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Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge-based design of cancer-targeting molecular tools, such as pH-low insertion peptide (pHLIP). Here, we explore the effects of PS, Ca2+ , and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond-timescale all-atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide-lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH-induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+ -induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to linking cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+ -dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid-coupled regulation of protein-membrane insertion by divalent cations.

Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit "a" has four isoforms, a1–a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KXnK(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa. [ABSTRACT FROM AUTHOR]

Protein-lipid interactions are crucial for various cellular biological processes like intracellular signaling, membrane transport, and cytoskeletal dynamics. Therefore, studying these interactions is essential to understand and unravel their specific functions. Nevertheless, the interacting proteins of many lipids are poorly understood and still require systematic study. Liposomes are the most well-known and familiar biomimetic systems used to study protein-lipid interactions. Although liposomes have been widely used for studying protein-lipid interactions in classical methods such as the co-flotation assay (CFA), co-sedimentation assay (CSA), and flow cytometric assay (FCA), an overview of their current applications and developments in high-throughput methods is not yet available. Here, we summarize the liposome development in low and high-throughput methods to study protein-lipid interactions. Besides, a constructive comment for each platform is presented to stimulate the advancement of these technologies in the future. [ABSTRACT FROM AUTHOR]

Abstract Background TMC1, which encodes transmembrane channel-like protein 1, forms the mechanoelectrical transduction (MET) channel in auditory hair cells, necessary for auditory function. TMC1 variants are known to cause autosomal dominant (DFNA36) and autosomal recessive (DFNB7/11) non-syndromic hearing loss, but only a handful of TMC1 variants underlying DFNA36 have been reported, hampering analysis of genotype-phenotype correlations. Methods In this study, we retrospectively reviewed 338 probands in an in-house database of genetic hearing loss, evaluating the clinical phenotypes and genotypes of novel TMC1 variants associated with DFNA36. To analyze the structural impact of these variants, we generated two structural models of human TMC1, utilizing the Cryo-EM structure of C. elegans TMC1 as a template and AlphaFold protein structure database. Specifically, the lipid bilayer-embedded protein database was used to construct membrane-embedded models of TMC1. We then examined the effect of TMC1 variants on intramolecular interactions and predicted their potential pathogenicity. Results We identified two novel TMC1 variants related to DFNA36 (c.1256T > C:p.Phe419Ser and c.1444T > C:p.Trp482Arg). The affected subjects had bilateral, moderate, late-onset, progressive sensorineural hearing loss with a down-sloping configuration. The Phe419 residue located in the transmembrane domain 4 of TMC1 faces outward towards the channel pore and is in close proximity to the hydrophobic tail of the lipid bilayer. The non-polar-to-polar variant (p.Phe419Ser) alters the hydrophobicity in the membrane, compromising protein-lipid interactions. On the other hand, the Trp482 residue located in the extracellular linker region between transmembrane domains 5 and 6 is anchored to the membrane interfaces via its aromatic rings, mediating several molecular interactions that stabilize the structure of TMC1. This type of aromatic ring-based anchoring is also observed in homologous transmembrane proteins such as OSCA1.2. Conversely, the substitution of Trp with Arg (Trp482Arg) disrupts the cation-π interaction with phospholipids located in the outer leaflet of the phospholipid bilayer, destabilizing protein-lipid interactions. Additionally, Trp482Arg collapses the CH-π interaction between Trp482 and Pro511, possibly reducing the overall stability of the protein. In parallel with the molecular modeling, the two mutants degraded significantly faster compared to the wild-type protein, compromising protein stability. Conclusions This results expand the genetic spectrum of disease-causing TMC1 variants related to DFNA36 and provide insight into TMC1 transmembrane protein-lipid interactions.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

Background: TMC1, which encodes transmembrane channel-like protein 1, forms the mechanoelectrical transduction (MET) channel in auditory hair cells, necessary for auditory function. TMC1 variants are known to cause autosomal dominant (DFNA36) and autosomal recessive (DFNB7/11) non-syndromic hearing loss, but only a handful of TMC1 variants underlying DFNA36 have been reported, hampering analysis of genotype-phenotype correlations. Methods: In this study, we retrospectively reviewed 338 probands in an in-house database of genetic hearing loss, evaluating the clinical phenotypes and genotypes of novel TMC1 variants associated with DFNA36. To analyze the structural impact of these variants, we generated two structural models of human TMC1, utilizing the Cryo-EM structure of C. elegans TMC1 as a template and AlphaFold protein structure database. Specifically, the lipid bilayer-embedded protein database was used to construct membrane-embedded models of TMC1. We then examined the effect of TMC1 variants on intramolecular interactions and predicted their potential pathogenicity. Results: We identified two novel TMC1 variants related to DFNA36 (c.1256T > C:p.Phe419Ser and c.1444T > C:p.Trp482Arg). The affected subjects had bilateral, moderate, late-onset, progressive sensorineural hearing loss with a down-sloping configuration. The Phe419 residue located in the transmembrane domain 4 of TMC1 faces outward towards the channel pore and is in close proximity to the hydrophobic tail of the lipid bilayer. The non-polar-to-polar variant (p.Phe419Ser) alters the hydrophobicity in the membrane, compromising protein-lipid interactions. On the other hand, the Trp482 residue located in the extracellular linker region between transmembrane domains 5 and 6 is anchored to the membrane interfaces via its aromatic rings, mediating several molecular interactions that stabilize the structure of TMC1. This type of aromatic ring-based anchoring is also observed in homologous transmembrane proteins such as OSCA1.2. Conversely, the substitution of Trp with Arg (Trp482Arg) disrupts the cation-π interaction with phospholipids located in the outer leaflet of the phospholipid bilayer, destabilizing protein-lipid interactions. Additionally, Trp482Arg collapses the CH-π interaction between Trp482 and Pro511, possibly reducing the overall stability of the protein. In parallel with the molecular modeling, the two mutants degraded significantly faster compared to the wild-type protein, compromising protein stability. Conclusions: This results expand the genetic spectrum of disease-causing TMC1 variants related to DFNA36 and provide insight into TMC1 transmembrane protein-lipid interactions. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.

