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Background Daptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum. DAP resistance (DAP-R) has been associated with activation of the LiaFSR system resulting in redistribution of APL microdomains (likely containing cardiolipin, CL) away from the septum. E. faecalis (Efs) possess two CL synthase genes, (cls1 and cls2) and changes in Cls1 are associated with DAP-R. However, the roles of each enzyme are unknown. Here, we characterize the roles of cls genes in DAP-R in the context of LiaFSR activation. Methods cls1 and/or cls2 were deleted from Efs OG117 and OG117ΔliaX (DAP-R strain with an activated LiaFSR response). qRT-PCR was used to study gene expression of cls1 and cls2 in the cls mutants. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry. Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange. Results cls1 and cls2 are upregulated in exponential phase of DAP-R Efs OG117ΔliaX relative to DAP-S Efs OG117, with only cls1 upregulated in stationary phase. Deletion of cls1 or cls2 resulted in upregulation of the other cls gene, independent of activation of LiaFSR. Lipidomics analysis confirmed that deletion of both cls resulted in complete absence of cell membrane CL content. When comparing CL profiles of Δcls1 relative to Δcls2 in both DAP-S and DAP-R, both strains produced similar levels and species of CL. However, development of DAP-R caused a change in membrane lipid content, namely, an increase in CL with no significant difference in phosphatidylglycerol compared to DAP-S strain. Evaluation of CL species in DAP-R shows a shift towards species containing longer fatty acid chains and higher saturation. Independent deletions of cls1 or cls2 did not revert the DAP phenotype. In contrast, deletion of both cls genes decreased the DAP MIC (2-3 fold) relative to the parent strain and restored septal localization of APL microdomains. DAP MIC was restored upon trans complementation of either cls1 or cls2 into the double deletion mutant. Conclusion Our results support a major role of Cls in changes in cell membrane architecture and DAP-R in enterococci, with overlapping roles for Cls1 and Cls2. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support.

Rationale The nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) and its primary inhibitor, tuberin (TSC2), are cues for the development of cardiac hypertrophy. The phenotype of mTORC1 induced hypertrophy is unknown. Objective To examine the impact of sustained mTORC1 activation on metabolism, function, and structure of the adult heart. Methods and results We developed a mouse model of inducible, cardiac-specific sustained mTORC1 activation (mTORC1iSA) through deletion of Tsc2. Prior to hypertrophy, rates of glucose uptake and oxidation, as well as protein and enzymatic activity of glucose 6-phosphate isomerase (GPI) were decreased, while intracellular levels of glucose 6-phosphate (G6P) were increased. Subsequently, hypertrophy developed. Transcript levels of the fetal gene program and pathways of exercise-induced hypertrophy increased, while hypertrophy did not progress to heart failure. We therefore examined the hearts of wild-type mice subjected to voluntary physical activity and observed early changes in GPI, followed by hypertrophy. Rapamycin prevented these changes in both models. Conclusion Activation of mTORC1 in the adult heart triggers the development of a non-specific form of hypertrophy which is preceded by changes in cardiac glucose metabolism.

The lipid environment in which membrane proteins are embedded can influence their structure and function. Lipid−protein interactions and lipid-induced conformational changes necessary for protein function remain intractable in vivo using high-resolution techniques. Using Escherichia coli strains in which the normal phospholipid composition can be altered or foreign lipids can be introduced, we established the importance of membrane lipid composition for the proper folding, assembly, and function of E. coli lactose (LacY) and sucrose (CscB) permeases. However, the molecular mechanism underlying the lipid dependence for active transport remains unknown. Herein, we demonstrate that the structure and function of CscB and LacY can be modulated by the composition of the lipid environment. Using a combination of assays (transport activity of the substrate, protein topology, folding, and assembly into the membrane), we found that alterations in the membrane lipid composition lead to lipid-dependent structural changes in CscB and LacY. These changes affect the orientation of residues involved in LacY proton translocation and impact the rates of protonation and deprotonation of E325 by affecting the arrangement of transmembrane domains in the vicinity of the R302-E325 charge pair. Furthermore, the structural changes caused by changes in membrane lipid composition can be altered by a single-point mutation, highlighting the adaptability of these transporters to their environment. Altogether, our results demonstrate that direct interactions between a protein and its lipid environment uniquely contribute to membrane protein organization and function. Because members of the major facilitator superfamily present with well-conserved functional architecture, we anticipate that our findings can be extrapolated to other membrane protein transporters.

Posttranslational modifications of proteins, such as phosphorylation and dephosphorylation, play critical roles in cellular functions through diverse cell signaling pathways. Protein kinases and phosphatases have been described early on as key regulatory elements of the phosphorylated state of proteins. Tight spatial and temporal regulation of protein kinase and phosphatase activities has to be achieved in the cell to ensure accurate signal transduction. We demonstrated previously that phosphorylation of a membrane protein can lead to its topological rearrangement. Additionally, we found that both the rate and extent of topological rearrangement upon phosphorylation are lipid charge– and lipid environment–dependent. Here, using a model membrane protein (the bacterial lactose permease LacY reconstituted in proteoliposomes) and a combination of real-time measurements and steady-state assessments of protein topology, we established a set of experimental conditions to dissect the effects of phosphorylation and dephosphorylation of a membrane protein on its topological orientation. We also demonstrate that the phosphorylation-induced topological switch of a membrane protein can be reversed upon protein dephosphorylation, revealing a new regulatory role for phosphorylation/dephosphorylation cycles. Furthermore, we determined that the rate of topological rearrangement reversal is correlated with phosphatase activity and is influenced by the membrane's lipid composition, presenting new insights into the spatiotemporal control of the protein phosphorylation state. Together, our results highlight the importance of the compartmentalization of phosphorylation/dephosphorylation cycles in controlling membrane protein topology and, therefore, function, which are influenced by the local lipid environment of the membrane protein.

