRACT

Antimicrobial and cell-penetrating peptides have been the object of extensive studies for more than 60 years. Initially these two families were studied separately, and more recently parallels have been drawn. These studies have given rise to numerous methodological developments both in terms of observation techniques and membrane models. This review presents some of the most recent original and innovative developments in this field, namely droplet interface bilayers (DIBs), new fluorescence approaches, force measurements, and photolabelling.


Subject(s)
Antimicrobial Cationic Peptides/metabolism , Cell Membrane/metabolism , Cell-Penetrating Peptides/metabolism , Amino Acid Sequence , Antimicrobial Cationic Peptides/chemistry , Cell Membrane/chemistry , Cell-Penetrating Peptides/chemistry , Fluorescent Dyes/chemistry , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Microscopy, Fluorescence/methods , Photoaffinity Labels/chemistry , Spectrometry, Fluorescence/methods
10.
Bioconjug Chem ; 31(6): 1611-1615, 2020 06 17.
Article in English | MEDLINE | ID: mdl-32378884

ABSTRACT

Membrane curvature plays a pivotal role in cellular life, including cellular uptake and membrane trafficking. The modulation of membrane curvature provides a novel means of manipulating cellular events. In this report, we show that a nine-residue amphiphilic peptide (R6W3) stimulates endocytic uptake by inducing membrane curvature. Curvature formation on cell membranes was confirmed by observing the cellular distribution of the curvature-sensing protein amphiphysin fused with a yellow fluorescent protein (Amp-YFP). Dot-like signals of Amp-YFP were visible following the addition of R6W3, suggesting curvature formation in cell membranes, leading to endocytic cup and vesicle formation. The promotion of endocytic uptake was confirmed using the endocytosis marker polydextran. Treatment of cells with R6W3 yielded a 4-fold dextran uptake compared with untreated cells. The amphiphilic helical structure of R6W3 was also crucial for R6W3-stimulated endocytic uptake.


Subject(s)
Cell Membrane/drug effects , Cell Membrane/metabolism , Endocytosis/drug effects , Hydrophobic and Hydrophilic Interactions , Peptides/chemistry , Peptides/pharmacology , Bacterial Proteins/metabolism , HeLa Cells , Humans , Luminescent Proteins/metabolism
11.
ACS Infect Dis ; 6(2): 224-236, 2020 02 14.
Article in English | MEDLINE | ID: mdl-31855415

ABSTRACT

We have developed a nanocarrier consisting of large unilamellar vesicles (LUVs) for combined delivery of two human immunodeficiency virus type 1 (HIV-1) entry inhibitors, enfuvirtide (ENF) and protoporphyrin IX (PPIX). The intrinsic lipophilicity of ENF and PPIX, a fusion inhibitor and an attachment inhibitor, respectively, leads to their spontaneous incorporation into the lipid bilayer of the LUVs nanocarrier. Both entry inhibitors partition significantly toward LUVs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a 9:1 mixture of POPC:1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), representative of conventional and immune-evasive drug delivery formulations, respectively. These colocalize in the core of lipid membranes. Dual-loaded nanocarriers are monodispersed and retain the size distribution, thermotropic behavior, and surface charge of the unloaded form. Combination of the two entry inhibitors in the nanocarrier resulted in improved synergy against HIV-1 entry compared to combination in free form, strongly when immune-evasive formulations are used. We propose that the improved action of the entry inhibitors when loaded into the nanocarriers results from their slow release at the site of viral entry. Overall, liposomes remain largely unexplored platforms for combination of viral entry inhibitors, with potential for improvement of current antiretroviral therapy drug safety and application. Our work calls for a reappraisal of the potential of entry inhibitor combinations and delivery for clinical use in antiretroviral therapy.