The aggregation of human Islet Amyloid Polypeptide (hIAPP) on cell membranes is linked to amyloid diseases. However, the physio-chemical mechanisms of how these hIAPP aggregates trigger membrane damage are unclear. Using coarse-grained and all-atom molecular dynamics simulations, we investigated the role of lipid nanodomains in the presence or absence of anionic lipids, phosphatidylserine (PS), and a ganglioside (GM1), in the membrane disruption and protein folding behaviors of hIAPP aggregates on phase-separated raft membranes. Our raft membranes contain liquid-ordered (Lo), liquid-disordered (Ld), mixed Lo/Ld (Lod), PS-cluster, and GM1-cluster nanosized domains. We observed that hIAPP aggregates bound to the Lod domain in the absence of anionic lipids, but also to the GM1-cluster- and PS-cluster-containing domains, with stronger affinity in the presence of anionic lipids. We discovered that L16 and I26 are the lipid anchoring residues of hIAPP binding to the Lod and PS-cluster domains. Finally, significant lipid acyl chain order disruption in the annular lipid shells surrounding the membrane-bound hIAPP aggregates and protein folding, particularly beta-sheet formation, in larger protein aggregates were evident. We propose that the interactions of hIAPP and both non-anionic and anionic lipid nanodomains represent key molecular events of membrane damage associated with the pathogenesis of amyloid diseases. [ABSTRACT FROM AUTHOR]

The gene encoding the GTPase KRAS is frequently mutated in pancreatic, lung, and colorectal cancers. The KRAS fraction in the plasma membrane (PM) correlates with activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent cellular proliferation. Understanding KRAS's interaction with the PM is challenging given the complexity of the cellular environment. To gain insight into key components necessary for KRAS signal transduction at the PM, we used synthetic membranes such as liposomes and giant unilamellar vesicles. Using surface plasmon resonance (SPR) spectroscopy, we demonstrated that KRAS and Raf-1 proto-oncogene Ser/Thr kinase (RAF1) domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. We found that the RAF1 region spanning RBD through CRD (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS partitioning to the membrane. We also found that calmodulin and phosphodiesterase 6 delta (PDE6δ), but not galectin3 previously proposed to directly interact with KRAS, passively sequester KRAS and prevent it from partitioning into the PM. RAF1 RBDCRD interacted with membranes preferentially at nonraft lipid domains. Moreover, a C-terminal O-methylation was crucial for KRAS membrane localization. These results contribute to a better understanding of how the KRAS-membrane interaction is tuned by multiple factors whose identification could inform drug discovery efforts to disrupt this critical interaction in diseases such as cancer.

Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K234IKK237, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P2, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P2 by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P2 binding capacity is involved in a4 V-ATPase plasma membrane retention. [ABSTRACT FROM AUTHOR]

Gap junction channels and ATP release channels are called “large pore channels” because these have pore sizes that allow the permeation of various sizes of solutes, from ions to metabolites. We investigated the structures of C. elegans innexin-6 gap junction channels and human pannexin-1 channels with cryo-EM. Using nanodisc reconstitution, we found double-layer densities in the pore and lipid acyl chains in the intersubunit spaces. Together with the molecular dynamics simulation, these studies suggest a lipid gating mechanism for large pore channels in that phospholipids are diffused to the pore for channel closure. [ABSTRACT FROM AUTHOR]

The molecular cause of transmissible spongiform encephalopathies (TSEs) involves the conversion of the cellular prion protein (PrPC) into its pathogenic form, called prion scrapie (PrPSc), which is prone to the formation of amorphous and amyloid aggregates found in TSE patients. Although the mechanisms of conversion of PrPC into PrPSc are not entirely understood, two key points are currently accepted: (i) PrPSc acts as a seed for the recruitment of native PrPC, inducing the latter’s conversion to PrPSc; and (ii) other biomolecules, such as DNA, RNA, or lipids, can act as cofactors, mediating the conversion from PrPC to PrPSc. Interestingly, PrPC is anchored by a glycosylphosphatidylinositol molecule in the outer cell membrane. Therefore, interactions with lipid membranes or alterations in the membranes themselves have been widely investigated as possible factors for conversion. Alone or in combination with RNA molecules, lipids can induce the formation of PrP in vitro-produced aggregates capable of infecting animal models. Here, we discuss the role of lipids in prion conversion and infectivity, highlighting the structural and cytotoxic aspects of lipid-prion interactions. Strikingly, disorders like Alzheimer’s and Parkinson’s disease also seem to be caused by changes in protein structure and share pathogenic mechanisms with TSEs. Thus, we posit that comprehending the process of PrP conversion is relevant to understanding critical events involved in a variety of neurodegenerative disorders and will contribute to developing future therapeutic strategies for these devastating conditions.