About 20-30% of cancer-associated deaths are due to complications from cachexia which is characterized by skeletal muscle atrophy. Metabolic reprogramming in cancer cells causes body-wide metabolic and proteomic remodeling, which remain poorly understood. Here, we present evidence that the oncometabolite D-2-hydroxylgutarate (D2-HG) impairs NAD+ redox homeostasis in skeletal myotubes, causing atrophy via deacetylation of LC3-II by the nuclear deacetylase Sirt1. Overexpression of p300 or silencing of Sirt1 abrogate its interaction with LC3, and subsequently reduced levels of LC3 lipidation. Using RNA-sequencing and mass spectrometry-based metabolomics and proteomics, we demonstrate that prolonged treatment with the oncometabolite D2-HG in mice promotes cachexia in vivo and increases the abundance of proteins and metabolites, which are involved in energy substrate metabolism, chromatin acetylation and autophagy regulation. We further show that D2-HG promotes a sex-dependent adaptation in skeletal muscle using network modeling and machine learning algorithms. Our multi-omics approach exposes new metabolic vulnerabilities in response to D2-HG in skeletal muscle and provides a conceptual framework for identifying therapeutic targets in cachexia.

Cancer cells rewire metabolism to support tumor growth and proliferation. In isocitrate dehydrogenase (IDH) 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. D2-HG causes inhibition of alpha-ketoglutarate dehydrogenase (AKGDH), which is associated with reduced cardiac contractile function. How tumor cells influence the metabolism of cardiomyocytes remains mostly unknown. Specific cancer cells use glutamine-dependent reductive carboxylation to circumvent defective mitochondrial metabolism by producing citrate and acetyl-CoA for lipid synthesis, which tumors require for growth. Here, we explore the hypothesis that inhibition of AKGDH by the oncometabolite D2-HG increases glutamine-dependent reductive carboxylation in the heart. We combined ex vivo rat heart perfusions with mass-spectrometry-based stable isotope tracer studies and in silico metabolic flux analysis. In response to D2-HG-mediated inhibition of AKGDH, we observed an increased reductive carboxylation of alpha-ketoglutarate to citrate rather than oxidative decarboxylation. This pathway increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. When we perfused rat hearts with 13C-labelled D2-HG, we observed a similarly increased formation of citrate. To identify which IDH isoform is responsible for redirecting carbon flux, we modulated IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes using siRNAs. Reduced expression of IDH1 impaired reductive formation of citrate. Importantly, we observed a significant correlation between reductive citrate formation and epigenetic modifications of histones, including increased histone 3 lysine 9 acetylation and di-methylation. To explore these observations, we conducted ChIP-sequencing and identified distinct transcriptional remodeling. Taken together, we demonstrate how oncometabolic stress in the heart causes redirection of central carbon metabolism via reductive carboxylation, and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.

Translocation of preproteins across the Escherichia coli inner membrane requires anionic lipids by virtue of their negative head-group charge either in vivo or in situ. However, available results do not differentiate between the roles of monoanionic phosphatidylglycerol and dianionic cardiolipin (CL) in this essential membrane-related process. To define in vivo the molecular steps affected by the absence of CL in protein translocation and insertion, we analyzed translocon activity, SecYEG stability and its interaction with SecA in an E. coli mutant devoid of CL. Although no growth defects were observed, co- and post-translational translocation of α-helical proteins across inner membrane and the assembly of outer membrane β-barrel precursors were severely compromised in CL-lacking cells. Components of proton-motive force which could impair protein insertion into and translocation across the inner membrane, were unaffected. However, stability of the dimeric SecYEG complex and oligomerization properties of SecA were strongly compromised while the levels of individual SecYEG translocon components, SecA and insertase YidC were largely unaffected. These results demonstrate that CL is required in vivo for the stability of the bacterial translocon and its efficient function in co-translational insertion into and translocation across the inner membrane of E. coli.

Background: Effective treatment options for triple-negative inflammatory breast cancer (TN-IBC), the most aggressive form of breast cancer, are currently lacking. We previously reported that mediators of inflammation promote the growth of TN-IBC xenografts. Eicosapentaenoic acid (EPA), an omega-3 fatty acid (fish oil) with anti-inflammatory properties, is an emerging FDA-approved therapeutic with a favorable toxicology profile. Here we aimed to develop a novel approach to enhance EPA efficacy against TN-IBC by identifying a kinase inhibitor that synergizes with EPA's antitumor activity. Methods and Results: Using a high-throughput siRNA screen in the TN-IBC cell line SUM149PT, we identified inhibition of ephrin type-A receptor 2 (EPHA2), an oncogenic receptor tyrosine kinase, as a target that sensitizes TN-IBC cells to EPA therapy. To determine the clinical relevance of EPHA2, we investigated a meta-analysis of breast cancer mRNA expression data sets and found that high EPHA2 tumor expression, compared with low expressing, correlated significantly with poor overall survival in TN-IBC patients (P = 0.01), while not with other subtypes. Similar findings were observed in vitro, were EPHA2 protein and mRNA overexpression occurred predominantly in the TN subtypes among 49 and 51 breast cancer cell lines (63% and 47%, respectively), highlighting EPHA2 translational potential. Functional expression studies using proliferation and apoptosis assays in vitro, and xenografts in vivo, were performed in two EPHA2-expressing TN-IBC cell lines, SUM149PT and BCX010, to validate EPHA2 as a synergistic combinational target with EPA. EPHA2 gene silencing in combination with EPA significantly reduced cell growth, and enhanced apoptosis, compared with untreated and monotherapy in vitro (P < 0.05), and in vivo (P < 0.001). To translate our findings to the clinic, we validated dasatinib, an FDA-approved small molecule inhibitor of EPHA2, in combination to EPA to significantly enhance apoptosis of TN-IBC cells in vitro (P < 0.05) and in vivo (P < 0.05), compared with untreated and monotherapies. Using membrane fluidity assessment and cholesterol quantification we determined that apoptosis induction after combination therapy was due to increased membrane rigidity and cholesterol concentrations in the plasma membrane of TN-IBC cells (P < 0.05, compared with monotherapies). Finally, we discovered by western blot and gain/loss-of-expression studies that combination therapy inhibited the cholesterol efflux protein ATP-binding cassette sub-family A member 1 (ABCA1), which plays a significant role mediating increased cellular cholesterol (P < 0.05), cell membrane rigidity (P < 0.05), and induction of apoptosis (P < 0.05) in TN-IBC after EPA and EPHA2-targeting combination therapy. Conclusions: This is the first study demonstrating that EPA can enhance conventional targeted therapy against breast cancer. Our study provides molecular and preclinical evidence to support the development of an EPA/EPHA2-inhibition–based phase I clinical trial for patients with EPHA2-positive TN-IBC; our study further suggests the use of EPHA2 and ABCA1 protein expression as biomarkers for patient selection and therapeutic response. Citation Format: Torres-Adorno AM, Vitrac H, Qi Y, Tan L, Levental KR, Fan Y-Y, Yang P, Chapkin RS, Eckhardt BL, Ueno NT. EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-09.

