RESUMO
SARS-CoV-2 is the third known coronavirus (CoV) that has crossed the animal-human barrier in the last two decades. However, little structural information exists related to the close genetic species within the SARS-related coronaviruses. Here, we present three novel SARS-related CoV spike protein structures solved by single particle cryo-electron microscopy analysis derived from bat (bat SL-CoV WIV1) and civet (cCoV-SZ3, cCoV-007) hosts. We report complex glycan trees that decorate the glycoproteins and density for water molecules which facilitated modeling of the water molecule coordination networks within structurally important regions. We note structural conservation of the fatty acid binding pocket and presence of a linoleic acid molecule which are associated with stabilization of the receptor binding domains in the "down" conformation. Additionally, the N-terminal biliverdin binding pocket is occupied by a density in all the structures. Finally, we analyzed structural differences in a loop of the receptor binding motif between coronaviruses known to infect humans and the animal coronaviruses described in this study, which regulate binding to the human angiotensin converting enzyme 2 receptor. This study offers a structural framework to evaluate the close relatives of SARS-CoV-2, the ability to inform pandemic prevention, and aid in the development of pan-neutralizing treatments.
Assuntos
Quirópteros , Microscopia Crioeletrônica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , SARS-CoV-2/química , Animais , Humanos , Quirópteros/virologia , COVID-19/virologia , Sítios de Ligação , Betacoronavirus , Motivos de Aminoácidos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Modelos Moleculares , Ligação ProteicaRESUMO
Male and female Plasmodium falciparum gametocytes are the parasite lifecycle stage responsible for transmission of malaria from the human host to the mosquito vector. Not only are gametocytes able to survive in radically different host environments, but they are also precursors for male and female gametes that reproduce sexually soon after ingestion by the mosquito. Here, we investigate the sex-specific lipid metabolism of gametocytes within their host red blood cell. Comparison of the male and female lipidome identifies cholesteryl esters and dihydrosphingomyelin enrichment in female gametocytes. Chemical inhibition of each of these lipid types in mature gametocytes suggests dihydrosphingomyelin synthesis but not cholesteryl ester synthesis is important for gametocyte viability. Genetic disruption of each of the two sphingomyelin synthase genes points towards sphingomyelin synthesis contributing to gametocytogenesis. This study shows that gametocytes are distinct from asexual stages, and that the lipid composition is also vastly different between male and female gametocytes, reflecting the different cellular roles these stages play. Taken together, our results highlight the sex-specific nature of gametocyte lipid metabolism, which has the potential to be targeted to block malaria transmission. This article has an associated First Person interview with the first author of the paper.
Assuntos
Malária Falciparum , Plasmodium falciparum , Animais , Feminino , Humanos , Estágios do Ciclo de Vida/fisiologia , Metabolismo dos Lipídeos , Masculino , Mosquitos Vetores , Plasmodium falciparum/metabolismo , Esfingomielinas/metabolismoRESUMO
Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.
Assuntos
Proteínas de Bactérias , Reparo do DNA , Escherichia coli , Fatores de Transcrição , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Fatty acid receptors have been recognized as important players in glycaemic control. This study is the first to describe a role for the medium-chain fatty acid (MCFA) receptor G-protein-coupled receptor (Gpr) 84 in skeletal muscle mitochondrial function and insulin secretion. We are able to show that Gpr84 is highly expressed in skeletal muscle and adipose tissue. Mice with global deletion of Gpr84 [Gpr84 knockout (KO)] exhibit a mild impairment in glucose tolerance when fed a MCFA-enriched diet. Studies in mice and pancreatic islets suggest that glucose intolerance is accompanied by a defect in insulin secretion. MCFA-fed KO mice also exhibit a significant impairment in the intrinsic respiratory capacity of their skeletal muscle mitochondria, but at the same time also exhibit a substantial increase in mitochondrial content. Changes in canonical pathways of mitochondrial biogenesis and turnover are unable to explain these mitochondrial differences. Our results show that Gpr84 plays a crucial role in regulating mitochondrial function and quality control.-Montgomery, M. K., Osborne, B., Brandon, A. E., O'Reilly, L., Fiveash, C. E., Brown, S. H. J., Wilkins, B. P., Samsudeen, A., Yu, J., Devanapalli, B., Hertzog, A., Tolun, A. A., Kavanagh, T., Cooper, A. A., Mitchell, T. W., Biden, T. J., Smith, N. J., Cooney, G. J., Turner, N. Regulation of mitochondrial metabolism in murine skeletal muscle by the medium-chain fatty acid receptor Gpr84.
