RESUMO
Staphylococcus aureus is the leading cause of infections worldwide, and methicillin-resistant strains (MRSA) are emerging. New strategies are urgently needed to overcome this threat. Using a cell-based screen of ~45,000 diverse synthetic compounds, we discovered a potent bioactive, MAC-545496, that reverses ß-lactam resistance in the community-acquired MRSA USA300 strain. MAC-545496 could also serve as an antivirulence agent alone; it attenuates MRSA virulence in Galleria mellonella larvae. MAC-545496 inhibits biofilm formation and abrogates intracellular survival in macrophages. Mechanistic characterization revealed MAC-545496 to be a nanomolar inhibitor of GraR, a regulator that responds to cell-envelope stress and is an important virulence factor and determinant of antibiotic resistance. The small molecule discovered herein is an inhibitor of GraR function. MAC-545496 has value as a research tool to probe the GraXRS regulatory system and as an antibacterial lead series of a mechanism to combat drug-resistant Staphylococcal infections.
Assuntos
Antibacterianos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Piperidinas/farmacologia , Piridinas/farmacologia , Resistência beta-Lactâmica/efeitos dos fármacos , Animais , Biofilmes/efeitos dos fármacos , Larva/microbiologia , Lepidópteros/microbiologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Testes de Sensibilidade Microbiana , Células RAW 264.7 , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Fatores de Virulência/antagonistas & inibidoresRESUMO
Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA). In humans, the alpha isoform (DGKα) has emerged as a potential target in the treatment of cancer due to its anti-tumor and pro-immune responses. However, its mechanism of action at a molecular level is not fully understood. In this work, a systematic investigation of the role played by the membrane in the regulation of the enzymatic properties of human DGKα is presented. By using a cell-free system with purified DGKα and model membranes of variable physical and chemical properties, it is shown that membrane physical properties determine human DGKα substrate acyl chain specificity. In model membranes with a flat morphology; DGKα presents high enzymatic activity, but it is not able to differentiate DAG molecular species. Furthermore, DGKα enzymatic properties are insensitive to membrane intrinsic curvature. However, in the presence of model membranes with altered morphology, specifically the presence of physically curved membrane structures, DGKα bears substrate acyl chain specificity for palmitic acid-containing DAG. The present results identify changes in membrane morphology as one possible mechanism for the depletion of specific pools of DAG as well as the production of specific pools of PA by DGKα, adding an extra layer of regulation on the interconversion of these two potent lipid-signaling molecules. It is proposed that the interplay between membrane physical (shape) and chemical (lipid composition) properties guarantee a fine-tuned signal transduction system dependent on the levels and molecular species of DAG and PA.
Assuntos
Membrana Celular/química , Diacilglicerol Quinase/química , Diglicerídeos/química , Ácidos Fosfatídicos/química , Domínio Catalítico , Membrana Celular/metabolismo , Diacilglicerol Quinase/metabolismo , Diglicerídeos/metabolismo , Humanos , Ácidos Fosfatídicos/metabolismo , Fosforilação , Especificidade por SubstratoRESUMO
Barth syndrome (BTHS) is a rare inherited metabolic disease resulting from mutations in the gene of the enzyme tafazzin, which catalyzes the acyl chain remodeling of the mitochondrial-specific lipid cardiolipin (CL). Tissue samples of individuals with BTHS present abnormalities in the level and the molecular species of CL. In addition, in tissues of a tafazzin knockdown mouse as well as in cells derived from BTHS patients it has been shown that plasmalogens, a subclass of glycerophospholipids, also have abnormal levels. Likewise, administration of a plasmalogen precursor to cells derived from BTHS patients led to an increase in plasmalogen and to some extent CL levels. These results indicate an interplay between CL and plasmalogens in BTHS. This interdependence is supported by the concomitant loss in these lipids in different pathological conditions. However, currently the molecular mechanism linking CL and plasmalogens is not fully understood. Here, a review of the evidence showing the linkage between the levels of CL and plasmalogens is presented. In addition, putative mechanisms that might play a role in this interplay are proposed. Finally, the opportunity of therapeutic approaches based on the regulation of plasmalogens as new therapies for the treatment of BTHS is discussed.