Membrane proteins play key roles in cellular functions, their activity mainly depending on their topological arrangement in membranes. Structural studies of membrane proteins have long adopted a protein-centric view regarding the determinants of membrane protein topology and function. Several studies have shown that the orientation of transmembrane domains of polytopic membrane proteins with respect to the plane of the lipid bilayer can be largely determined by membrane lipid composition. However, the mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is still unknown. Here we show that lipid-dependent structural and functional assessment of a membrane protein can be conducted in detergent micelles, opening the possibility for the determination of lipid-dependent high-resolution crystal structures. We found that the lactose permease purified from Escherichia coli cells exhibiting varied phospholipid compositions exhibits the same topology and similar function as in its membrane of origin. Furthermore, we found several conditions, including protein mutations and micelle lipid composition, that lead to increased protein stability, correlating with a higher yield of two-dimensional crystal formation. Altogether, our results demonstrate how the membrane lipid environment influences membrane protein topology and arrangement, both in native membranes and in mixed detergent micelles.

Metabolic rewiring is a hallmark of cancer and muscle cells. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. Our recent in vivo work showed that increased D2-HG supply by IDH-mutant cells causes heart and skeletal muscle atrophy, and decreases cellular ATP and NADH. Although heart failure and cachexia in cancer are commonly associated with chemotherapy, cancer survivors have a 5-fold increased risk of heart failure independent of any cytostatic treatment. The connection between metabolic and proteomic remodeling in this context remain poorly understood. We hypothesize that D2-HG-mediated alpha-ketoglutarate dehydrogenase (AKGDH) inhibition in myocytes results in metabolomic perturbations, increases autophagy and proteomic remodeling. Here, we report that LC3, a key regulator of autophagy, is activated in the nucleus of myocytes in presence of D2-HG through deacetylation by the nuclear deacetylase Sirt1. Activation of Sirt1 is driven by increased NAD + levels through D2-HG-mediated AKGDH inhibition. We used LC3 mutants with arginine and glutamine replacements at lysine residues to show that deacetylation of LC3 at K49 and K51 by Sirt1 shifts LC3 distribution from the nucleus into the cytosol, where it is able to undergo lipidation at pre-autophagic membranes. Live cell imaging with GFP-tagged LC3 in L6 myocytes indicated that the cycle of acetylation-deacetylation allows LC3 to redistribute from the nucleus to the cytosol within less than 24 h. Co-immunoprecipitation of LC3 followed by proteomics analysis revealed that LC3 binds to dynein in presence of D2-HG. Furthermore, D2-HG promoted skeletal muscle atrophy and reduced grip strength in wild-type C57BL/J6 mice in vivo. Using LC-MS/MS-based proteomics and metabolomics combined with RNA-sequencing, we assessed the effect of D2-HG on a systems level in skeletal muscle. Pathway-enrichment analysis revealed that D2-HG induces upregulation of key metabolic enzymes involved in glycolysis and the pentose phosphate pathway. In short, autophagy activation supports proteome remodeling in muscle cells during IDH-mutant leukemia.

Due to the heterogenous lipid environment in which integral membrane proteins are embedded, they should follow a set of assembly rules, which govern transmembrane protein folding and topogenesis accordingly to a given lipid profile. Recombinant strains of bacteria have been engineered to have different membrane phospholipid compositions by molecular genetic manipulation of endogenous and foreign genes encoding lipid biosynthetic enzymes. Such strains provide a means to investigate the in vivo role of lipids in many different aspects of membrane function, folding and biogenesis. In vitro and in vivo studies established a function of lipids as molecular chaperones and topological determinants specifically assisting folding and topogenesis of membrane proteins. These results led to the extension of the Positive Inside Rule to Charge Balance Rule, which incorporates a role for lipid-protein interactions in determining membrane protein topological organization at the time of initial membrane insertion and dynamically after initial assembly. Membrane protein topogenesis appears to be a thermodynamically driven process in which lipid-protein interactions affect the potency of charged amino acid residues as topological signals. Dual topology for a membrane protein can be established during initial assembly where folding intermediates in multiple topological conformations are in rapid equilibrium (thus separated by a low activation energy), which is determined by the lipid environment. Post-assembly changes in lipid composition or post-translational modifications can trigger a reorganization of protein topology by inducing destabilization and refolding of a membrane protein. The lipid-dependent dynamic nature of membrane protein organization provides a novel means of regulating protein function.

Background Daptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum, and resistance (DAP-R) has been linked to mutations in genes encoding i) the LiaFSR stress response system or its effector LiaX, and ii) cardiolipin synthase (Cls). Activation of the E. faecalis (Efs) LiaFSR response is associated with DAP-R and redistribution of APL microdomains away from the septum, and cardiolipin is predicted to be a major component of these APL microdomains. Efs harbors two putative cls genes, cls1 and cls2. While changes in Cls1 have been implicated in DAP-R, the exact roles of each enzyme in resistance are unknown. We aim to characterize the contributions of Cls1 and Cls2 in the development of DAP-R. Methods cls1 and cls2 were deleted individually and in tandem from DAP-S Efs OG117 and DAP-R Efs OG117∆liaX (a DAP-R derivative strain with an activated LiaFSR response). Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test on Mueller-Hinton II agar and localization of APL microdomains with 10-N-nonyl-acridine orange staining. Quantitative PCR (qRT-PCR) was used to study gene expression profiles of cls1 and cls2 in Efs OG117∆liaX relative to Efs OG117 across the cell growth cycle. Results qRT-PCR revealed differential expression profiles of cls1 and cls2 associated with DAP-R. cls1 was highly upregulated in stationary phase concurrent with a decrease in cls2 expression. However, independent deletion of cls1 or cls2 in the DAP-R background resulted in no significant changes in DAP MICs or localization of APL microdomains (remaining non-septal). Further studies revealed that cls2 expression is upregulated upon deletion of cls1 in both the DAP-S and DAP-R background, suggesting a potential compensatory role for Cls2. Double deletion of both cls genes in the DAP-R strain decreased DAP MIC and restored the septal localization of APL microdomains. Conclusion Cls1 is the major and predominant enzyme involved in cell membrane adaptation associated with the development of DAP-R in E. faecalis. However, we describe a novel compensatory and overlapping role for cardiolipin synthases to ensure bacterial survival upon attack from antimicrobial peptides and related antibiotics. Disclosures Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