Assuntos
Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Composição Corporal , Glucose/metabolismo , Resistência à Insulina , Lipídeos/análise , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/química , Receptores Acoplados a Proteínas G/genéticaRESUMO
Honey bees have evolved a system in which fertilised eggs transit through the same developmental stages but can become either workers or queens. This difference is determined by their diet through development. Whereas workers live for weeks (normally 2-6â weeks), queens can live for years. Unfertilised eggs also develop through the same stages but result in a short-lived male caste (drones). Workers and drones are fed pollen throughout their late larval and adult life stages, while queens are fed exclusively on royal jelly and do not eat pollen. Pollen has a high content of polyunsaturated fatty acids (PUFA) while royal jelly has a negligible amount of PUFA. To investigate the role of dietary PUFA lipids and their oxidation in the longevity difference of honey bees, membrane fatty acid composition of the three castes was characterised at six different life-history stages (larva, pupa, emergent and different adult stages) through mass spectrometry. All castes were found to share a similar membrane phospholipid composition during early larval development. However, at pupation, drones and workers increased their level of PUFA, whilst queens increased their level of monounsaturated fatty acids. After emergence, worker bees further increased their level of PUFA by 5-fold across most phospholipid classes. In contrast, the membrane phospholipids of adult queens remained highly monounsaturated throughout their adult life. We postulate that this diet-induced increase in membrane PUFA results in more oxidative damage and is potentially responsible for the much shorter lifespan of worker bees compared with long-lived queens.
Assuntos
Abelhas/fisiologia , Metabolismo dos Lipídeos , Longevidade , Animais , Abelhas/crescimento & desenvolvimento , Abelhas/metabolismo , Feminino , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lipidômica , Masculino , Espectrometria de Massas , Pupa/crescimento & desenvolvimento , Pupa/metabolismoRESUMO
The (O-acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.
Assuntos
Ácidos Graxos/química , Ácidos Graxos/síntese química , Espectrometria de Massas , Técnicas de Química Sintética , Ésteres/química , Humanos , Glândulas Tarsais/química , EstereoisomerismoRESUMO
Apolipoprotein-D is a 25â¯kDa glycosylated member of the lipocalin family that folds into an eight-stranded ß-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-ß burden in Alzheimer's disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a â¼95 to â¼100â¯kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.
Assuntos
Doença de Alzheimer/genética , Apolipoproteínas D/química , Conformação Proteica , Multimerização Proteica , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Animais , Apolipoproteínas D/líquido cefalorraquidiano , Apolipoproteínas D/genética , Cisto Mamário/química , Cristalografia por Raios X , Modelos Animais de Doenças , Humanos , Ligantes , Lipocalinas/química , Camundongos , Ligação Proteica , Espalhamento a Baixo ÂnguloRESUMO
In a recent study, we showed that in response to high fat feeding C57BL/6, 129X1, DBA/2 and FVB/N mice all developed glucose intolerance, while BALB/c mice displayed minimal deterioration in glucose tolerance and insulin action. Lipidomic analysis of livers across these five strains has revealed marked strain-specific differences in ceramide (Cer) and sphingomyelin (SM) species with high-fat feeding; with increases in C16-C22 (long-chain) and reductions in C>22 (very long-chain) Cer and SM species observed in the four strains that developed HFD-induced glucose intolerance. Intriguingly, the opposite pattern was observed in sphingolipid species in BALB/c mice. These strain-specific changes in sphingolipid acylation closely correlated with ceramide synthase 2 (CerS2) protein content and activity, with reduced CerS2 levels/activity observed in glucose intolerant strains and increased content in BALB/c mice. Overexpression of CerS2 in primary mouse hepatocytes induced a specific elevation in very long-chain Cer, but despite the overall increase in ceramide abundance, there was a substantial improvement in insulin signal transduction, as well as decreased ER stress and gluconeogenic markers. Overall our findings suggest that very long-chain sphingolipid species exhibit a protective role against the development of glucose intolerance and hepatic insulin resistance.