The endoplasmic reticulum is organized into ordered regions enriched in cholesterol and sphingomyelin, and disordered microdomains characterized by more fluidity. Rabbit CYP1A1 and CYP1A2 localize into disordered and ordered microdomains, respectively. Previously, a CYP1A2 chimera containing the first 109 amino acids of CYP1A1 showed altered microdomain localization. The goal of this study was to identify specific residues responsible for CYP1A microdomain localization. Thus, CYP1A2 chimeras containing substitutions from homologous regions of CYP1A1 were expressed in HEK 293T/17 cells, and the localization was examined after solubilization with Brij 98. A CYP1A2 mutant with the three amino acids from CYP1A1 (VAG) at positions 27 to 29 of CYP1A2 was generated that showed a distribution pattern similar to those of CYP1A1/1A2 chimeras containing both the first 109 amino acids and the first 31 amino acids of CYP1A1 followed by remaining amino acids of CYP1A2. Similarly, the reciprocal substitution of three amino acids from CYP1A2 (AVR) into CYP1A1 resulted in a partial redistribution of the chimera into ordered microdomains. Molecular dynamic simulations indicate that the positive charges of the CYP1A1 and CYP1A2 linker regions between the N termini and catalytic domains resulted in different depths of immersion of the N termini in the membrane. The overlap of the distribution of positively charged residues in CYP1A2 (AVR) and negatively charged phospholipids was higher in the ordered than in the disordered microdomain. These findings identify three residues in the CYP1AN terminus as a novel microdomain-targeting motif of the P450s and provide a mechanistic explanation for the differential microdomain localization of CYP1A., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Toxoplasma gondii, the causative agent of toxoplasmosis, infects cells and replicates inside via the secretion of factors stored in specialized organelles (rhoptries, micronemes, and dense granules) and the capture of host materials. The genesis of the secretory organelles and the processes of secretion and endocytosis depend on vesicular trafficking events whose molecular bases remain poorly known. Notably, there is no characterization of the BAR (Bin/Amphiphysin/Rvs) domain-containing proteins expressed by T. gondii and other apicomplexans, although such proteins are known to play critical roles in vesicular trafficking in other eukaryotes. Here, by combining structural analyses with in vitro assays and cellular observations, we have characterized TgREMIND (regulators of membrane interacting domains), involved in the genesis of rhoptries and dense granules, and TgBAR2 found at the parasite cortex. We establish that TgREMIND comprises an F-BAR domain that can bind curved neutral membranes with no strict phosphoinositide requirement and exert a membrane remodeling activity. Next, we establish that TgREMIND contains a new structural domain called REMIND, which negatively regulates the membrane-binding capacities of the F-BAR domain. In parallel, we report that TgBAR2 contains a BAR domain with an extremely basic membrane-binding interface able to deform anionic membranes into very narrow tubules. Our data show that T. gondii codes for two atypical BAR domain-containing proteins with very contrasting membrane-binding properties, allowing them to function in two distinct regions of the parasite trafficking system., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge‐based design of cancer‐targeting molecular tools, such as pH‐low insertion peptide (pHLIP). Here, we explore the effects of PS, Ca2+, and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond‐timescale all‐atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide‐lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH‐induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+‐induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to linking cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+‐dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid‐coupled regulation of protein‐membrane insertion by divalent cations. [ABSTRACT FROM AUTHOR]

Cytoglobin is a hemoprotein widely expressed in fibroblasts and related cell lineages with yet undefined physiological function. Cytoglobin, as other heme proteins, can reduce nitrite to nitric oxide (NO) providing a route to generate NO in vivo in low oxygen conditions. In addition, cytoglobin can also bind lipids such as oleic acid and cardiolipin with high affinity. These two processes are potentially relevant to cytoglobin function. Little is known about how specific amino acids contribute to nitrite reduction and lipid binding. Here we investigate the role of the distal histidine His81 (E7) and several surface residues on the regulation of nitrite reduction and lipid binding. We observe that the replacement of His81 (E7) greatly increases heme reactivity towards nitrite, with nitrite reduction rate constants of up to 1100 M−1s−1 for the His81Ala mutant. His81 (E7) mutation causes a small decrease in lipid binding affinity, however experiments on the presence of imidazole indicate that His81 (E7) does not compete with the lipid for the binding site. Mutations of the surface residues Arg84 and Lys116 largely impair lipid binding. Our results suggest that dissociation of His81 (E7) from the heme mediates the formation of a hydrophobic cavity in the proximal heme side that can accommodate the lipid, with important contributions of the hydrophobic patch around residues Thr91, Val105, and Leu108, whereas the positive charges from Arg84 and Lys116 stabilize the carboxyl group of the fatty acid. Gain and loss-of-function mutations described here can serve as tools to study in vivo the physiological role of these putative cytoglobin functions. • Replacement of cytoglobin distal histidine increases reactivity towards nitrite. • Mutations of the cytoglobin surface residues Arg84 and Lys116 impair lipid binding. • Dissociation of His81 (E7) from the heme is required for lipid binding. • The hydrophobic patch formed by Thr91, Val105, and Leu108 mediates lipid binding. [ABSTRACT FROM AUTHOR]

Food structure is governed by the assembly and interactions between macromolecules such as carbohydrates, proteins, and lipids. A deeper understanding on the science governing food structure is integral for manufacturers seeking opportunities to develop food and food-based products with enhanced performance, safety, sustainability, and nutritional profiles. While interactions between protein and polysaccharides are generally well characterised for a range of applications, much less is known of the factors governing protein-lipid interactions (PLI) for food and food-based applications. This review examines the factors and mechanism of PLI for food and food-based applications. The influence of factors such as processing method, type of lipids and type of proteins on PLI and/or protein-lipid structures are summarised. In addition, we report a link between PLI and protein-lipid structures on techno-functional properties in food and food-based applications. Based on the analytical data on protein and lipid structures, we postulate a mechanism involved in PLI at the emulsion interfacial region. The findings from this study provides a starting point on understanding PLI and highlights the potential of PLI to influence techno-functional properties in food and food-based applications. [Display omitted] • Effect of protein-lipid interaction on lipid and protein structures. • Correlation of protein microenvironment with protein-lipid interaction. • Mechanism of protein-lipid interactions at emulsion interface. • Influence of processing, lipid and protein types on protein-lipid structures and interactions. • Role of protein-lipid structures and interactions on texture, physical and oxidative stability. [ABSTRACT FROM AUTHOR]

