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1.
Nat Struct Mol Biol ; 27(3): 249-259, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32157247

RESUMO

Aggregation of human α-synuclein (αSyn) is linked to Parkinson's disease (PD) pathology. The central region of the αSyn sequence contains the non-amyloid ß-component (NAC) crucial for aggregation. However, how NAC flanking regions modulate αSyn aggregation remains unclear. Using bioinformatics, mutation and NMR, we identify a 7-residue sequence, named P1 (residues 36-42), that controls αSyn aggregation. Deletion or substitution of this 'master controller' prevents aggregation at pH 7.5 in vitro. At lower pH, P1 synergises with a sequence containing the preNAC region (P2, residues 45-57) to prevent aggregation. Deleting P1 (ΔP1) or both P1 and P2 (ΔΔ) also prevents age-dependent αSyn aggregation and toxicity in C. elegans models and prevents αSyn-mediated vesicle fusion by altering the conformational properties of the protein when lipid bound. The results highlight the importance of a master-controller sequence motif that controls both αSyn aggregation and function-a region that could be targeted to prevent aggregation in disease.


Assuntos
Neurônios/química , Doença de Parkinson/metabolismo , Agregados Proteicos , alfa-Sinucleína/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans , Clonagem Molecular , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fosfatidilserinas/química , Multimerização Proteica , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
2.
Int J Mol Sci ; 20(18)2019 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-31500345

RESUMO

BACKGROUND: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. METHODS: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. RESULTS: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. CONCLUSIONS: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteolipídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transporte Biológico , Fibroblastos , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/isolamento & purificação , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Riboflavina/metabolismo , Relação Estrutura-Atividade
3.
Elife ; 82019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31339488

RESUMO

The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation.


Assuntos
Antiporters/metabolismo , Antiporters/ultraestrutura , Cloretos/metabolismo , Microscopia Crioeletrônica , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/ultraestrutura , Animais , Antiporters/química , Sítios de Ligação , Células HEK293 , Humanos , Transporte de Íons , Camundongos , Modelos Moleculares , Domínios Proteicos , Proteolipídeos/metabolismo , Eletricidade Estática , Especificidade por Substrato , Transportadores de Sulfato/química
4.
Protein J ; 38(3): 229-235, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31144202

RESUMO

To identify the translocation components in cells, and to understand how they function in protein transport and membrane insertion, a variety of techniques have been used such as genetics, biochemistry, structural biology and single molecule methods. In particular, site-directed crosslinking between the client proteins and components of the translocation machineries have contributed significantly in the past and will do so in the future. One advantage of this technology is that it can be applied in vivo as well as in vitro and a comparison of the two approaches can be made. Also, the in vivo techniques allow time-dependent protocols which are essential for studying cellular pathways. Protein purification and reconstitution into proteoliposomes are the gold standard for studying membrane-based transport and translocation systems. With these biochemically defined approaches the function of each component in protein transport can be addressed individually with a plethora of biophysical techniques. Recently, the use of nanodiscs for reconstitution has added another extension of this reductionistic approach. Fluorescence based studies, cryo-microscopy and NMR spectroscopy have significantly added to our understanding how proteins move into and across membranes and will do this also in future.


Assuntos
Sistemas de Translocação de Proteínas/química , Transporte Proteico , Bactérias/metabolismo , Microscopia Crioeletrônica/métodos , Eucariotos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Espectroscopia de Ressonância Magnética/métodos , Microscopia de Fluorescência/métodos , Proteolipídeos/metabolismo , Imagem Individual de Molécula/métodos
5.
Chem Biol Drug Des ; 93(5): 712-723, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30737896

RESUMO

AgrC, as an integral membrane receptor protein with histidine kinase activity, is an important component of the agr quorum-sensing system of Staphylococcus aureus. AgrC acts as a sensor for the recognition of environmental signals and transduction of the signals into the cytoplasm. Therefore, AgrC is considered to be a compelling target for the development of novel quorum-sensing inhibitors. Here, we constructed a proteoliposome-based model for screening inhibitors targeting AgrC by incorporating AgrC into liposomes. We demonstrated that the dissolution state of the liposome was a critical factor in the reconstruction of the AgrC proteoliposome, in which AgrC maintained similar orientation and function as those in natural biological membranes. Two monomers, namely, rhein and aloeemodin, were successfully screened out as inhibitors targeting AgrC by the proteoliposome-based model from 14 traditional Chinese medicine monomers. The inhibitory effects of these compounds on the growth of suspended bacteria was dose dependent, and subinhibitory concentrations of these compounds significantly reduced the expression of three virulence factors (hla, clfA, and clpP), that are regulated by the agr system. The results preliminarily indicated that rhein and aloeemodin can inhibit the agr signaling pathway and also indirectly confirmed the feasibility and effectiveness of the AgrC proteoliposome as a drug screening model.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteolipídeos/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Antraquinonas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Emodina/química , Emodina/metabolismo , Emodina/farmacologia , Regulação Bacteriana da Expressão Gênica , Medicina Tradicional Chinesa , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/fisiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
6.
Methods Mol Biol ; 1949: 181-199, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790257