Assuntos
Síndrome de Barth/metabolismo , Cardiolipinas/metabolismo , Plasmalogênios/metabolismo , Aciltransferases/genética , Animais , Síndrome de Barth/genética , Humanos , Camundongos , Mitocôndrias/metabolismo , MutaçãoRESUMO
Diacylglycerol kinase ε (DGKε) is a membrane-bound enzyme that catalyzes the ATP-dependent phosphorylation of diacylglycerol to form phosphatidic acid (PA) in the phosphatidylinositol cycle. DGKε lacks a putative regulatory domain and has recently been reported to be regulated by highly curved membranes. To further study the effect of other membrane properties as a regulatory mechanism of DGKε, our work reports the effect of negatively charged phospholipids on DGKε activity and substrate acyl chain specificity. These studies were conducted using purified DGKε and detergent-free phospholipid aggregates, which present a more suitable model system to access the impact of membrane physical properties on membrane-active enzymes. The structural properties of the different model membranes were studied by means of differential scanning calorimetry and 31P-NMR. It is shown that the enzyme is inhibited by a variety of negatively charged phospholipids. However, PA, which is a negatively charged phospholipid and the product of DGKε catalyzed reaction, showed a varied regulatory effect on the enzyme from being an activator to an inhibitor. The type of feedback regulation of DGKε by PA depends on the particular PA molecular species as well as the physical properties of the membrane that the enzyme binds to. In the presence of highly packed PA-rich domains, the enzyme is activated. However, its acyl chain specificity is only observed in liposomes containing 1,2-dioleoyl PA in the presence of Ca2+. It is proposed that to endow the enzyme with its substrate acyl chain specificity, a highly dehydrated (hydrophobic) membrane interface is needed. The presence of an overlap of mechanisms to regulate DGKε ensures proper phosphatidylinositol cycle function regardless of the trigged stimulus and represents a sophisticated and specialized manner of membrane-enzyme regulation.
Assuntos
Diacilglicerol Quinase , Fosfolipídeos , Diacilglicerol Quinase/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Fosfatidilinositóis , Especificidade por SubstratoRESUMO
Phosphatidylinositol (PI) lipids have a predominance of a single molecular species present through the organism. In healthy mammals this molecular species is 1-stearoyl-2-arachidonoyl (18:0/20:4) PI. Although the importance of PI lipids for cell physiology has long been appreciated, less is known about the biological role of enriching PI lipids with 18:0/20:4 acyl chains. In conditions with dysfunctional lipid metabolism, the predominance of 18:0/20:4 acyl chains is lost. Recently, molecular mechanisms underpinning the enrichment or alteration of these acyl chains in PI lipids have begun to emerge. In the majority of the cases a common feature is the presence of enzymes bearing substrate acyl chain specificity. However, in cancer cells, it has been shown that one (not the only) of the mechanisms responsible for the loss in this acyl chain enrichment is mutation on the transcription factor p53 gene, which is one of the most highly mutated genes in cancers. There is a compelling need for a global picture of the specificity of the acyl chain composition of PIs. This can be possible once high-resolution spatio-temporal information is gathered in a cellular context; which can ultimately lead to potential novel targets to combat conditions with altered PI acyl chain profiles.
Assuntos
Aciltransferases/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Acilação , Animais , Humanos , Metabolismo dos Lipídeos , Especificidade por SubstratoRESUMO
Signaling events at membranes are often mediated by membrane lipid composition or membrane physical properties. These membrane properties could act either by favoring the membrane binding of downstream effectors or by modulating their activity. Several proteins can sense/generate membrane physical curvature (i.e. shape). However, the modulation of the activity of enzymes by a membrane's shape has not yet been reported. Here, using a cell-free assay with purified diacylglycerol kinase ϵ (DGKϵ) and liposomes, we studied the activity and acyl-chain specificity of an enzyme of the phosphatidylinositol (PI) cycle, DGKϵ. By systematically varying the model membrane lipid composition and physical properties, we found that DGKϵ has low activity and lacks acyl-chain specificity in locally flat membranes, regardless of the lipid composition. On the other hand, these enzyme properties were greatly enhanced in membrane structures with a negative Gaussian curvature. We also found that this is not a consequence of preferential binding of the enzyme to those structures, but rather is due to a curvature-mediated allosteric regulation of DGKϵ activity and acyl-chain specificity. Moreover, in a fine-tuned interplay between the enzyme and the membrane, DGKϵ favored the formation of structures with greater Gaussian curvature. DGKϵ does not bear a regulatory domain, and these findings reveal the importance of membrane curvature in regulating DGKϵ activity and acyl-chain specificity. Hence, this study highlights that a hierarchic coupling of membrane physical property and lipid composition synergistically regulates membrane signaling events. We propose that this regulatory mechanism of membrane-associated enzyme activity is likely more common than is currently appreciated.