P2Y12 receptors on the platelet plasma membrane are targeted by several antiplatelets drugs. Although oligomerization and functioning of P2Y12 receptors depend on the membrane environment, little is known about their preferred membrane localization and the role of surrounding lipid composition, especially the arachidonic acids (ARA), which are abundant in platelets. Coarse-grained molecular dynamics simulations of platelet plasma membrane based on the lipidomics data were used to investigate the P2Y12 lipid environment and the involvement of ARA in its oligomerization in platelet plasma membranes. The platelet plasma membrane contains two types of lipids nanodomains: ordered, enriched in SM and cholesterol, and disordered, enriched in ARA-containing lipids. P2Y12 receptors prefer to localize in these ARA-rich domains and induce the sorting of the ARA-containing lipids in their vicinity. This ARA-rich environment promotes the oligomerization of P2Y12 receptors and stabilizes the protein-protein interfaces of oligomers. As summary, oligomerization of P2Y12 receptors is promoted in ARA-rich nano-domains of the platelet plasma membrane. [Display omitted] • Platelet plasma membrane contains ordered and disordered nanodomains. • Disordered nanodomains are enriched with arachidonic acid-containing lipids. • P2Y12 receptors localize to arachidonic acid-enriched disordered nanodomains. • Arachidonic acid-containing lipids promote oligomerization of P2Y12 receptors. [ABSTRACT FROM AUTHOR]

Sturgeon, with 4 times higher lipid content than silver carp (ubiquitously applied for surimi production in China), affects surimi gelling properties. However, how the flesh lipids affect gelling properties remains unclear. This study investigated how flesh lipids impact surimi gelling properties and elucidated the interaction mechanism between lipids and proteins. Results revealed yellow meat contains 7 times higher lipids than white meat. Stronger ionic protein-protein interactions were replaced by weaker hydrophobic forces and hydrogen bonds in protein-lipid interaction. Protein-lipid interaction zones encapsulated lipid particles, changing protein structure from α-helix to β-sheet structure thereby gel structure becomes flexible and disordered, significantly diminishing surimi gel strength. Docking analysis validated fatty acid mainly binding at Ala577, Ile461, Arg231, Phe165, His665, and His663 of myosin. This study first reported the weakened surimi gelling properties from the perspective of free fatty acids and myosin interactions, offering a theoretical basis for sturgeon surimi production. • Lipid-rich sturgeon yellow meat reduced surimi gel strength. • Lipids decreased the stronger ionic bonds in protein-protein interactions. • Lipids occupy the hydrophobic core of protein-protein interactions. • Protein conformation shifted from α-helix to β-sheet, indicating more flexibility. [ABSTRACT FROM AUTHOR]

In this study we provide the first evidence of the interaction of a truncated-TRAF2 with lipid raft microdomains. We have analyzed this interaction by measuring the diffusion coefficient of the protein in large and giant unilamellar vesicles (LUVs and GUVs, respectively) obtained both from synthetic lipid mixtures and from natural extracts. Steady-state fluorescence measurements performed with synthetic vesicles indicate that this truncated form of TRAF2 displays a tighter binding to raft-like LUVs with respect to the control (POPC-containing LUVs), and that this process depends on the protein oligomeric state. Generalized Polarization measurements and spectral phasor analysis revealed that truncated-TRAF2 affects the membrane fluidity, especially when vesicles are heated up at physiological temperature. The addition of nanomolar concentration of TRAF2 in GUVs also seems to exert a mechanical action, as demonstrated by the formation of intraluminal vesicles, a process in which ganglioside GM1 plays a crucial role.

The aggregation of human Islet Amyloid Polypeptide (hIAPP) on cell membranes is linked to amyloid diseases. However, the physio-chemical mechanisms of how these hIAPP aggregates trigger membrane damage are unclear. Using coarse-grained and all-atom molecular dynamics simulations, we investigated the role of lipid nanodomains in the presence or absence of anionic lipids, phosphatidylserine (PS), and a ganglioside (GM1), in the membrane disruption and protein folding behaviors of hIAPP aggregates on phase-separated raft membranes. Our raft membranes contain liquid-ordered (Lo), liquid-disordered (Ld), mixed Lo/Ld (Lod), PS-cluster, and GM1-cluster nanosized domains. We observed that hIAPP aggregates bound to the Lod domain in the absence of anionic lipids, but also to the GM1-cluster- and PS-cluster-containing domains, with stronger affinity in the presence of anionic lipids. We discovered that L16 and I26 are the lipid anchoring residues of hIAPP binding to the Lod and PS-cluster domains. Finally, significant lipid acyl chain order disruption in the annular lipid shells surrounding the membrane-bound hIAPP aggregates and protein folding, particularly beta-sheet formation, in larger protein aggregates were evident. We propose that the interactions of hIAPP and both non-anionic and anionic lipid nanodomains represent key molecular events of membrane damage associated with the pathogenesis of amyloid diseases.