Autophagy “scavenges” proteins and yields amino acids under conditions of metabolic stress to support cell survival and growth. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including dilated cardiomyopathy. Our recent in vivo work showed that increased D2-HG supply by IDH2-mutant hematopoetic stem cells causes heart and skeletal muscle atrophy, and decreases cellular ATP and NADH. While heart failure in cancer is commonly associated with chemotherapy, cancer survivors have a five-fold increased risk of heart failure independent of any cytostatic treatment. The connection between metabolic changes and proteomic remodeling in this context remain poorly understood. We hypothesize that D2-HG-mediated alpha-ketoglutarate dehydrogenase inhibition in myocytes results in metabolomic pertubations, proteomic remodeling, and increased autophagy. We measured autophagic flux and remodeling of the stable proteome upon D2-HG treatment in vivo using wild-type C57BL/J6 mice, and in vitro using both cultured L6 myocytes and adult mouse ventricular cardiomyocytes. We observed increases in the LC3-II/LC3-I ratio and p62 expression in heart and skeletal muscle from mice treated with D2-HG, indicating activation of autophagy. Live cell imaging with GFP-tagged LC3 indicated that D2-HG (1 mM) increased LC3-II lipidation and flux within 24 h. Furthermore, we observed increased phosphorylation and activation of AMPK, while phosphorylation of mTOR and p70S6K were decreased in presence of D2-HG. In vitro exposure to D2-HG resulted in the formation of a molecular complex between Sirt1 and LC3, indicating that increased NAD+ in presence of D2-HG promotes Sirt1 activation in myocytes. Finally, we used LC-MS/MS to assess the effect of D2-HG on the stable proteome and metabolome in heart and skeletal muscle. Myocytes exposed to D2-HG showed proteomic remodeling and metabolomic changes within 24 h. Integrating multi-omics data in a network-level context revealed upregulation of glycolysis and the pentose phosphate pathway. In short, autophagy activation may support proteome remodeling in muscle cells during IDH-mutant leukemia.

Triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, currently lacks effective targeted therapy options. Eicosapentaenoic acid (EPA), an omega-3 fatty acid and constituent of fish oil, is a common supplement with anti-inflammatory properties. Although it is not a mainstream treatment, several preclinical studies have demonstrated that EPA exerts anti-tumor activity in breast cancer. However, against solid tumors, EPA as a monotherapy is clinically ineffective; thus, we sought to develop a novel targeted drug combination to bolster its therapeutic action against TNBC. Using a high-throughput functional siRNA screen, we identified Ephrin type-A receptor 2 (EPHA2), an oncogenic cell-surface receptor tyrosine kinase, as a therapeutic target that sensitizes TNBC cells to EPA. EPHA2 expression was uniquely elevated in TNBC cell lines and patient tumors. In independent functional expression studies in TNBC models, EPHA2 gene-silencing combined with EPA significantly reduced cell growth and enhanced apoptosis compared with monotherapies, both in vitro and in vivo. EPHA2 specific inhibitors similarly enhanced the therapeutic action of EPA. Finally, we identified that therapy-mediated apoptosis was attributed to a lethal increase in cancer cell membrane polarity due to ABCA1 inhibition and subsequent dysregulation of cholesterol homeostasis. This study provides new molecular and pre-clinical evidence to support a clinical evaluation of EPA combined with EPHA2 inhibition in patients with TNBC.

A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids.

Bacteria have evolved multiple strategies to sense and rapidly adapt to challenging and ever-changing environmental conditions. The ability to alter membrane lipid composition, a key component of the cellular envelope, is crucial for bacterial survival and adaptation in response to environmental stress. However, the precise roles played by membrane phospholipids in bacterial physiology and stress adaptation are not fully elucidated. The goal of this study was to define the role of membrane phospholipids in adaptation to stress and maintenance of bacterial cell fitness. By using genetically modified strains in which the membrane phospholipid composition can be systematically manipulated, we show that alterations in major Escherichia coli phospholipids transform these cells globally. We found that alterations in phospholipids impair the cellular envelope structure and function, the ability to form biofilms, and bacterial fitness and cause phospholipid-dependent susceptibility to environmental stresses. This study provides an unprecedented view of the structural, signaling, and metabolic pathways in which bacterial phospholipids participate, allowing the design of new approaches in the investigation of lipid-dependent processes involved in bacterial physiology and adaptation. IMPORTANCE In order to cope with and adapt to a wide range of environmental conditions, bacteria have to sense and quickly respond to fluctuating conditions. In this study, we investigated the effects of systematic and controlled alterations in bacterial phospholipids on cell shape, physiology, and stress adaptation. We provide new evidence that alterations of specific phospholipids in Escherichia coli have detrimental effects on cellular shape, envelope integrity, and cell physiology that impair biofilm formation, cellular envelope remodeling, and adaptability to environmental stresses. These findings hold promise for future antibacterial therapies that target bacterial lipid biosynthesis.

Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have co-evolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid–protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Background Daptomycin (DAP) is a lipopeptide antibiotic that targets the cell membrane (CM) at the division septum. DAP resistance (DAP-R) in E. faecalis (Efs) has been linked to mutations in genes encoding the LiaFSR stress response system and lipid biosynthetic enzymes, including cardiolipin synthase (Cls). The signature phenotype of DAP-R is redistribution of CM anionic phospholipid (APL) microdomains. Using a genetic approach, we have identified a transmembrane protein (LiaY) as a major mediator of cell membrane APL redistribution associated with DAP-R. Here, we explore the mechanism of LiaY-mediated changes in the CM under the hypothesis that CM remodeling occurs through interactions with Cls. Methods Efs encodes two cls genes (cls1 and cls2). Deletion mutants of both cls genes were generated using the Crispr/cas9 system in the daptomycin-sensitive strain Efs OG117 and Efs OG117∆liaX (a DAP-R derivative of OG117). DAP minimum inhibitory concentration (MIC) was determined using E-test on Mueller–Hinton II agar. Visualization of APL microdomains was performed by staining mid-logarithmic phase cells with 1 µM of 10-N-nonyl-acridine orange (NAO) and fluorescence microscopy. Bacterial two-hybrid system was used to study interactions between LiaY with Cls1 or Cls2. Results Single or double deletion of cls1 or cls2 in Efs OG117 did not affect DAP MIC, and no changes in CM architecture were seen by NAO staining. In contrast,deletion of cls1 (alone or in conjunction with a deletion of cls2) in a DAP-R derivative of OG117 OG117∆liaX, resulted in a marked decrease in DAP MIC, and NAO staining of Efs OG117∆liaX∆cls1∆cls2 shows a restoration of septal APL microdomain localization.In the same DAP-R background, deletion of cls2 alone did not have any effect on DAP MIC or APL microdomain distribution. Additionally, bacterial two-hybrid assays showed a positive interaction of LiaY with Cls1 but not with Cls2. Conclusion We have identified the biochemical basis for DAP-R associated CM remodeling. In a proposed model, the LiaR-mediated activation of the LiaY triggers specific interactions with Cls1 displacing the protein away from the septum, resulting in local generation of APL microdomains that prevents DAP-mediated damage to the CM. Disclosures All authors: No reported disclosures.

Lipids of the Escherichia coli membrane are mainly composed of 70–80% phosphatidylethanolamine (PE) and 20–25% phosphatidylglycerol (PG). Biochemical studies indicate that the depletion of PE causes inversion of the N-terminal helix bundle of the lactose permease (LacY), and helix VII becomes extramembraneous. Here we study this phenomenon using single-molecule force spectroscopy, which is sensitive to the structure of membrane proteins. In PE and PG at a ratio of 3:1, ~95% of the LacY molecules adopt a native structure. However, when PE is omitted and the membrane contains PG only, LacY almost equally populates a native and a perturbed conformation. The most drastic changes occur at helices VI and VII and the intervening loop. Since helix VII contains Asp237 and Asp240, zwitterionic PE may suppress electrostatic repulsion between LacY and PG in the PE:PG environment. Thus, PE promotes a native fold and prevents LacY from populating a functionally defective, non-native conformation.

The final topology of membrane proteins is thought to be dictated primarily by the encoding sequence. However, according to the Charge Balance Rule the topogenic signals within nascent membrane proteins are interpreted in agreement with the Positive Inside Rule as influenced by the protein phospholipid environment. The role of long-range protein-lipid interactions in establishing a final uniform or dual topology is unknown. In order to address this role, we determined the positional dependence of the potency of charged residues as topological signals within Escherichia coli sucrose permease (CscB) in cells in which the zwitterionic phospholipid phosphatidylethanolamine (PE), acting as topological determinant, was either eliminated or tightly titrated. Although the position of a single or paired oppositely charged amino acid residues within an extramembrane domain (EMD), either proximal, central or distal to a transmembrane domain (TMD) end, does not appear to be important, the oppositely charged residues exert their topogenic effects separately only in the absence of PE. Thus, the Charge Balance Rule can be executed in a retrograde manner from any cytoplasmic EMD or any residue within an EMD most likely outside of the translocon. Moreover, CscB is inserted into the membrane in two opposite orientations at different ratios with the native orientation proportional to the mol % of PE. The results demonstrate how the cooperative contribution of lipid-protein interactions affects the potency of charged residues as topological signals, providing a molecular mechanism for the realization of single, equal or different amounts of oppositely oriented protein within the same membrane.

Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.

Hematologic malignancies are frequently associated with cardiac pathologies. Mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a subset of acute myeloid leukemia patients, causing metabolic and epigenetic derangements. We have now discovered that altered metabolism in leukemic cells has a profound effect on cardiac metabolism. Combining mathematical modeling and in vivo as well as ex vivo studies, we found that increased amounts of the oncometabolite d-2-hydroxyglutarate (D2-HG), produced by IDH2 mutant leukemic cells, cause contractile dysfunction in the heart. This contractile dysfunction is associated with impaired oxidative decarboxylation of α-ketoglutarate, a redirection of Krebs cycle intermediates, and increased ATP citrate lyase (ACL) activity. Increased availability of D2-HG also leads to altered histone methylation and acetylation in the heart. We propose that D2-HG promotes cardiac dysfunction by impairing α-ketoglutarate dehydrogenase and induces histone modifications in an ACL-dependent manner. Collectively, our results highlight the impact of cancer cell metabolism on function and metabolism of the heart.

Submit Manuscript | http://medcraveonline.com J Bacteriol Mycol Open Access 2015, 1(1): 00001 The development of antimicrobial resistance is inevitable and inherent to the remarkable mechanisms of bacterial adaptation to adverse environmental conditions in order to survive. However, many human actions have contributed to the currently observed acceleration and spread of antimicrobial resistance (poor hygiene, over prescription of antibiotics, intensive use of antibiotics in farming, etc.) and battling the antimicrobial resistance threat will require scientific, societal, and political actions to be taken. In addition to changes in human and veterinarian health care practices and new policy development for better risk assessment, there is still a crucial need for the development of new antimicrobial therapies. The number of FDA-approved antibiotics has been constantly decreasing since the 1990s and biopharmaceuticals companies are less willing to invest in the drug pipeline [4], especially for a product that bacterial resistance will eventually render obsolete. According to a Pew Charitable Trusts study, only 38 new antibiotics were in development by pharmaceutical companies in 2014 [5]. Despite the gap in the drug development pipeline, there are very promising approaches currently being applied that have led to encouraging findings in the recent years.

The N-terminal six-transmembrane domain (TM) bundle of lactose permease of Escherichia coli is uniformly inverted when assembled in membranes lacking phosphatidylethanolamine (PE). Inversion is dependent on the net charge of cytoplasmically exposed protein domains containing positive and negative residues, net charge of the membrane surface, and low hydrophobicity of TM VII acting as a molecular hinge between the two halves of lactose permease (Bogdanov, M., Xie, J., Heacock, P., and Dowhan, W. (2008) J. Cell Biol. 182, 925–935). Net neutral lipids suppress the membrane translocation potential of negatively charged amino acids, thus increasing the cytoplasmic retention potential of positively charged amino acids. Herein, TM organization of sucrose permease (CscB) and phenylalanine permease (PheP) as a function of membrane lipid composition was investigated to extend these principles to other proteins. For CscB, topological dependence on PE only becomes evident after a significant increase in the net negative charge of the cytoplasmic surface of the N-terminal TM bundle. High negative charge is required to overcome the thermodynamic block to inversion due to the high hydrophobicity of TM VII. Increasing the positive charge of the cytoplasmic surface of the N-terminal TM hairpin of PheP, which is misoriented in PE-lacking cells, favors native orientation in the absence of PE. PheP and CscB also display co-existing dual topologies dependent on changes in the charge balance between protein domains and the membrane lipids. Therefore, the topology of both permeases is dependent on PE. However, CscB topology is governed by thermodynamic balance between opposing lipid-dependent electrostatic and hydrophobic interactions.