Assuntos
Diacilglicerol Quinase/química , Lipossomos/química , Fosfatidilinositóis/química , Animais , Linhagem Celular , Colesterol/química , Diglicerídeos/química , Ensaios Enzimáticos , Humanos , Fusão de Membrana , Micelas , Estrutura Molecular , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Spodoptera , Propriedades de SuperfícieRESUMO
The low aqueous solubility of phospholipids makes necessary the use of lipid carriers in studies ranging from lipid traffic and metabolism to the engineering of model membranes bearing lipid transverse asymmetry. One particular lipid carrier that has proven to be particularly useful is methyl-ß-cyclodextrin (MßCD). To assess the interaction of MßCD with structurally different phospholipids, the present work reports the results of isothermal titration calorimetry in conjunction with dynamic light scattering measurements. The results showed that the interaction of MßCD with large unilamellar vesicles composed of a single type of lipid led to the solubilization of the lipid vesicle and, consequently, the complexation of MßCD with the lipids. This interaction is dependent on the nature of the lipid headgroup, with a preferable interaction with phosphatidylglycerol in comparison to phosphatidylcholine. It was also possible to show a role played by the phospholipid backbone in this interaction. In many cases, the differences in the transfer energy between one lipid and another in going from a bilayer to a cyclodextrin-bound state can be qualitatively explained by the energy required to extract the lipid from a bilayer. In all cases, the data showed that the solubilization of the vesicles is entropically driven with a large negative ΔCp, suggesting a mechanism dependent on the hydrophobic effect.
Assuntos
beta-Ciclodextrinas/química , Bicamadas Lipídicas , Lipídeos , Fosfatidilcolinas , TermodinâmicaRESUMO
Specific ion effects in surfactant solutions affect the properties of micelles. Dodecyltrimethylammonium chloride (DTAC), bromide (DTAB), and methanesulfonate (DTAMs) micelles are typically spherical, but some organic anions can induce shape or phase transitions in DTA(+) micelles. Above a defined concentration, sodium triflate (NaTf) induces a phase separation in dodecyltrimethylammonium triflate (DTATf) micelles, a phenomenon rarely observed in cationic micelles. This unexpected behavior of the DTATf/NaTf system suggests that DTATf aggregates have unusual properties. The structural properties of DTATf micelles were analyzed by time-resolved fluorescence quenching, small-angle X-ray scattering, nuclear magnetic resonance, and electron paramagnetic resonance and compared with those of DTAC, DTAB, and DTAMs micelles. Compared to the other micelle types, the DTATf micelles had a higher average number of monomers per aggregate, an uncommon disk-like shape, smaller interfacial hydration, and restricted monomer chain mobility. Molecular dynamic simulations supported these observations. Even small water-soluble salts can profoundly affect micellar properties; our data demonstrate that the -CF3 group in Tf(-) was directly responsible for the observed shape changes by decreasing interfacial hydration and increasing the degree of order of the surfactant chains in the DTATf micelles.
Assuntos
Mesilatos/química , Micelas , Compostos de Amônio Quaternário/química , Cátions/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Tensoativos/químicaRESUMO
Cancer immunotherapy has revolutionized the oncology field. Despite the success, new molecular targets are needed to increase the percentage of patients that benefits from this therapy. Diacylglycerol kinase α (DGKα) has gathered great attention as a potential molecular target in immunotherapy because of its role in cancer proliferation and immunosuppression. DGKα catalyzes the ATP-dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA). Since both lipids are potent signaling messengers, DGKα acts as a switch between different signaling pathways. Its role in cancer and immunosuppression has long been ascribed to the regulation of DAG/PA levels. However, this paradigm has been challenged with the identification of DGKα substrate acyl chain specificity, which suggests its role in signaling could be specific to DAG/PA molecular species. In several biological processes where DGKα plays a role, large membrane morphological changes take place. DGKα substrate specificity depends on the shape of the membrane that the enzyme binds to. Hence, DGKα can act as a bridge between large membrane morphological changes and the regulation of specific molecular species of DAG/PA. Bearing in mind the potential therapeutic benefits of targeting DGKα, here, the role of DGKα in cancer and T cell biology with a focus on the modulation of its enzymatic properties by membrane shape is reviewed. The goal is to contribute to a global understanding of the molecular mechanisms governing DGKα biology. This will pave the way for future experimentation and, consequently, the design of better, more potent therapeutic strategies aiming at improving the health outcomes of cancer patients.