Assuntos
Ceramidas/metabolismo , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Esfingolipídeos/metabolismo , Acilação , Animais , Dieta Hiperlipídica , Diglicerídeos/metabolismo , Estresse do Retículo Endoplasmático , Comportamento Alimentar , Hepatócitos/enzimologia , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Oxirredutases/metabolismo , Transdução de Sinais , Especificidade da Espécie , Esfingomielinas/metabolismoRESUMO
We have discovered that a small cationic molecule, GW4869, is cytotoxic to a subset of myeloma cell lines and primary myeloma plasma cells. Biochemical analysis revealed that GW4869 binds to anionic phospholipids such as phosphatidylserine - a lipid normally confined to the intracellular side of the cell membrane. However, interestingly, phosphatidylserine was expressed on the surface of all myeloma cell lines tested (n = 12) and 9/15 primary myeloma samples. Notably, the level of phosphatidylserine expression correlated well with sensitivity to GW4869. Inhibition of cell surface phosphatidylserine exposure with brefeldin A resulted in resistance to GW4869. Finally, GW4869 was shown to delay the growth of phosphatidylserine-high myeloma cells in vivo. To the best of our knowledge, this is the first example of using a small molecule to target phosphatidylserine on malignant cells. This study may provide the rationale for the development of phosphatidylserine-targeting small molecules for the treatment of surface phosphatidylserine-expressing cancers.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Compostos de Benzilideno/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Fosfatidilserinas/metabolismo , Compostos de Anilina/administração & dosagem , Compostos de Anilina/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Compostos de Benzilideno/administração & dosagem , Compostos de Benzilideno/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Camundongos SCID , Mieloma Múltiplo/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The development of differentiated sexual stages (gametocytes) within human red blood cells is essential for the propagation of the malaria parasite, since only mature gametocytes will survive in the mosquito's midgut. Hence gametocytogenesis is a pre-requisite for transmission of the disease. Physiological changes involved in sexual differentiation are still enigmatic. In particular the lipid metabolism-despite being central to cellular regulation and development-is not well explored. METHODS: Here the lipid profiles of red blood cells infected with the five different sexual stages of Plasmodium falciparum were analysed by mass spectrometry and compared to those from uninfected and asexual trophozoite infected erythrocytes. RESULTS: Fundamental differences between erythrocytes infected with the different parasite stages were revealed. In mature gametocytes many lipids that decrease in the trophozoite and early gametocyte infected red blood cells are regained. In particular, regulators of membrane fluidity, cholesterol and sphingomyelin, increased significantly during gametocyte maturation. Neutral lipids (serving mainly as caloriometric reserves) increased from 3 % of total lipids in uninfected to 27 % in stage V gametocyte infected red blood cells. The major membrane lipid class (phospholipids) decreased during gametocyte development. CONCLUSIONS: The lipid profiles of infected erythrocytes are characteristic for the particular parasite life cycle and maturity stages of gametocytes. The obtained lipid profiles are crucial in revealing the lipid metabolism of malaria parasites and identifying targets to interfere with this deadly disease.
Assuntos
Lipídeos/análise , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Animais , Eritrócitos/parasitologia , Humanos , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/metabolismoRESUMO
PURPOSE: To determine whether tear collection by flushing the ocular surface with saline (flush tears) or collection by stimulation (reflex tears) can be used as an alternative to basal tear collection for the identification and quantification of lipids in the tear film. METHODS: Tear samples were collected from 10 participants with no history of ocular surface disease or contact lens wear. Up to 10 µl of basal, reflex, and flush tear samples were collected from each eye using a microcapillary tube on three occasions with the order of methods randomized and allowing at least 24 hours between each collection method. Lipids were quantified from each tear sample using nano-electrospray ionization tandem mass spectrometry. RESULTS: Total lipids significantly differed in their concentration (pmol/µl) and mole % with each collection technique. Cholesterol esters [mean % (SE)] formed the major component of the total lipidome in basal [54.8% (3.1)], reflex [35.7% (6.4)], and flush [33.0% (3.1)] tear samples. However, the mole % of each lipid class substantially varied with each tear collection method. Nonpolar lipids, including cholesterol, wax esters, and triacylglycerols, dominated the tear lipidome in basal [92.8% (1.9)], reflex [71.8% (7.9)], and flush [83.6% (3.8)] tear samples. However, the mole % of phospholipids in reflex [27.5% (8.1)] and flush [15.8% (3.8)] tear samples was higher (p = 0.005) than that in basal tears [5.4% (2.0)]. CONCLUSIONS: Flush or reflex tears did not have similar lipid profiles in either concentration or in mole % to basal tears. It is recommended that basal tears are used for tear lipid analysis as the reflex or flush tears contain very low levels of most lipid components.