The viral entry consists of several sequential events that ensure the attachment of the virus to the host cell and the introduction of its genetic material for the continuation of the replication cycle. Both cellular and viral lipids have gained a wider focus in recent years in the field of viral entry, as they are found to play key roles in different steps of the process. The specific role is summarized that lipids and lipid membrane nanostructures play in viral attachment, fusion, and immune evasion and how they can be targeted with antiviral therapies. Finally, some of the limitations of techniques commonly used for protein–lipid interactions studies are discussed, and new emerging tools are reviewed that can be applied to this field. [ABSTRACT FROM AUTHOR]

α‐Synuclein is a central player in Parkinson's disease (PD) pathology. Various point mutations in α‐synuclein have been identified to alter the protein‐phospholipid binding behavior and cause PD. Therefore, exploration of α‐synuclein‐phospholipid interaction is important for understanding the PD pathogenesis and helping the early diagnosis of PD. Herein, a phospholipid‐decorated liquid crystal (LC)‐aqueous interface is constructed to investigate the binding between α‐synucleins (wild‐type and six familial mutant A30P, E46K, H50Q, G51D, A53E and A53T) and phospholipid. The application of deep learning analyzes and reveals distinct LC signatures generated by the binding of α‐synuclein and phospholipid. This system allows for the identification of single point mutant α‐synucleins with an average accuracy of 98.3±1.3% in a fast and efficient manner. We propose that this analytical methodology provides a new platform to understand α‐synuclein‐lipid interactions, and can be potentially developed for easy identification of α‐synuclein mutations in common clinic. [ABSTRACT FROM AUTHOR]

• Hybrid resolution molecular dynamics simulation method is described. • Hybrid resolution molecular dynamics simulations allow characterization of interactions of amyloid proteins with lipid membranes on long time scales. • The hybrid resolution method was applied to computer modeling of the interactions of α-synuclein protein with a mixed lipid bilayer. • Interaction of α -syn with the lipid bilayer leads to dramatic changes in the conformation of the protein and to a smaller degree its structure. A broad range of human diseases, including Alzheimer's and Parkinson's diseases, arise from or have as key players intrinsically disordered proteins. The aggregation of these amyloid proteins into fibrillar aggregates are the key events of such diseases. Characterizing the conformation dynamics of the proteins involved is crucial for understanding the molecular mechanisms of aggregation, which in turn is important for drug development efforts against these diseases. Computational approaches have provided extensive detail about some steps of the aggregation process, however the biologically relevant elements responsible for the aggregation and or aggregation propagation have not been fully characterized. Here we describe a hybrid resolution molecular dynamics simulation method that can be employed to investigate the interaction of amyloid proteins with lipid membranes, shown to dramatically accelerate the aggregation propensity of amyloid proteins. The hybrid resolution method enables routine and accurate simulation of multi-protein and complex membrane systems, mimicking biologically relevant lipid membranes, on microsecond time scales. The hybrid resolution method was applied to computer modeling of the interactions of α -synuclein protein with a mixed lipid bilayer. [ABSTRACT FROM AUTHOR]

Glycosphingolipids (GSLs) are abundant glycolipids on cells and essential for cell recognition, adhesion, signal transduction, and so on. However, their lipid anchors are not long enough to cross the membrane bilayer. To transduce transmembrane signals, GSLs must interact with other membrane components, whereas such interactions are difficult to investigate. To overcome this difficulty, bifunctional derivatives of II 3 -β-N-acetyl-D-galactosamine-GA2 (GalNAc-GA2) and β-N-acetyl-D-glucosamine-ceramide (GlcNAc-Cer) were synthesized as probes to explore GSL-interacting membrane proteins in live cells. Both probes contain photoreactive diazirine in the lipid moiety, which can crosslink with proximal membrane proteins upon photoactivation, and clickable alkyne in the glycan to facilitate affinity tag addition for crosslinked protein pull-down and characterization. The synthesis is highlighted by the efficient assembly of simple glycolipid precursors followed by on-site lipid remodeling. These probes were employed to profile GSL-interacting membrane proteins in HEK293 cells. The GalNAc-GA2 probe revealed 312 distinct proteins, with GlcNAc-Cer probe-crosslinked proteins as controls, suggesting the potential influence of the glycan on GSL functions. Many of the proteins identified with the GalNAc-GA2 probe are associated with GSLs, and some have been validated as being specific to this probe. The versatile probe design and experimental protocols are anticipated to be widely applicable to GSL research., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human Atg3 (hAtg3) is an E2-like enzyme that catalyzes the conjugation of LC3 family proteins to phosphatidylethanolamine (PE) lipids in the autophagosomal membrane during autophagy. The reaction product, LC3-PE, acts as a marker for autophagic cargo and is required for the effective construction of functional autophagosomes. However, the structural and molecular basis of this conjugation reaction remains elusive, at least in part, because of the absence of lipid bilayers in structural studies conducted to date. Here, we report a sequential resonance assignment for an hAtg3 construct both in aqueous solution and in bicelles. hAtg3 has 314 residues, and our construct lacks the unstructured region from residues 90 to 190. Our results demonstrate a structural rearrangement of hAtg3 N-terminus when it interacts with membranes. [ABSTRACT FROM AUTHOR]

Islet amyloid polypeptide (IAPP) is a 37-amino acid amyloid protein intimately associated with pancreatic islet β-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy, we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for human IAPP than for the less toxic rat IAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rat IAPP and human IAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well.

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg(995))-β3(Asp(723)) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg(995)) contacts with β3(Asp(723)) but paradoxically do not diminish the contribution of αIIb(Arg(995))-β3(Asp(723)) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.