RESUMO
Diacylglycerol kinases (DGKs) are important enzymes in molecular membrane biology, as they can lower the concentration of diacylglycerol through phosphorylation while at the same time producing phosphatidic acid. Dysfunction of DGK is linked with multiple diseases including cancer and autoimmune disorders. Currently, the high-resolution structures have not been determined for any of the 10 human DGK paralogs, which has made it difficult to gain a more complete understanding of the enzyme's mechanism of action and regulation. In the present study, we have taken advantage of the significant developments in protein structural prediction technology by artificial intelligence (i.e., Alphafold 2.0), to conduct a comprehensive investigation on the properties of all 10 human DGK paralogs. Structural alignment of the predictions reveals that the C1, catalytic, and accessory domains are conserved in their spatial arrangement relative to each other, across all paralogs. This suggests a critical role played by this domain architecture in DGK function. Moreover, docking studies corroborate the existence of a conserved ATP-binding site between the catalytic and accessory domains. Interestingly, the ATP bound to the interdomain cleft was also found to be in proximity of the conserved glycine-rich motif, which in protein kinases has been suggested to function in ATP binding. Lastly, the spatial arrangement of DGK, with respect to the membrane, reveals that most paralogs possess a more energetically favorable interaction with curved membranes. In conclusion, AlphaFold predictions of human DGKs provide novel insights into the enzyme's structural and functional properties while also paving the way for future experimentation.
Assuntos
Diacilglicerol Quinase , Diglicerídeos , Trifosfato de Adenosina , Inteligência Artificial , Diacilglicerol Quinase/química , Diacilglicerol Quinase/metabolismo , Diglicerídeos/química , Glicina , Humanos , Ácidos Fosfatídicos/química , Ácidos Fosfatídicos/metabolismo , Proteínas QuinasesRESUMO
Diacylglycerol kinase ε (DGKε), an enzyme of the phosphatidylinositol (PI) cycle, bears a highly conserved hydrophobic N-terminal segment, which was proposed to anchor the enzyme into the membrane. However, the importance of this segment to the DGKε function remains to be determined. To address this question, it is here reported an in silico and in vitro combined research strategy. Capitalizing on the AlphaFold 2.0 predicted structure of human DGKε, it is shown that its hydrophobic N-terminal segment anchors it into the membrane via a transmembrane α-helix. Coarse-grained based elastic network model studies showed that a conformational change in the hydrophobic N-terminal segment determines the proximity between the active site of DGKε and the membrane-water interface, likely regulating its kinase activity. In vitro studies with a purified DGKε construct lacking the hydrophobic N-terminal segment (His-SUMO*-Δ50-DGKε) corroborated the role of the N-terminus in regulating DGKε enzymatic properties. The comparison between the enzymatic properties of DGKε and His-SUMO*-Δ50-DGKε showed that the conserved N-terminal segment markedly inhibits the enzyme activity and its sensitivity to membrane intrinsic negative curvature, while also playing a role in the modulation of the enzyme by phosphatidylserine. On the other hand, this segment did not strongly affect its diacylglycerol acyl chain specificity, the modulation of the enzyme by membrane morphological changes, or the activation by phosphatidic acid-rich lipid domains. Hence, these results suggest that the conservation of the hydrophobic N-terminal segment of DGKε throughout evolution guaranteed not only membrane anchorage but also an efficient and elegant manner to regulate the rate of the PI cycle.