Assuntos
Lipídeos/análise , Manejo de Espécimes , Lágrimas/química , Adulto , Voluntários Saudáveis , Humanos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
PURPOSE: Alterations in tear film lipids may be important in modulating discomfort during contact lens wear. This study investigates associations between clinical and biological components of the lipid layer and seeks to determine the effect of lipid supplementation on contact lens wear comfort. METHODS: Participants were grouped into symptomatic (n = 10) and asymptomatic (n = 10) contact lens wearers according to the Contact Lens Dry Eye Questionnaire. After 6 hours of lens wear, noninvasive surface drying time (NISDT) and the lipid layer grade were assessed using a Tearscope. Basal tears were collected using a microcapillary tube and assayed for concentration and activity of the secretory phospholipase A2 enzyme and concentration of the lipid aldehyde malondialdehyde. Mass spectrometry was used to characterize the tear lipidome. In the second phase, a liposomal spray (Tears Again, BioRevive) or a saline spray was sprayed over the upper eyelids of each subject during their down gaze and during lens wear. Noninvasive surface drying time and ocular comfort were obtained soon after spraying and again at 2 and 6 hours after the initial spray. Statistical tests included the Student t test, repeated-measures analysis of variance, and the Pearson correlation test where appropriate. RESULTS: Noninvasive surface drying time was lower (p = 0.01) in symptomatic (4.5 ± 0.6 seconds) than in asymptomatic (9.9 ± 3.1 seconds) contact lens wearers. The mole percentage of wax esters in the total lipidome increased with NISDT (R = 0.70, p = 0.01). Secretory phospholipase A2 enzyme activity in tears was associated with higher levels of malondialdehyde (R = 0.65, p = 0.01) and shorter NISDT (R = 0.84, p = 0.001). Noninvasive surface drying time reduced over the time course for the saline spray (p = 0.01) but did not reduce until the 6-hour time point with the liposomal spray. With liposomal spray, NISDT was higher (p = 0.03) immediately after instillation compared with 6 hours later (9.5 ± 1.9 vs. 5.2 ± 2.1 seconds). A longer NISDT was associated with improved ocular comfort for those using the liposomal spray (R = 0.25, p = 0.005) but not with saline. CONCLUSIONS: Degraded lipids and a lower mole percentage of wax esters in the tear film may be associated with a lower NISDT. Lipid supplements may improve ocular comfort during lens wear by increasing NISDT.
Assuntos
Lentes de Contato Hidrofílicas , Lipídeos/análise , Malondialdeído/análise , Fosfolipases A2 Secretórias/análise , Lágrimas/química , Adulto , Feminino , Humanos , Lubrificantes Oftálmicos/administração & dosagem , Masculino , Espectrometria de Massas , Inquéritos e Questionários , Adulto JovemRESUMO
F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalytic mechanism and isolated F1-ATPase subcomplexes can also hydrolyze ATP to generate rotation of their central γ rotor subunit. As ATP is hydrolyzed, the F1-ATPase cycles through a series of conformational states that mediates unidirectional rotation of the rotor. However, even in the absence of a rotor, the α and ß subunits are still able to pass through a series of conformations, akin to those that generate rotation. Here, we use cryoelectron microscopy to establish the structures of these rotorless states. These structures indicate that cooperativity in this system is likely mediated by contacts between the ß subunit lever domains, irrespective of the presence of the γ rotor subunit. These findings provide insight into how long-range information may be transferred in large biological systems.
Assuntos
Adenosina Trifosfatases , Trifosfato de Adenosina , Hidrólise , Microscopia Crioeletrônica , Subunidades Proteicas/química , Conformação Proteica , RotaçãoRESUMO
F1Fo ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F1-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central γ rotor subunit and the opening and closing of the catalytic ß subunits. Cooperativity in F1-ATPase has long thought to be conferred through the γ subunit, with three key interaction sites between the γ and ß subunits being identified. Single molecule studies have demonstrated that the F1 complexes lacking the γ axle still "rotate" and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F1-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less γ and the open ß subunit suggests that the complete γ subunit is important for coordinating efficient ATP binding of F1-ATPase.