Subject(s)
Enfuvirtide/pharmacology , HIV-1/drug effects , HIV-1/physiology , Protoporphyrins/pharmacology , Virus Internalization/drug effects , Cell Line , Drug Synergism , Humans , Inhibitory Concentration 50 , Liposomes/chemistry , Nanoparticles/chemistry , Polyethylene Glycols
12.
Biochim Biophys Acta Biomembr ; 1862(2): 183098, 2020 02 01.
Article in English | MEDLINE | ID: mdl-31676372

ABSTRACT

Cell-penetrating peptides (CPPs) internalization occurs both by endocytosis and direct translocation through the cell membrane. These different entry routes suggest that molecular partners at the plasma membrane, phospholipids or glycosaminoglycans (GAGs), bind CPPs with different affinity or selectivity. The analysis of sequence-dependent interactions of CPPs with lipids and GAGs should lead to a better understanding of the molecular mechanisms underlying their internalization. CPPs are short sequences generally containing a high number of basic arginines and lysines and sometimes aromatic residues, in particular tryptophans. Tryptophans are crucial residues in membrane-active peptides, because they are important for membrane interaction. Membrane-active peptides often present facial amphiphilicity, which also promote the interaction with lipid bilayers. To study the role of Trp and facial amphiphilicity in cell interaction and penetration of CPPs, a nonapeptide series containing only Arg, Trp or D-Trp residues at different positions was designed. Our quantitative study indicates that to maintain/increase the uptake efficiency, Arg can be advantageously replaced by Trp in the nonapeptides. The presence of Trp in oligoarginines increases the uptake in cells expressing GAGs at their surface, while it compensates for the loss of charge interactions from Arg and maintains similar peptide uptake in GAG-deficient cells. In addition, we show that facial amphiphilicity is not required for efficient uptake of these nonapeptides. Thermodynamic analyses point towards a key role of Trp that highly contributes to the binding enthalpy of complexes formation. Density functional theory (DFT) analysis highlights that salt bridge-π interactions play a crucial role for the GAG-dependent entry mechanisms.


Subject(s)
Cell Membrane/metabolism , Cell-Penetrating Peptides/chemistry , Amino Acid Sequence , Animals , Arginine , CHO Cells , Cell-Penetrating Peptides/pharmacokinetics , Cricetinae , Cricetulus , Endocytosis , Glycosaminoglycans/metabolism , Humans , Protein Transport , Thermodynamics , Tryptophan
13.
Anal Chem ; 91(14): 9102-9110, 2019 07 16.
Article in English | MEDLINE | ID: mdl-31251038

ABSTRACT

Affinity photo-cross-linking coupled to mass spectrometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ9), 18:1 (trans Δ9) PG, and DLoPG (18:2 cis Δ9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: C═C double bond formation on saturated fatty acids, covalent adducts formation via classical C-C bond, and Paternò-Büchi oxetane formation followed or not by fragmentation (retro-Paternò-Büchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction.


Subject(s)
Benzophenones/chemistry , Cell-Penetrating Peptides/chemistry , Cross-Linking Reagents/chemistry , Fatty Acids/analysis , Lipid Bilayers/chemistry , Amino Acid Sequence , Benzophenones/radiation effects , Cholesterol/chemistry , Cross-Linking Reagents/radiation effects , Fatty Acids/chemistry , Oxidation-Reduction/radiation effects , Phospholipids/chemistry , Tandem Mass Spectrometry , Ultraviolet Rays
14.
J Phys Chem Lett ; 10(7): 1523-1529, 2019 Apr 04.
Article in English | MEDLINE | ID: mdl-30864805

ABSTRACT

We introduce a method for the detection of weak interactions of small molecules such as metabolites or medicaments that contain deuterated methyl groups with proteins in solution. The technique relies on long-lived imbalances of spin state populations, which are generated by dissolution dynamic nuclear polarization (D-DNP) and feature lifetimes that depend on the frequency of internal rotation of deuterated methyl groups. We demonstrate the technique for interactions between deuterated dimethyl sulfoxide (DMSO- d6) and bovine serum albumin (BSA) or trypsin, where the methyl group rotation is slowed down upon protein binding, which causes a marked reduction in the lifetime of the population imbalances.


Subject(s)
Dimethyl Sulfoxide/chemistry , Serum Albumin, Bovine/chemistry , Animals , Cattle , Deuterium/chemistry , Dimethyl Sulfoxide/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Serum Albumin, Bovine/metabolism , Trypsin/chemistry , Trypsin/metabolism
15.
PLoS One ; 13(10): e0205727, 2018.
Article in English | MEDLINE | ID: mdl-30325956

ABSTRACT

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.