Assuntos
Diacilglicerol Quinase , Diglicerídeos , Diacilglicerol Quinase/química , Diglicerídeos/química , Humanos , Fosfatidilinositóis , Fosfatidilserinas , ÁguaRESUMO
It is becoming widely acknowledged that lipids play key roles in cellular function, regulating a variety of biological processes. Lately, a subclass of glycerophospholipids, namely plasmalogens, has received increased attention due to their association with several degenerative and metabolic disorders as well as aging. All these pathophysiological conditions involve chronic inflammatory processes, which have been linked with decreased levels of plasmalogens. Currently, there is a lack of full understanding of the molecular mechanisms governing the association of plasmalogens with inflammation. However, it has been shown that in inflammatory processes, plasmalogens could trigger either an anti- or pro-inflammation response. While the anti-inflammatory response seems to be linked to the entire plasmalogen molecule, its pro-inflammatory response seems to be associated with plasmalogen hydrolysis, i.e., the release of arachidonic acid, which, in turn, serves as a precursor to produce pro-inflammatory lipid mediators. Moreover, as plasmalogens comprise a large fraction of the total lipids in humans, changes in their levels have been shown to change membrane properties and, therefore, signaling pathways involved in the inflammatory cascade. Restoring plasmalogen levels by use of plasmalogen replacement therapy has been shown to be a successful anti-inflammatory strategy as well as ameliorating several pathological hallmarks of these diseases. The purpose of this review is to highlight the emerging role of plasmalogens in chronic inflammatory disorders as well as the promising role of plasmalogen replacement therapy in the treatment of these pathologies.
RESUMO
Plasmalogens, a subclass of glycerophospholipids containing a vinyl-ether bond, are one of the major components of biological membranes. Changes in plasmalogen content and molecular species have been reported in a variety of pathological conditions ranging from inherited to metabolic and degenerative diseases. Most of these diseases have no treatment, and attempts to develop a therapy have been focusing primarily on protein/nucleic acid molecular targets. However, recent studies have shifted attention to lipids as the basis of a therapeutic strategy. In these pathological conditions, the use of plasmalogen replacement therapy (PRT) has been shown to be a successful way to restore plasmalogen levels as well as to ameliorate the disease phenotype in different clinical settings. Here, the current state of PRT will be reviewed as well as a discussion of future perspectives in PRT. It is proposed that the use of PRT provides a modern and innovative molecular medicine approach aiming at improving health outcomes in different conditions with clinically unmet needs.
RESUMO
Membrane lipids play a role in the modulation of a variety of biological processes. This is often achieved through fine-tuned changes in membrane physical and chemical properties. While some membrane physical properties (e.g., curvature, lipid domains, fluidity) have received increased scientific attention over the years, only recently has membrane shape emerged as an active modulator of protein properties. Biological membranes are mostly found organized into a lipid bilayer arrangement, in which the spontaneous shape is an intrinsically flat, planar morphology (in relation to the size of proteins). However, it is known that many cells and organelles have non-planar morphologies. In addition, perturbations in membrane morphology occur in a variety of biological processes. Recent studies have shown that membrane shape can modulate a variety of biological processes by determining protein properties. While membrane shape generation modulates proteins via changes in membrane mechanical properties, membrane shape recognition regulates proteins by providing the optimal surface for interaction. Hence, membranes have evolved an elegant mechanism to couple mesoscopic perturbations to molecular properties and vice-versa. In this review, the regulation of the enzymatic properties of two isoforms of mammalian diacylglycerol kinase, which play important roles in cellular signal transductions, will be used to exemplify the recent advancements in the field of membrane shape recognition, as well as future challenges and perspectives.
Assuntos
Diacilglicerol Quinase/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Diacilglicerol Quinase/química , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Lipídeos de Membrana/química , Proteínas de Membrana/química , Transdução de SinaisRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease. Currently, PD has no treatment. The neuronal protein α-synuclein (αS) plays an important role in PD. However, the molecular mechanisms governing its physiological and pathological roles are not fully understood. It is becoming widely acknowledged that the biological roles of αS involve interactions with biological membranes. In these biological processes there is a fine-tuned interplay between lipids affecting the properties of αS and αS affecting lipid metabolism, αS binding to membranes, and membrane damage. In this review, the intricate interactions between αS and membranes will be reviewed and a discussion of the relationship between αS and neuronal membrane structural plasticity in health and disease will be made. It is proposed that in healthy neurons the conformational flexibilities of αS and the neuronal membranes are coupled to assist the physiological roles of αS. However, in circumstances where their conformational flexibilities are decreased or uncoupled, there is a shift toward cell toxicity. Strategies to modulate toxic αS-membrane interactions are potential approaches for the development of new therapies for PD. Future work using specific αS molecular species as well as membranes with specific physicochemical properties should widen our understanding of the intricate biological roles of αS which, in turn, would propel the development of new strategies for the treatment of PD.