31 Laboratoire de Spectrome´trie de Masse des Prote´ines, Institut de Biologie Structurale (CEA, CNRS, UJF, UMR 5075), Grenoble, France2 Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vi´denˇska´ 1083,Prague 4, Czech Republic3 CEA; DSV; iRTSV; Laboratoire de Chimie et Biologie des Me´taux (UMR 5249); CEA-Grenoble, Grenoble, France4 Commissariat a` l’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Service d’Inge´nierie Mole´culaire desProte´ines (SIMOPRO), F-91191 Gif sur Yvette, FranceKeywordsdiphtheriatoxin;hydrogen⁄deuteriumexchanges;massspectrometry;protein⁄membraneinteractions;translocationdomainCorrespondenceD. Gillet, Commissariat a` l’Energie Atomique(CEA), Institut de Biologie et Technologiesde Saclay (iBiTecS), Service d’Inge´nierieMole´culaire des Prote´ines (SIMOPRO),F-91191 Gif sur Yvette, FranceFax: +33 1 69 08 94 30Tel: +33 1 69 08 76 46E-mail: daniel.gillet@cea.frE. Forest, Laboratoire de Spectrome´trie deMasse des Prote´ines, Institut de BiologieStructurale (CEA-CNRS-UJF), 41 rue JulesHorowitz, 38027 Grenoble, FranceFax: +33 4 38 78 54 94Tel: +33 4 38 78 34 03E-mail: eric.forest@ibs.frV. Forge, CEA; DSV; iRTSV; Laboratoire deChimie et Biologie des Me´taux (UMR 5249);CEA-Grenoble, 17 rue des martyrs, 38054Grenoble, FranceFax: +33 4 38 78 54 87Tel: +33 4 38 78 94 05E-mail: vincent.forge@cea.fr*These authors contributed equally to this work(Received 7 August 2009, revised 6November2009,accepted23November2009)doi:10.1111/j.1742-4658.2009.07511.x

International audience; Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels. The mechanism of heteromeric channel regulation has not been clearly defined. Tissue ischemia leads to intracellular acidification and closure of Cx43 and Cx40 homomeric channels. However, coexpression of Cx40 and Cx43 in Xenopus oocytes enhances the pH sensitivity of the channel. This phenomenon requires the carboxyl-terminal (CT) part of both connexins. In this study we used different biophysical methods to determine the structure of the Cx40CT and characterize the Cx40CT/Cx43CT interaction. Our results revealed that the Cx40CT is an intrinsically disordered protein similar to the Cx43CT and that the Cx40CT and Cx43CT can interact. Additionally, we have identified an interaction between the Cx40CT and the cytoplasmic loop of Cx40 as well as between the Cx40CT and the cytoplasmic loop of Cx43 (and vice versa). Our studies support the “particle-receptor” model for pH gating of Cx40 and Cx43 gap junction channels and suggest that interactions between cytoplasmic regulatory domains (both homo- and hetero-connexin) could be important for the regulation of heteromeric channels.

Large unilamellar vesicles of 1-hexanoyl-2-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (PLPC) have been used as model membrane to investigate the effect of increasing amount of cardiolipin (1',3'-bis-[1,2-Di-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phospho]-sn-glycerol, CL) on the peroxidizability of the lipid phase. Hydroxyl radicals generated by gamma radiolysis of water initiated the lipid peroxidation. Both peroxidation products (conjugated dienes and hydroperoxides of PLPC, mono- and dihydroperoxides of CL) and disappearance of CL and PLPC were assessed as a function of the radiation dose (25 to 400 Gy, I = 10 Gy min(-1)). Our results show that the addition of 5% to 15% CL to large unilamellar vesicles (concentration ratio) produces almost complete inhibition of PLPC peroxidation. Thus, for 15% CL (known to be the proportion of CL in the inner mitochondrial membrane), the radiolytic yield of formation of PLPC hydroperoxides is reduced to zero, whereas it is equal to (3.1 +/- 0.2) x 10(-7) mol J(-1) for CL hydroperoxides, showing the importance of the targeted CL. For this concentration ratio (CL/ PLPC 15%), we have established the balance equation between the consumption of CL [G(-CL) = (2.8 +/- 0.1) x 10(-7) mol J(-1)] and the formation of CL hydroperoxides [G(CLOOH(T)) = (3.1 +/- 0.2) x 10(-7) mol J(-1)]. In addition, the radiolytic yields of disappearance of PLPC and CL have been determined [(1.5 +/- 0.1) x 10(-7) mol J(-1) and (2.8 +/- 0.1) x 10(-7) mol J(-1), respectively], their sum [(4.3 +/- 0.2) x 10(-7) mol J(-1)] being higher than G(HO.) (2.8 x 10(-7) mol J(-1)). However, there is no balance between the radiolytic yield of formation of PLPC hydroperoxides [G (PCOOH(T)) approximately 0] and the yield of disappearance of PLPC [(1.5 +/- 0.1) x 10(-7) mol J(-1)], likely because lipid fragments (not measured in this work) could be generated from HO(.) reaction on the polar head of PLPC. These results have been interpreted by assuming that the hydroxyl radicals attack in competition both lipid targets, i.e. PLPC and CL, with a higher sensitivity to CL oxidation. It can be concluded that a little amount of CL (10-15% CL/ PLPC concentration ratio) may exert a strong protective effect against the HO(.)-induced peroxidation of PLPC.