Subject(s)
Amphibian Proteins/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Anura/metabolism , Enterococcus faecium/drug effects , Membranes/drug effects , Peptidoglycan/pharmacology , Skin/chemistry , Staphylococcus aureus/drug effects , A549 Cells/drug effects , Amphibian Proteins/genetics , Amphibian Proteins/isolation & purification , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/isolation & purification , Circular Dichroism , Drug Synergism , Humans , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests
16.
Acc Chem Res ; 50(12): 2968-2975, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29172443

ABSTRACT

Living organisms have to maintain a stable balance in molecules and ions in the changing environment in which they are living, a process known as homeostasis. At the level of cells, the plasma membrane has a major role in homeostasis, since this hydrophobic film prevents passive diffusion of large and hydrophilic molecules between the extracellular and intracellular milieu. Living organisms have evolved with highly sophisticated transport systems to control exchanges across this barrier: import of nutrients and fuel essential for their survival; recognition of chemical or physical messengers allowing information interchanges with surrounding cells. Besides specialized proteins, endocytosis mechanisms at the level of the lipid bilayer can transport molecules from the outside across the cell membrane, in an energy-dependent manner. The cell membrane is highly heterogeneous in its molecular composition (tens of different lipids, proteins, polysaccharides, and combinations of these) and dynamic with bending, deformation, and elastic properties that depend on the local composition of membrane domains. Many viruses, microorganisms, and toxins exploit the plasma membrane to enter into cells. Chemists develop strategies to target the plasma membrane with molecules capable of circumventing this hydrophobic barrier, in particular to transport and deliver nonpermeable drugs in cells for biotechnological or pharmaceutical purposes. Drug delivery systems are numerous and include lipid-, sugar-, protein-, and peptide-based delivery systems, since these biomolecules generally have good biocompatibility, biodegradability, environmental sustainability, cost effectiveness, and availability. Among those, cell-penetrating peptides (CPPs), reported for the first time in the early 1990s, are attracting major interest not only as potential drug delivery systems but also at the level of fundamental research. It was indeed demonstrated very early that these peptides, which generally correspond to highly cationic sequences, can still cross the cell membrane at 4 °C, a temperature at which all active transport and endocytosis pathways are totally inhibited. Therefore, how these charged hydrophilic peptides cross the hydrophobic membrane barrier is of utmost interest as a pure basic and physicochemical question. In this Account, we focus on cationic cell-penetrating peptides (CPPs) and the way they cross cell membranes. We summarize the history of this field that emerged around 20 years ago. CPPs were indeed first identified as protein-transduction domains from the human immunodeficiency virus (HIV) TAT protein and the Antennapedia homeoprotein, a transcription factor from Drosophila. We highlight our contribution to the elucidation of CPP internalization pathways, in particular translocation, which implies perturbation and reorganization of the lipid bilayer, and endocytosis depending on sulfated glycosaminoglycans. We show a particular role of Trp (indole side chain) and Arg (guanidinium side chain), which are essential amino acids for CPP internalization. Interactions with the cell-surface are not only Coulombic; H-bonds and hydrophobic interactions contribute also significantly to CPP entry. The capacity of CPPs to cross cell membrane is not related to their strength of membrane binding. Finally, we present optimized methods based on mass spectrometry and fluorescence spectroscopy that allow unequivocal quantification of CPPs inside cells or bound to the outer leaflet of the membrane, and discuss some limitations of the technique of flow cytometry that we have recently highlighted.


Subject(s)
Cell Membrane/metabolism , Cell-Penetrating Peptides/metabolism , Oligopeptides/metabolism , Amino Acid Sequence , Animals , Cell Membrane/chemistry , Cell-Penetrating Peptides/chemistry , Endocytosis , Fluorometry , Glycosaminoglycans/chemistry , Kinetics , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Oligopeptides/chemistry , Protein Transport , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thermodynamics
17.
J Cell Biol ; 216(11): 3745-3765, 2017 11 06.
Article in English | MEDLINE | ID: mdl-28923975

ABSTRACT

The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP-WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.