RESUMO

The distribution of different lipid species between the two leaflets is tightly regulated and underlies the concerted action of distinct catalytic entities. While flippases and floppases establish membrane asymmetry, scramblases randomize the lipid distribution and play pivotal roles during blood clotting, apoptosis, and in processes such as N-linked glycosylation of proteins. The recent discovery of TMEM16 family members acting as scramblases has led to an increasing demand for developing protocols tailored for TMEM16 proteins to enable functional investigations of their scrambling activity. Here we describe a protocol for the expression, purification, and functional reconstitution of TMEM16 proteins into preformed liposomes and measurement of their scrambling activity using fluorescence-labeled lipid derivatives. The reconstitution involves extrusion of liposomes through a membrane, destabilization of liposomes using Triton X-100, and stepwise detergent removal by adsorption on styryl-beads. The scrambling assay is based on the selective bleaching of nitrobenzoxadiazol fluorescent lipids on the outer leaflet of liposomes by the membrane-impermeant reducing agent sodium dithionite. The assay allows conclusions on the substrate specificity and on the kinetics of the transported lipids as shown with the example of a Ca2+-activated TMEM16 scramblase from the fungus Nectria haematococca (nhTMEM16).


Assuntos
Anoctaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteolipídeos/metabolismo , Anoctaminas/isolamento & purificação , Proteínas de Transferência de Fosfolipídeos/isolamento & purificação , Proteolipídeos/química
7.
Structure ; 27(4): 669-678.e5, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30799077

RESUMO

PglK is a lipid-linked oligosaccharide (LLO) flippase essential for asparagine-linked protein glycosylation in Campylobacter jejuni. Previously we have proposed a non-alternating-access LLO translocation mechanism, where postulated outward-facing states play a primary role. To investigate this unusual mechanistic proposal, we have determined a high-resolution structure of PglK that displays an outward semi-occluded state with the two nucleotide binding domains forming an asymmetric closed dimer with two bound ATPγS molecules. Based on this structure, we performed extensive molecular dynamics simulations to investigate LLO recognition and flipping. Our results suggest that PglK may employ a "substrate-hunting" mechanism to locally increase the LLO concentration and facilitate its jump into the translocation pathway, for which sugars from the LLO head group are essential. We further conclude that the release of LLO to the outside occurs before ATP hydrolysis and is followed by the closing of the periplasmic cavity of PglK.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/química , Campylobacter jejuni/química , Glicosiltransferases/química , Lipopolissacarídeos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hidrólise , Cinética , Lipopolissacarídeos/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica
8.
J Cell Sci ; 132(4)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670475

RESUMO

The endoplasmic reticulum (ER) is a major membrane-bound organelle in all eukaryotic cells. This organelle comprises morphologically distinct domains, including the nuclear envelope and peripheral sheets and tubules. The tubules are connected by three-way junctions into a network. Several membrane proteins have been implicated in network formation; curvature-stabilizing proteins generate the tubules themselves, and membrane-anchored GTPases fuse tubules into a network. Recent experiments have shown that a tubular network can be formed with reconstituted proteoliposomes containing the yeast membrane-fusing GTPase Sey1 and a curvature-stabilizing protein of either the reticulon or REEP protein families. The network forms in the presence of GTP and is rapidly disassembled when GTP hydrolysis of Sey1 is inhibited, indicating that continuous membrane fusion is required for its maintenance. Atlastin, the ortholog of Sey1 in metazoans, forms a network on its own, serving both as a fusion and curvature-stabilizing protein. These results show that the reticular ER can be generated by a surprisingly small set of proteins, and represents an energy-dependent steady state between formation and disassembly. Models for the molecular mechanism by which curvature-stabilizing proteins cooperate with fusion GTPases to form a reticular network have been proposed, but many aspects remain speculative, including the function of additional proteins, such as the lunapark protein, and the mechanism by which the ER interacts with the cytoskeleton. How the nuclear envelope and peripheral ER sheets are formed remain major unresolved questions in the field. Here, we review reconstitution experiments with purified curvature-stabilizing proteins and fusion GTPases, discuss mechanistic implications and point out open questions.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteolipídeos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Fenômenos Biomecânicos , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Fusão de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Membrana Nuclear/ultraestrutura , Proteolipídeos/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo
9.
Methods Mol Biol ; 1925: 59-63, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30674016