Heterotrimeric G proteins are responsible for signal transduction from G-protein-coupled receptors (GPCRs) to intracellular effectors. This process is only possible when G proteins are located on the inner side of the cell membrane due to the specific localization of GPCR receptors. The Gα subunit is directed to the cell membrane through several signals, including modification by fatty acid moieties, interaction with the Gβγ complex, and, as observed in some Gα proteins, the presence of basic amino acid residues in the N-terminal region. In this work, we focused on investigating the influence of the polybasic region on the localization and function of a representative member of the Gαi family, Gαi 3. Through the use of confocal microscopy and fluorescence lifetime microscopy, we showed that, in the case of this protein, neutralizing the positive charge does not significantly affect its abundance in the cell membrane. However, it does affect its spatial arrangement concerning the dopamine D 2 receptor and influences inhibitory effect of Gαi 3 on intracellular cAMP production triggered by D 2 receptor stimulation. Moreover, in this work, we have shown, for the first time, that nonlipidated Gαi 3 binds to negatively charged lipids through electrostatic interactions, and membrane fluidity plays a significant role in this interaction. • Neutralizing N-terminal basic residues in Gαi 3 doesn't alter its cellular localization. • Basic amino acids in Gαi 3 N-terminus impact membrane interactions and signalling. • Cell membrane lipids determine Gαi 3 localization and function. [ABSTRACT FROM AUTHOR]

Boundary lipids surrounding membrane proteins play an essential role in protein function and structure. These protein–lipid interactions are mainly divided into electrostatic interactions between the polar amino acids of proteins and polar heads of phospholipids, and hydrophobic interactions between protein transmembrane sites and phospholipid acyl chains. Our previous report (Kawatake et al., Biochim. Biophys. Acta 1858 [2016] 2106–2115) covered a method for selectively analyzing boundary lipid interactions and showed differences in membrane protein–peripheral lipid interactions due to differences in their head group. Interactions in the hydrophobic acyl chains of phospholipids are relatively consistent among proteins, but the details of these interactions have not been elucidated. In this study, we reconstituted bacteriorhodopsin as a model protein into phospholipid membranes labeled with 2H and 13C for solid-state NMR measurement to investigate the depth-dependent effect of the head group structure on the lipid bilayer. The results showed that the position of the phospholipid near the carbonyl carbon was affected by the head group in terms of selectivity for protein surfaces, whereas in the deep interior of the bilayer near the leaflet interface, there was little difference between the head groups, indicating that the dependence of their interactions on the head group was much reduced. [Display omitted] • Depth-dependent changes in protein–lipid interactions were evaluated. • Phospholipid ester showed head group dependence in the protein interaction. • Near the bilayer center, there was little difference between the head groups. [ABSTRACT FROM AUTHOR]

Desmin, an intermediate filament protein expressed in muscle cells, plays a key role in the integrity and regulation of the contractile system. Furthermore, the distribution of desmin in cells and its interplay with plasma and organelle membranes are crucial for cell functions; however, the fundamental properties of lipid-desmin interactions remain unknown. Using a water-in-oil method for a limited space system in vitro , we examined the distribution of desmin in three types of phospholipid droplets: 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl- sn -glycero-3-phosphoserine (DOPS). When fluorescent-labeled desmin was observed for 60 min after desmin assembly was initiated by adding 25 mM KCl, desmin accumulated on both the DOPE and DOPS layers; however, it did not accumulate on the DOPC layer of droplets. An increase in salt concentration did not moderate the accumulation. The initial form of either oligomer or mature filament affected the accumulation on each lipid layer. When liposomes were included in the droplets, desmin was associated with DOPE but not on DOPC liposomes. These results suggest that desmin has the potential for association with phospholipids concerning desmin form and lipid shape. The behavior and composition of living membranes may affect the distribution of desmin networks. [Display omitted] • Desmin was associated with DOPE and DOPS layers. • Maturation of desmin filaments weakened the affinity for phospholipid layers. • Desmin could accumulate around hybrid DOPE liposomes. [ABSTRACT FROM AUTHOR]

Knowing the topology and location of protein segments at water-membrane interfaces is critical for rationalizing their functions, but their characterization is challenging under physiological conditions. Here, we debut a unique spectroscopic approach by using the hydration dynamics gradient found across the phospholipid bilayer as an intrinsic ruler for determining the topology, immersion depth, and orientation of protein segments in lipid membranes, particularly at water-membrane interfaces. This is achieved through the site-specific quantification of translational diffusion of hydration water using an emerging tool, (1)H Overhauser dynamic nuclear polarization (ODNP)-enhanced NMR relaxometry. ODNP confirms that the membrane-bound region of α-synuclein (αS), an amyloid protein known to insert an amphipathic α-helix into negatively charged phospholipid membranes, forms an extended α-helix parallel to the membrane surface. We extend the current knowledge by showing that residues 90-96 of bound αS, which is a transition segment that links the α-helix and the C terminus, adopt a larger loop than an idealized α-helix. The unstructured C terminus gradually threads through the surface hydration layers of lipid membranes, with the beginning portion residing within 5-15 Å above the phosphate level, and only the very end of C terminus surveying bulk water. Remarkably, the intrinsic hydration dynamics gradient along the bilayer normal extends to 20-30 Å above the phosphate level, as demonstrated with a peripheral membrane protein, annexin B12. ODNP offers the opportunity to reveal previously unresolvable structure and location of protein segments well above the lipid phosphate, whose structure and dynamics critically contribute to the understanding of functional versatility of membrane proteins.