The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

This study assessed the location of melatonin (N-acetyl-5-methoxytryptamine) and of a pinoline derivative (GWC22) [6-ethyl-1-(3-methoxyphenyl)-2-propyl-1,2,3,4-tetrahydro-beta-carboline], when present in lipid assemblies such as linoleate micelles, phosphatidylcholine liposomes or low density lipoproteins (LDL). The efficiency of radical scavenging by these compounds is highly dependent on their partitioning between the lipidic and aqueous phases. We determined the proportion of melatonin or GWC22 in the aqueous and lipid phases of each system (concentrations of the antioxidants ranging between 3 x 10(-5) and 10(-4) m) by assaying melatonin or GWC22 by HPLC/UV detection, or by fluorescence for melatonin in micelles. Our results show that melatonin and GWC22 were preferentially located in the aqueous phase of micelles (68.4% and 59.0%, respectively), whereas only 30.5% of melatonin and 39.0% of GWC22 were found in the lipid phase. By contrast, in phosphatidylcholine liposomes, both compounds were essentially present in the lipid phase (73.5% for melatonin and 79.1% for GWC22, versus 25.9% and 19.5% in the aqueous phase, respectively). In the case of LDL, 99.9% of the melatonin added was found in the methanol/water extracting phase containing phospholipids, unesterified cholesterol and apolipoprotein B100. The partitioning of melatonin and GWC22 in linoleate micelles gave new insights on the marked protective effect of GWC22 towards radiation-induced lipid peroxidation and allowed us to determine more accurately the lower limit values of the reaction rate constants of the two molecules studied with lipid peroxyl radicals, i.e. k(LOO.+melatonin)) >or= 9.0 x 10(4)m(-1)s(-1) and k(LOO.+GWC22) >or= 3.5 x 10(5)m(-1)s(-1).

The present study was aimed at determining the peroxidation of model membranes constituted of liposomes of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) submitted to hydroxyl free radicals (generated by γ-radiolysis) attack. Liposomes of PLPC were prepared using the sonication technique, and dynamic light-scattering (DLS) measurements allowed characterization of the liposomal dispersions. Irradiation damages in sonication-generated liposomes were assessed by monitoring several oxidation products, such as conjugated dienes (by means of UV–visible spectrophotometry) and hydroperoxides (using reverse phase high-performance liquid chromatography (HPLC) associated with chemiluminescence detection). It has been shown that three different families of hydroperoxides are formed: the first one (at low radiation doses) results from HO· attack on the linoleyl chain of PLPC, giving phosphatidylcholine hydroperoxides possessing a conjugated dienic structure; the two others (at high radiation doses) are obtained by the secondary HO· attack on the primary hydroperoxide family. The quantification of these products associated with the comparison of their radiation-dose-dependent formation has provided valuable information concerning the mechanisms of their formation. Analysis by HPLC – mass spectrometry has confirmed the presence of hydroperoxides and underlined various other products, like chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl chain residues. Structural assignment proposals of some oxidation products have been proposed.Key words: radiolysis, phospholipids, peroxidation, hydroperoxides, liposomes.

Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. We have previously reported that mediators of inflammation, such as COX-2, promote the growth of Triple-Negative receptor (TN) IBC xenografts; therefore, inflammation in TN-IBC has a unique opportunity as a therapeutic strategy. Eicosapentaenoic acid (EPA), a non-toxic omega-3 fatty acid with anti-inflammatory properties, has partially reduced tumor growth in pre-clinical models of TN-IBC. Therefore, our goal is to develop a novel non-toxic approach that enhances EPA efficacy against TN-IBC in combination with targeted therapy. Methods and Results: Using a high-throughput, siRNA screen (939 genes) in the TN-IBC cell line SUM149PT, we identified Ephrin type-A receptor 2 (EPHA2), an oncogenic cell-surface receptor tyrosine kinase, as a target that modulates the sensitivity of TN-IBC cells to EPA treatment. To determine the clinical relevance of EPHA2, we interrogated a meta-analysis of breast cancer mRNA expression data sets, and found that high EPHA2 tumor expression was significantly correlated with poor overall survival in TN-IBC patients, compared to low EPHA2 expressing tumors (P = 0.01). We observed no significant correlations to other breast cancer subtypes. Similar findings were observed in vitro were EPHA2 expression predominantly occurred in the TN-IBC subtypes (19 of 30) among 49 breast cancer cell lines. Gain/loss-of-expression studies were performed to functionally validate EPHA2 as a synergistic combinational target with EPA in two EPHA2-expressing TN-IBC models, SUM149PT and BCX010, using proliferation and apoptosis assays in vitro and established tumor xenografts in vivo. EPHA2 gene silencing significantly reduced cell growth and induced apoptosis in combination with EPA when compared with untreated control and monotherapy in vitro (P < 0.05) and in vivo (P < 0.001), while vector-induced EPHA2 expression reversed cell growth reduction and apoptosis induction following combination treatment with EPA in vitro (P < 0.05). To translate our findings to the clinic, we validated that dasatinib, a small molecule inhibitor of EPHA2, in combination with EPA significantly enhanced cell death of SUM149PT and BCX010 cells in vitro when compared to non-treated and monotherapy (P < 0.05). Finally, using membrane fluidity assessment and reverse-phase protein array (300 antibodies), we determined that combination treatment efficacy depended on EPA/EPHA2 inhibition-mediated increase in cell membrane rigidity (P < 0.001, compared to monotherapy), which subsequently inhibited receptor tyrosine kinase signaling activity, potentially resulting in induction of apoptosis. Conclusions: Our preclinical findings provide a rationale for the development of a phase 1 clinical trial investigating combination EPA and EPHA2-inhibitors in patients with EPHA2-positive TN-IBC. Citation Format: Angie M. Torres-Adorno, Heidi Vitrac, Yuan Qi, Yiwen Yang, Peiying Yang, Bedrich L. Eckhardt, Naoto T. Ueno. EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2017-1235

Phospholipids could exert their effect on membrane protein topology either directly by interacting with topogenic signals of newly inserted proteins or indirectly by influencing the protein assembly machinery. In vivo lactose permease (LacY) of Escherichia coli displays a mixture of topological conformations ranging from complete inversion of the N-terminal helical bundle to mixed topology and then to completely native topology as phosphatidylethanolamine (PE) is increased from 0% to 70% of membrane phospholipids. These topological conformers are interconvertible by postassembly synthesis or dilution of PE in vivo. To investigate whether coexistence of multiple topological conformers is dependent solely on the membrane lipid composition, we determined the topological organization of LacY in an in vitro proteoliposome system in which lipid composition can be systematically controlled before (liposomes) and after (fliposomes) reconstitution using a lipid exchange technique. Purified LacY reconstituted into preformed liposomes of increasing PE content displayed inverted topology at low PE and then a mixture of inverted and proper topologies with the latter increasing with increasing PE until all LacY adopted its native topology. Interconversion between topological conformers of LacY was observed in a PE dose-dependent manner by either increasing or decreasing PE levels in proteoliposomes postreconstitution of LacY, clearly demonstrating that membrane protein topology can be changed simply by changing membrane lipid composition independent of other cellular factors. The results provide a thermodynamic-based lipid-dependent model for shifting the equilibrium between different conformational states of a membrane protein.