RESUMO

The mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uniporter dominant negative b- subunit (MCUb) are pore-forming components of the uniporter complex. We expressed these MCU subunits in cell-free transcription/translation systems, and we studied them, at the single molecule level, using the electrophysiological technique of planar lipid bilayer.We showed that MCU gives rise to single-channel Ca2+ currents. In contrast, MCUb alone does not display calcium-permeable channel activity, while the co-expression of MCUb:MCU drastically alters the calcium permeation mediated by MCU subunit.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Bicamadas Lipídicas/metabolismo , Animais , Sinalização do Cálcio , Cátions Monovalentes/metabolismo , Humanos , Transporte de Íons , Subunidades Proteicas/metabolismo , Proteolipídeos/metabolismo
10.
Methods Mol Biol ; 1925: 65-73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30674017

RESUMO

Numerous researchers tried to identify the key players of calcium signaling in mitochondria using molecular and cell biology techniques for more than five decades. However, only an integrated approach involving also electrophysiological techniques has finally allowed to define the components of the protein complex responsible for the uptake of this ion into mitochondria.Here we describe the protocol used for the electrophysiological characterization of the mitochondrial calcium uniporter (MCU) complex: the following outline indicates step-by-step the setup of planar lipid bilayer experiments.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Bicamadas Lipídicas/metabolismo , Animais , Sinalização do Cálcio , Humanos , Proteolipídeos/metabolismo
11.
Microbiol Spectr ; 7(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30681067

RESUMO

Outer membrane vesicles (OMVs) are nanosized proteoliposomes derived from the outer membrane of Gram-negative bacteria. They are ubiquitously produced both in culture and during infection and are now recognized to play crucial roles during host-microbe interactions. OMVs can transport a broad range of chemically diverse cargoes, including lipids and lipopolysaccharides, membrane-embedded and associated proteins and small molecules, peptidoglycan, and nucleic acids. Particularly, virulence factors such as adhesins and toxins are often enriched in OMVs. Here we discuss a variety of ways in which OMVs facilitate host-microbe interactions, including their contributions to biofilm formation, nutrient scavenging, and modulation of host cell function. We particularly examine recent findings regarding OMV-host cell interactions in the oral cavity and the gastrointestinal tract.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Bactérias Gram-Negativas/metabolismo , Proteolipídeos/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Transporte Biológico/fisiologia , Bactérias Gram-Negativas/imunologia , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Proteolipídeos/imunologia
12.
Am J Physiol Endocrinol Metab ; 316(3): E432-E442, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30601702

RESUMO

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump is a major contributor to skeletal muscle Ca2+ homeostasis and metabolic rate. SERCA activity can become adaptively uncoupled by its regulator sarcolipin (SLN) to increase the energy demand of Ca2+ pumping, preventing excessive obesity and glucose intolerance in mice. Several other SERCA regulators bear structural and functional resemblance to SLN, including phospholamban (PLN). Here, we sought to examine whether endogenous levels of skeletal muscle PLN control SERCA Ca2+ pumping efficiency and whole body metabolism. Using PLN-null mice ( Pln-/-), we found that soleus (SOL) muscle's SERCA pumping efficiency (measured as an apparent coupling ratio: Ca2+ uptake/ATP hydrolysis) was unaffected by PLN. Expression of Ca2+-handling proteins within the SOL, including SLN, were comparable between Pln-/- and wild-type (WT) littermates, as were fiber-type characteristics. Not surprisingly then, Pln-/- mice developed a similar degree of diet-induced obesity and glucose intolerance as WT controls when given a "Western" high-fat diet. Lack of an excessively obesogenic phenotype of Pln-/- could not be explained by compensation from skeletal muscle SLN or brown adipose tissue uncoupling protein-1 content. In agreement with several other reports, our study lends support to the notion that PLN serves a functionally distinct role from that of SLN in skeletal muscle physiology.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Intolerância à Glucose/genética , Músculo Esquelético/metabolismo , Obesidade/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Dieta Ocidental , Intolerância à Glucose/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/metabolismo , Obesidade/metabolismo , Proteolipídeos/metabolismo , Proteína Desacopladora 1/metabolismo
13.
Prog Lipid Res ; 73: 92-100, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30611882

RESUMO

To study membrane fusion mediated by synaptic proteins, proteoliposomes have been widely used for in vitro ensemble measurements with limited insights into the fusion mechanism. Single-particle techniques have proven to be powerful in overcoming the limitations of traditional ensemble methods. Here, we summarize current single-particle methods in biophysical and biochemical studies of fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and other synaptic proteins, together with their advantages and limitations.