Assuntos
Membrana Celular/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , alfa-Sinucleína/metabolismo , Membrana Celular/química , Humanos , Conformação Molecular , Neurônios/química , alfa-Sinucleína/químicaRESUMO
Biological membranes define and determine the architecture, i.e., shape, of cells and organelles. While most membranes present a planar morphology on the nanometer length scale, their shape could change in a wide range of length and time scales, leading to more intricate shapes that could be (transient) short- or long-lived. The change in membrane shape from the energetically more stable planar one is accomplished by bending it away (curving) from this morphology, a process that is determined by the lipid bilayer structural properties and/or the application of forces by proteins. For a long time, the membrane shape was believed to play a passive role. However, recently this view has started changing by examples of biological processes controlled by the membrane shape and/or its curved structures, which poses membrane shapes as active modulators of signaling in biological processes. The ability of membrane shape and/or its curved structures to regulate biological processes usually occurs either by a preferential binding of proteins to membranes or the allosteric regulation of enzymes by membrane shape changes. Here, the current knowledge of the roles of membrane shape on the regulation of biological processes will be discussed. While the role of membrane shape is usually tied up with the bilayer bending properties, recent reports showed that some proteins prefer a planar membrane shape instead of curved structures. Hence, it is here proposed that membrane shape recognition is a trigger for signaling events. We present examples in which different membrane shapes stimulate protein binding and/or enzyme activity.
Assuntos
Membrana Celular/fisiologia , Proteínas de Membrana/metabolismo , Regulação Alostérica , Fenômenos Biofísicos , Ligação ProteicaRESUMO
It is becoming widely acknowledged that many biological processes are dependent on specific lipid molecular species. In healthy humans, two important lipid molecular species for cell physiology are tetralinoleoyl cardiolipin (in the heart) and 1-stearoyl-2-arachidonoyl phosphatidylinositols (throughout the organism). The predominance of these lipid molecular species is in part due to the presence of enzymes along their biosynthetic pathways that favor their enrichment with specific acyl chains. In cardiolipin biosynthesis, one example is the reaction catalyzed by the enzyme tafazzin, while for the biosynthesis of phosphatidylinositols the epsilson isoform of diacylglycerol kinase (DGKε) plays an important role. Here a discussion of the roles played by both enzyme structure and membrane environment on the production of specific lipid molecular species by these two membrane-acting enzymes will be made. It is proposed that the enrichment of certain lipid molecular species within the organism is a result of a fine-tuned interplay between enzyme structure and membrane environment.
Assuntos
Diacilglicerol Quinase/metabolismo , Lipídeos/química , Fatores de Transcrição/metabolismo , Aciltransferases , Animais , Catálise , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Diacilglicerol Quinase/química , Humanos , Conformação Proteica , Especificidade por Substrato , Fatores de Transcrição/químicaRESUMO
The presence of an asymmetric distribution of lipids in biological membranes was first described ca. 50 years ago. While various studies had reported the role of loss of lipid asymmetry on signaling processes, its effect on membrane physical properties and membrane-protein interactions lacks further understanding. The recent description of new technologies for the preparation of asymmetric model membranes has helped to fill part of this gap. However, the major effort so far has been on plasma membrane models. Here we describe the preparation of liposomes mimicking the mitochondria outer membrane (MOM) in regard to its lipid composition and asymmetry. By employing the methyl-ß-cyclodextrin-catalyzed lipid exchange technology and accurate quantification of lipid asymmetry with head group-specific probes we showed the successful preparation of a MOM model bearing a physiologically relevant lipid composition and asymmetry. In addition, by a direct comparison with its lipid symmetrical counterpart it is shown that asymmetric models were more resistant to tBid-promoted Bax-permeabilization, suggesting a role played by MOM lipid asymmetry on the mitochondria pathway of apoptosis. The barrier imposed by lipid asymmetry on membrane permeabilization was in part due to a decrease in the concentration of membrane-bound proteins, which was likely a consequence of the two mutually-dependent properties; i.e., the lower electrostatic surface potential and the higher molecular packing imposed by lipid asymmetry. It is proposed that MOM lipid asymmetry imparts different physical properties on the membrane and might add an additional component of regulation in intricate mitochondrial processes.