Subject(s)
Actins/metabolism , Clathrin-Coated Vesicles/metabolism , Clathrin/metabolism , Coated Pits, Cell-Membrane/metabolism , Endocytosis , Phosphatidylinositols/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Animals , CRISPR-Cas Systems , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , HeLa Cells , Humans , Hydrolysis , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Oculocerebrorenal Syndrome/genetics , Oculocerebrorenal Syndrome/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Protein Multimerization , RNA Interference , Retinal Pigment Epithelium/metabolism , Signal Transduction , Sorting Nexins/genetics , Sorting Nexins/metabolism , Time Factors , Transfection , Wiskott-Aldrich Syndrome Protein, Neuronal/genetics , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Xenopus laevis , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolism
18.
Biopolymers ; 107(12)2017 Dec.
Article in English | MEDLINE | ID: mdl-28944959

ABSTRACT

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.


Subject(s)
Anti-Infective Agents/metabolism , Escherichia coli/metabolism , Muramidase/metabolism , Anti-Infective Agents/pharmacology , Calorimetry, Differential Scanning , Cell Wall/metabolism , Circular Dichroism , Escherichia coli/drug effects , Escherichia coli/growth & development , Isoenzymes/chemistry , Isoenzymes/metabolism , Isoenzymes/pharmacology , Muramidase/chemistry , Muramidase/pharmacology , Spectrometry, Fluorescence , Succinimides/chemistry , Thermodynamics
19.
J Am Chem Soc ; 139(35): 12219-12227, 2017 09 06.
Article in English | MEDLINE | ID: mdl-28780862

ABSTRACT

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.


Subject(s)
Intrinsically Disordered Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Crystallography, X-Ray , DNA Ligase ATP/chemistry , Molecular Docking Simulation , Protein Binding , Protein Conformation , Protein Folding
20.
Methods Cell Biol ; 128: 125-47, 2015.
Article in English | MEDLINE | ID: mdl-25997346

ABSTRACT

Xenopus egg extracts are a powerful tool to reconstitute complex cell biological processes using a cell-free strategy. When used in conjunction with liposomes and supported lipid bilayers, they can recapitulate the biochemical activities occurring at the cytosol/plasma membrane interface of the cell that underlie remodeling of the actin cytoskeleton. We use these in vitro systems to elucidate how membranes and proteins collaborate to make the appropriate actin structure at a given time and place. We have recently broadened the types of membrane substrate used, and also optimized protocols for preparation of Xenopus egg extracts for actin assembly assays from membranes. Tuning the lipid composition and curvature appropriately demands an appreciation of the native phospholipid and curvature environments that can form transiently in cells. Supported lipid bilayers on glass coverslips that contain phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) make actin bundles termed filopodia-like structures that contain fascin and have vasodilator-stimulated phosphoprotein (VASP) at their growing tips, indicating that these resemble filopodia growing from the plasma membrane. The combination of PI(4,5)P2 and phosphatidylinositol 3-phosphate in curved liposomes or supported bilayers on glass nanospheres uses Snx9, Cdc42, N-WASP (neuronal-Wiskott-Aldrich syndrome protein), and Arp2/3 complex for actin polymerization suggesting that this membrane may mimic the progression from plasma membrane to endosomes. Here we describe how to prepare high-speed supernatant frog egg extracts and phosphoinositide-containing liposomes and supported lipid bilayers that can assemble actin structures. We also describe the methods we use to assay actin polymerization using microscopy and spectrofluorometry and our protocol for immunodepleting specific proteins from extracts.


Subject(s)
Actins/metabolism , Lipid Bilayers/chemical synthesis , Liposomes/chemical synthesis , Tissue Extracts/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Animals , Carrier Proteins/metabolism , Cell Membrane/metabolism , Cell-Free System , Female , Lipid Bilayers/metabolism , Liposomes/metabolism , Microfilament Proteins/metabolism , Monomeric GTP-Binding Proteins/metabolism , Nanospheres/metabolism , Ovum/cytology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphatidylserines/metabolism , Polymerization , Pseudopodia , Sorting Nexins/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Xenopus Proteins/metabolism , Xenopus laevis
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