International audience; The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis. Bcl-xL forms aggregates in three states, micelles, native-like fibrils, and amyloid fibers, and their biophysical characterization has been performed in detail. Bcl-xL remains in its native state within micelles and native-like fibrils, and our results suggest that native-like fibrils are formed by the association of micelles. Formation of amyloid structures, that is, nonnative intermolecular β-sheets, is favored by the proximity of proteins within fibrils at the expense of the Bcl-xL native structure. Finally, we provide evidence of a direct relationship between the amyloid character of the fibers and the tertiary-structure stability of the native Bcl-xL. The potential causality between the accumulation of Bcl-xL into amyloid deposits and abnormal apoptosis during neurodegenerative diseases is discussed.

International audience; The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the pH inside endosomes is acid. At that stage, the domain is in a partially folded state; this prevents the use of high-resolution methods for the characterization of its functional structure. On that purpose, we report here the use of hydrogen/deuterium exchange experiments coupled to mass spectrometry. The conformation changes during the different steps of insertion into lipid bilayer are monitored with a resolution of few residues. Three parts of the translocation domain can be distinguished. With a high protection against exchange, the C-terminal hydrophobic helical hairpin is embedded in the membrane. Despite a lower protection, a significant effect in the presence of lipid vesicles shows that the N-terminal part is in interaction with the membrane interface. The sensitivity to the ionic strength indicates that electrostatic interactions are important for the binding. The middle part of the domain has an intermediate protection; this suggests that this part of the domain can be embedded within the membrane but remains quite dynamic. These results provide unprecedented insight into the structure reorganization of the protein to go from a soluble state to a membrane-inserted one.

International audience; The last step of the folding reaction of myoglobin is the incorporation of a prosthetic group. In cells, myoglobin is soluble, while heme resides in the mitochondrial membrane. We report here an exhaustive study of the interactions of apomyoglobin with lipid vesicles. We show that apomyoglobin interacts with large unilamellar vesicles under acidic conditions, and that this requires the presence of negatively charged phospholipids. The pH dependence of apomyoglobin interactions with membranes is a two-step process, and involves a partially folded state stabilized at acidic pH. An evident role for the interaction of apomyoglobin with lipid bilayers would be to facilitate the uptake of heme from the outer mitochondrial membrane. However, heme binding to apomyoglobin is observed at neutral pH when the protein remains in solution, and slows down as the pH becomes more favorable to membrane interactions. The effective incorporation of soluble heme into apomyoglobin at neutral pH suggests that the interaction of apomyoglobin with membranes is not necessary for the heme uptake from the lipid bilayer. In vivo, however, the ability of apomyoglobin to interact with membrane may facilitate its localization in the vicinity of the mitochondrial membranes, and so may increase the yield of heme uptake. Moreover, the behavior of apomyoglobin in the presence of membranes shows striking similarities with that of other proteins with a globin fold. This suggests that the globin fold is well adapted for soluble proteins whose functions require interactions with membranes.

Archidonate peroxidation has been studied using HO* radicals radiolytically generated as initiators of this process. Irradiated aqueous solutions of arachidonate (between 0.01 and 25 mM at pH 10.5) have been characterised by means of conjugated dienes measurement (234 nm-absorption spectroscopy) and hydroperoxide detection (high-performance liquid chromatography coupled with a chemiluminescence detection). Radiation-induced peroxidation of arachidonate gives a different trend of peroxide products, depending on the degree of substrate interaction; endoperoxide and hydro-endoperoxide being favored at low concentrations (monomer/oligomer) and monohydroperoxide at high concentrations (micellar form). The experimental ratios G(Hydro2)/G(Hydro1) increase significantly only for arachidonate concentrations higher than 1 mM, i.e. in micellar medium. However, between 0.1 and 1?mM in arachidonate, G-values (for conjugated dienes, Hydro2 and Hydro1) remain nearly constant, meaning that the physical arrangement of the solution changes: Aggregation occurs. The experimental yields of conjugated dienes formation indicated that GDienesGHO for [arachidonate]2.5 mM, indicating that a chain propagation process had occurred. Radiolytic yields and structural identification (HPLC-MS analysis) of peroxidation products allowed us to propose a mechanism for the formation of both hydroperoxides.

Transmembrane domain (TMD) orientation within some membrane proteins is dependent on membrane lipid composition. When the lactose permease (LacY) is assembled in Escherichia coli membranes lacking the major phospholipid phosphatidylethanolamine (PE), the N-terminal TMD bundle is inverted with respect to the C-terminal TMD bundle and the plane of the membrane. This inversion is dependent on the interfacial net positive charge of the protein, the net negative charge of the membrane and a TMD of low hydrophobicity, acting as a molecular hinge between the two halves of the protein by exiting the membrane to the periplasm. Homologous E. coli sucrose permease (CscB) and non-homologous phenylalanine permease (PheP) were investigated to generalize these original observations.CscB function, like that of LacY, is dependent on the presence of PE but topological dependence on membrane lipid composition is less sensitive and only becomes evident after significant changes in the net positive charge of the cytoplasmic surface of the N-terminal bundle. The first cytoplasmic domains of PheP, which are misoriented in PE-lacking cells, have a net negative charge. Decreasing the negative charge density of the extramembrane domains flanking the N-terminal TMD hairpin of PheP favors a reorientation of the N-terminus and adjacent hairpin to their native orientation in the absence of PE as predicted by the above net charge balance rule.Polytopic membrane proteins containing competing opposite charges within their cytoplasmic domains may share a common mechanism for topogenesis dependent on PE. However the degree of sensitivity to phospholipid composition appears to be sequence-specific and might be a result of conformational flexibility, topological preference of individual domains or the availability of mechanical hinge region. Supported by NIH grant R37-GM20478.