RESUMO
Dietary intake of long-chain fatty acids (LCFAs) plays a causative role in insulin resistance and risk of diabetes. Whereas LCFAs promote lipid accumulation and insulin resistance, diets rich in medium-chain fatty acids (MCFAs) have been associated with increased oxidative metabolism and reduced adiposity, with few deleterious effects on insulin action. The molecular mechanisms underlying these differences between dietary fat subtypes are poorly understood. To investigate this further, we treated C2C12 myotubes with various LCFAs (16:0, 18:1n9, and 18:2n6) and MCFAs (10:0 and 12:0), as well as fed mice diets rich in LCFAs or MCFAs, and investigated fatty acid-induced changes in mitochondrial metabolism and oxidative stress. MCFA-treated cells displayed less lipid accumulation, increased mitochondrial oxidative capacity, and less oxidative stress than LCFA-treated cells. These changes were associated with improved insulin action in MCFA-treated myotubes. MCFA-fed mice exhibited increased energy expenditure, reduced adiposity, and better glucose tolerance compared with LCFA-fed mice. Dietary MCFAs increased respiration in isolated mitochondria, with a simultaneous reduction in reactive oxygen species generation, and subsequently low oxidative damage. Collectively our findings indicate that in contrast to LCFAs, MCFAs increase the intrinsic respiratory capacity of mitochondria without increasing oxidative stress. These effects potentially contribute to the beneficial metabolic actions of dietary MCFAs.
Assuntos
Ácidos Graxos/química , Ácidos Graxos/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Gorduras na Dieta/farmacologia , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Tolerância a Glucose , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/citologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The deposition of biological material (biofouling) onto polymeric contact lenses is thought to be a major contributor to lens discomfort and hence discontinuation of wear. We describe a method to characterize lipid deposits directly from worn contact lenses utilizing liquid extraction surface analysis coupled to tandem mass spectrometry (LESA-MS/MS). This technique effected facile and reproducible extraction of lipids from the contact lens surfaces and identified lipid molecular species representing all major classes present in human tear film. Our data show that LESA-MS/MS is a rapid and comprehensive technique for the characterization of lipid-related biofouling on polymer surfaces.
Assuntos
Lentes de Contato , Lipídeos/isolamento & purificação , Espectrometria de Massas em Tandem , Humanos , Propriedades de Superfície , Espectrometria de Massas em Tandem/métodos , Lágrimas/químicaRESUMO
F1Fo ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the Fo motor that is transferred to the F1 motor to catalyze ATP production, with flexible F1/Fo coupling required for efficient catalysis. F1Fo ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory ε subunit. Here we present cryo-EM data showing E. coli F1Fo ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory ε subunit induce torsional movement in the central stalk, thereby enabling its rotation within the Fο motor. This highlights the importance of the central rotor for flexible coupling of the F1 and Fo motors and provides further insight into the regulatory mechanism mediated by subunit ε.
Assuntos
Trifosfato de Adenosina , Escherichia coliRESUMO
Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.
Assuntos
Proteínas de Transporte de Cátions Orgânicos , Transportador 1 de Cátions Orgânicos , Humanos , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/química , Transportador 1 de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/química , Transporte Biológico , Transportador 2 de Cátion Orgânico/metabolismoRESUMO
Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Feminino , Animais , Camundongos , Anticorpos Amplamente Neutralizantes , Leucócitos Mononucleares , Anticorpos Antivirais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Epitopos , Glicoproteína da Espícula de Coronavírus/genética , Testes de NeutralizaçãoRESUMO
Recent developments in analytical technologies have driven significant advances in lipid science. The sensitivity and selectivity of modern mass spectrometers can now provide for the detection and even quantification of many hundreds of lipids in a single analysis. In parallel, increasing evidence from structural biology suggests that a detailed knowledge of lipid molecular structure including carbon-carbon double bond position, stereochemistry and acyl chain regiochemistry is required to fully appreciate the biochemical role(s) of individual lipids. Here we review the capabilities and limitations of tandem mass spectrometry to provide this level of structural specificity in the analysis of lipids present in complex biological extracts. In particular, we focus on the capabilities of a novel technology termed ozone-induced dissociation to identify the position(s) of double bonds in unsaturated lipids and discuss its possible role in efforts to develop workflows that provide for complete structure elucidation of lipids by mass spectrometry alone: so-called top-down lipidomics.