Advancements in medicine and pharmacology have led to the development of systems that deliver biologically active molecules inside cells, increasing drug concentrations at target sites. This improves effectiveness and duration of action and reduces side effects on healthy tissues. Cell-penetrating peptides (CPPs) show promise in this area. While traditional medicinal chemistry methods have been used to develop CPPs, machine learning techniques can speed up and reduce costs in the search for new peptides. A predictive algorithm based on machine learning models was created to identify novel CPP sequences using molecular descriptors using a combination of algorithms like k-nearest neighbors, gradient boosting, and random forest. Some potential CPPs were found and tested for cytotoxicity and penetrating ability. A new low-toxicity CPP was discovered from the Rhopilema esculentum venom proteome through this study.

The misfolding and fibrillization of the protein, α‐synuclein (αsyn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of αsyn misfolding is an important area of interest given that αsyn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate αsyn folding, there is little data pertaining to the mechanism(s) of this interaction. αSyn has previously been shown to associate with small lipid vesicles released by cells called extracellular vesicles (EVs) and it is postulated these interactions may assist in the spreading of pathological forms of this protein. Together, this presents the need for robust characterisation studies on αsyn fibrillization using biologically‐derived vesicles. In this study, we comprehensively characterised the ability of lipid‐rich small extracellular vesicles (sEVs) to alter the misfolding of αsyn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded αsyn were examined using a range of techniques including: Thioflavin T fluorescence, transmission electron microscopy, analytical centrifugation and western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation. We show that sEVs cause an acceleration in αsyn fibrillization and provide comprehensive evidence that this results in an increase in the abundance of mature insoluble fibrillar species. In order to elucidate the relevance of the lipid membrane to this interaction, sEV lipid membranes were modified by treatment with methanol, or a combination of methanol and sarkosyl. These treatments altered the ultrastructure of the sEVs without changing the protein cargo. Critically, these modified sEVs had a reduced ability to influence αsyn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of αsyn:EV interactions and demonstrates that sEVs are powerful modulators of αsyn fibrillization, which is mediated by the sEV membrane. In doing so, this work provides strong evidence for a role of sEVs in contributing directly to αsyn misfolding in the synucleinopathy disorders. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs), or oil bodies in plants, are specialized organelles that primarily serve as hubs of cellular metabolic energy storage and consumption. These ubiquitous cytoplasmic organelles are derived from the endoplasmic reticulum (ER) and consist of a hydrophobic neutral lipid core - mainly consisting of triglycerides and sterol esters - that is encircled by a phospholipid monolayer. The dynamic metabolic functions of the LDs are mainly executed and regulated by proteins on the monolayer surface. However, its unique architecture puts some structural constraints on the types of proteins that can associate with LDs. The lipid monolayer is decorated with either peripheral proteins or with integral membrane proteins that adopt a monotopic topology. Due to its oil-water interface, which is energetically costly, the LD surface happens to be favorable to the recruitment of many proteins involved in metabolic but also non-metabolic functions. We only started very recently to understand biophysical and biochemical principles controlling protein targeting to LDs. This review aims to summarize the most recent findings regarding this topic and proposes directions that will potentially lead to a better understanding of LD surface characteristics, as compared to bilayer membranes, and how that impacts protein-LD interactions. [ABSTRACT FROM AUTHOR]

Acyl‐CoA‐binding proteins (ACBPs) are a family of proteins that bind acyl‐CoA esters at a conserved acyl‐CoA‐binding domain. ACBPs maintain intracellular acyl‐CoA pools to regulate lipid metabolism. Here, we report on the structure of rice OsACBP2 in complex with C18:3‐CoA ester. The residues Y33, K34 and K56 of OsACBP2 play a crucial role in binding the CoA group, while residues N23, L27, K52 and Y55 in one molecule of OsACBP2 cooperate with L27, L28, A59 and A62 from another anchoring the fatty acyl group. Multiangle light scattering assays indicate that OsACBP2 binds C18:3‐CoA as a monomer. The first complex structure of a plant ACBP binding with C18:3‐CoA is therefore presented, providing a novel model for the interaction between an acyl‐CoA ester and the acyl‐CoA‐binding domain(s). [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is one of the major causes of viral encephalitis all around the globe. Approximately 3 billion people in endemic areas are at risk of Japanese encephalitis. To develop a wholistic understanding of the viral proteome, it is important to investigate both its ordered and disordered proteins. However, the functional and structural significance of disordered regions in the JEV proteome has not been systematically investigated as of yet. To fill this gap, we used here a set of bioinformatics tools to analyze the JEV proteome for the predisposition of its proteins for intrinsic disorder and for the presence of the disorder‐based binding regions (also known as molecular recognition features, MoRFs). We also analyzed all JEV proteins for the presence of the probable nucleic acid‐binding (DNA and RNA) sites. The results of these computational studies are experimentally validated using JEV capsid protein as an illustrative example. In agreement with bioinformatic analysis, we found that the N‐terminal region of the JEV capsid (residues 1–30) is intrinsically disordered. We showed that this region is characterized by the temperature response typical for highly disordered proteins. Furthermore, we have experimentally shown that this disordered N‐terminal domain of a capsid protein has a noticeable 'gain‐of‐structure' potential. In addition, using DOPS liposomes, we demonstrated the presence of pronounced membrane‐mediated conformational changes in the N‐terminal region of JEV capsid. In our view, this disorder‐centric analysis would be helpful for a better understanding of the JEV pathogenesis. [ABSTRACT FROM AUTHOR]