Assuntos
Lipídeos/química , Mitocôndrias/genética , Membranas Mitocondriais/química , Proteína X Associada a bcl-2/genética , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Lipossomos/química , Lipossomos/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/ultraestrutura , Fosfolipídeos/química , Fosfolipídeos/genéticaRESUMO
In Barth syndrome (BTHS) mutations in tafazzin leads to changes in both the quantities and the molecular species of cardiolipin (CL), which are the hallmarks of BTHS. Contrary to the well-established alterations in CL associated with BTHS; recently a marked decrease in the plasmalogen levels in Barth specimens has been identified. To restore the plasmalogen levels, the present study reports the effect of promotion of plasmalogen biosynthesis on the lipidome of lymphoblasts derived from Barth patients as well as on cell viability, mitochondria biogenesis, and mitochondrial membrane potential. High resolution 31P NMR phospholipidomic analysis showed an increase in the levels of plasmenylethanolamine (the major plasmalogen in lymphoblasts), which reached values comparable to the control and a compensatory decrease in the levels of its diacyl-PE counterpart. Importantly, 31P NMR showed a significant increase in the levels of CL, while not altering the levels of monolysocardiolipin. Mass spectrometry measurements showed that the promotion of plasmalogen biosynthesis did not change the molecular species profile of targeted phospholipids. In addition, promotion of plasmalogen biosynthesis did not impact on cellular viability, although it significantly decrease mitochondria copy number and restored mitochondrial membrane potential. Overall, the results showed the efficacy of the promotion of plasmalogen biosynthesis on increasing the CL levels in a BTHS cell model and highlight the potential beneficial effect of a diet supplemented with plasmalogen precursors to BTHS patients.
Assuntos
Síndrome de Barth/metabolismo , Cardiolipinas/metabolismo , Éteres de Glicerila/metabolismo , Linfócitos/metabolismo , Lisofosfolipídeos/metabolismo , Plasmalogênios/biossíntese , Aciltransferases , Síndrome de Barth/sangue , Síndrome de Barth/dietoterapia , Síndrome de Barth/genética , Cardiolipinas/análise , Sobrevivência Celular , Células Cultivadas , Criança , Pré-Escolar , Gorduras na Dieta , Suplementos Nutricionais , Éteres de Glicerila/administração & dosagem , Humanos , Lactente , Mutação com Perda de Função , Linfócitos/citologia , Lisofosfolipídeos/análise , Masculino , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Biogênese de Organelas , Cultura Primária de Células , Fatores de Transcrição/genéticaRESUMO
The extensive number of metabolic processes regulated by phosphatidylinositol (PI) phospholipids highlights their physiological importance. The major metabolic pathway for their biosynthesis in cells is the PI-cycle. Contrary to most metabolic cycles, reactions of the PI-cycle occur in two different locations; those are, the plasma membrane (PM) and endoplasmic reticulum (ER). Lipid movement between the two organelles is, therefore, a requirement of the cyclical process. Moreover, in mammals the PI-cycle yield PI molecular species enriched in specific acyl chains, namely 1-stearoyl-2-arachidonoyl acyl chains. Hence, to ensure cycle efficiency and specificity it should take place in specialized regions of PM and ER rather than being randomly distributed among those membranes. Along these lines, ER-PM contact sites have emerged as the location where a number of proteins related to the PI-cycle have been reported to localize. Of importance to this review is the presence of the epsilon isoform of diacylglycerol kinase (DGKε) at ER-PM contact sites. In the PI-cycle DGKε is in part responsible for the acyl chain enrichment of the PI molecular species. However, it has recently been shown that the enzyme can only engage in the PI-cycle upon membrane morphological changes. In this review we will discuss the PI-cycle at ER-PM contact sites and how the generation of membrane negative Gaussian curvature nearby those regions could regulate the cycle. We will focus our discussion on the hypothesis that actin polymerization provides the mechanical force needed to change membrane shape nearby ER-PM contact sites, which will transiently trigger DGKε and, therefore, link enzymatic catalysis and lipid transfer in the PI-cycle.