Dysregulation of lipid homeostasis leads to the development of metabolic disorders including obesity, diabetes, cardiovascular disease and cancer. Lipid droplets (LDs) are subcellular organelles vital in the maintenance of lipid homeostasis by coordinating lipid synthesis, lipid storage, lipid secretion and lipolysis. Under fed condition, free fatty acids (FFAs) are remodeled and esterified into neutral lipids by lipogenesis and stored in the LDs. The lipid storage capacity of LDs is controlled by its growth via local lipid synthesis or by LD fusion. During fasting, neutral lipids are hydrolyzed by lipolysis, released as FFAs and secreted to meet energy demand. Cell death‐inducing DNA fragmentation factor alpha (DFFA)‐like effector (CIDE) family proteins composed of Cidea, Cideb and Cidec/Fsp27 are ER‐ and LD‐associated proteins and have emerged as important regulators of lipid homeostasis. Notably, when localized on the LDs, CIDE proteins enrich at the LD‐LD contact sites (LDCSs) and control LD fusion and growth. Here, we summarize these recent advances made on the role of CIDE proteins in the regulation of lipid metabolism with a particular focus on the molecular mechanisms underlying CIDE‐mediated LD fusion and growth. [ABSTRACT FROM AUTHOR]

There are several examples of single mutations that lead to a well‐defined disease through a well‐known mechanism. In other cases, a collection of mutations of the same protein produces a pathology with different degrees of severity. The accompanying work by Uusitalo et al. studies several mutants of the fatty acid binding protein P2 of the peripheral nervous system myelin. They conserve the native tertiary structure but a remarkable difference in the capacity to interact with lipids. This could be a clue to unravel the complex way in which these mutations affect myelin structure and function in a variant of Charcot–Marie–Tooth disease. Comment on: https://doi.org/10.1111/febs.16079 [ABSTRACT FROM AUTHOR]

The protolytic and partition equilibria of the indicator dyes in the model lipid and protein-lipid systems have been analyzed. A methodological approach has been developed allowing the partition coefficients of the protonated and deprotonated dye forms to be derived from the spectrophotometric measurements. The partitioning of the indicator dye bromothymol blue into the model bilayer membranes composed of phosphatidylcholine and cardiolipin (9:1, mol:mol) has been examined. The partition coefficient of the protonated dye species into a lipid phase has been found to be 5 orders of magnitude higher than that of the deprotonated dye form. This effect has been interpreted in terms of the differences in the charge distribution over the protonated and deprotonated dye ions, preventing the hydrophobic dye-lipid interactions in the latter case. The reduction of the bromothymol blue partitioning into lipid bilayer in the presence of hemoglobin has been attributed to the protein-induced changes in the structure and physicochemical characteristics of the interfacial membrane region. In the practical aspect, the obtained findings may prove of significance in the design of hemosome-based blood substitutes and elucidating the role of hemoglobin in the molecular etiology of the amyloid disorders, particularly, Alzheimer's disease.

Plasma apoC-III levels correlate with triglyceride (TG) levels and are a strong predictor of CVD outcomes. ApoC-III elevates TG in part by inhibiting LPL. ApoC-III likely inhibits LPL by competing for lipid binding. To probe this, we used oil-drop tensiometry to characterize binding of six apoC-III variants to lipid/water interfaces. This technique monitors the dependence of lipid binding on surface pressure, which increases during TG hydrolysis by LPL. ApoC-III adsorption increased surface pressure by upward of 18 mN/m at phospholipid/TG/water interfaces. ApoC-III was retained to high pressures at these interfaces, desorbing at 21–25 mN/m. Point mutants, which substituted alanine for aromatic residues, impaired the lipid binding of apoC-III. Adsorption and retention pressures decreased by 1–6 mN/m in point mutants, with the magnitude determined by the location of alanine substitutions. Trp42 was most critical to mediating lipid binding. These results strongly correlate with our previous results, linking apoC-III point mutants to increased LPL binding and activity at lipid surfaces. We propose that aromatic residues in the C-terminal half of apoC-III mediate binding to TG-rich lipoproteins. Increased apoC-III expression in the hypertriglyceridemic state allows apoC-III to accumulate on lipoproteins and inhibit LPL by preventing binding and/or access to substrate.

Phosphoinositides are amphipathic lipid molecules derived from phosphatidylinositol that represent low abundance components of biological membranes. Rather than serving as mere structural elements of lipid bilayers, they represent molecular switches for a broad range of biological processes, including cell signaling, membrane dynamics and remodeling, and many other functions. Here, we focus on the molecular mechanisms that turn phosphoinositides into molecular switches and how the dysregulation of these processes can lead to disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 )-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P 2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling., Competing Interests: FL, JS, RS, DB, JF, SU, SK, PR, SW, HM, XS, SK, CF, MH, RŠ, PC, IV, WN No competing interests declared, (© 2023, Lolicato, Steringer et al.)

In vivo site-directed photocrosslinking provides a means to probe the vicinity of proteins in their native cellular environment. Because this method relies on the incorporation of unnatural amino acid analogs that are similar in size to natural amino acids, crosslink products are indicative of direct protein-protein interactions. Here, we present the use of this approach to monitor both transient and stable interactions of two proteins of the envelope of Escherichia coli. First, we describe a protocol to characterize the interactions of a secretory protein as it transverses the bacterial envelope with temporal and spatial resolution. We combine site-directed photocrosslinking with radiolabeling of proteins and lipids. Second, we describe a method to purify a photocrosslinked partner protein and to analyze it by mass spectrometry. We use in-gel protein digestion and peptide fragmentation by MALDI-TOF/TOF tandem mass spectrometry to determine the site of interaction on the photocrosslinked partner., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.