Assuntos
Fusão de Membrana , Transferência Ressonante de Energia de Fluorescência , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Imagem Individual de Molécula , Transmissão Sináptica , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo
14.
Neurosci Lett ; 696: 13-19, 2019 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-30528880

RESUMO

Hibernation in mammals is a whole-body phenotype that involves profound reductions in oxygen consumption, metabolic reactions, core body temperature, neural activity and heart rate. An important aspect of mammalian hibernation is the ability to reverse this state of hypothermic torpor by rewarming and subsequent arousal. Brown adipose tissue (BAT) and skeletal muscle shivering have been characterized as the predominant driving forces for thermogenesis during arousal. Conversely, the thermogenic contribution of these organs needs to be minimized as hibernating mammals enter torpor. Because skeletal muscle accounts for approximately 40% of the dry mass of the typical mammalian body, we aim to broaden the spotlight to include the importance of down-regulating skeletal muscle non-shivering thermogenesis during hibernation to allow for whole-body cooling and long-term maintenance of a depressed core body temperature when the animal is in torpor. This minireview will briefly describe the current understanding of thermoregulation in hibernating mammals and present new preliminary data on the importance of skeletal muscle and the micro-peptide sarcolipin as a major thermogenic target.


Assuntos
Regulação para Baixo , Hibernação/fisiologia , Temperatura Alta , Hipotermia/metabolismo , Proteínas Musculares/metabolismo , Proteolipídeos/metabolismo , Animais , Humanos , Músculo Esquelético/metabolismo
15.
J Bone Miner Metab ; 37(4): 607-613, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30324534

RESUMO

Tissue-nonspecific alkaline phosphatase (TNAP), a glycosylphosphatidylinositol-anchored ectoenzyme present on the membrane of matrix vesicles (MVs), hydrolyzes the mineralization inhibitor inorganic pyrophosphate as well as ATP to generate the inorganic phosphate needed for apatite formation. Herein, we used proteoliposomes harboring TNAP as MV biomimetics with or without nucleators of mineral formation (amorphous calcium phosphate and complexes with phosphatidylserine) to assess the role of the MVs' membrane lipid composition on TNAP activity by means of turbidity assay and FTIR analysis. We found that TNAP-proteoliposomes have the ability to induce mineralization even in the absence of mineral nucleators. We also found that the addition of cholesterol or sphingomyelin to TNAP-proteoliposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine reduced the ability of TNAP to induce biomineralization. Our results suggest that the lipid microenvironment is essential for the induction and propagation of minerals mediated by TNAP.


Assuntos
Fosfatase Alcalina/metabolismo , Calcificação Fisiológica , Microambiente Celular , Lipídeos/química , Proteolipídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Difusão Dinâmica da Luz , Humanos , Hidrólise , Cinética , Espectroscopia de Infravermelho com Transformada de Fourier
16.
Mol Pharmacol ; 95(2): 169-182, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30409791

RESUMO

Organic cation transporters OCT1 (SLC22A1) and OCT2 (SLC22A2) are critically involved in absorption and excretion of diverse cationic drugs. Because drug-drug interactions at these transporters may induce adverse drug effects in patients, in vitro testing during drug development for interaction with the human transporters is mandatory. Recent data performed with rat OCT1 (rOCT1) suggest that currently performed in vitro tests assuming one polyspecific binding site are insufficient. Here we measured the binding and transport of model substrate 1-methyl-4-phenylpyridinium+ (MPP+) by cell-free-expressed fusion proteins of rOCT1 and rOCT1 mutants with green fluorescent protein that had been reconstituted into nanodiscs or proteoliposomes. The nanodiscs were formed with major scaffold protein (MSP) and different phospholipids, whereas the proteoliposomes were formed with a mixture of cholesterol, phosphatidylserine, and phosphatidylcholine. In nanodiscs formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or cholesterol, phosphatidylserine, and phosphatidylcholine, two low-affinity MPP+ binding sites and one high-affinity MPP+ binding site per transporter monomer were determined. Mutagenesis revealed that tryptophan 218 and aspartate 475 in neighboring positions in the modeled outward-open cleft contribute to one low-affinity binding site, whereas arginine 440 located distantly in the cleft is critical for MPP+ binding to another low-affinity site. Comparing MPP+ binding with MPP+ transport suggests that the low-affinity sites are involved in MPP+ transport, whereas high-affinity MPP+ binding influences transport allosterically. The data will be helpful in the interpretation of future crystal structures and provides a rationale for future in vitro testing that is more sophisticated and reliable, leading to the generation of pharmacophore models with high predictive power.


Assuntos
1-Metil-4-fenilpiridínio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Animais , Sítios de Ligação , Proteínas de Fluorescência Verde/metabolismo , Mutagênese/fisiologia , Fosfolipídeos/metabolismo , Proteolipídeos/metabolismo , Ratos
17.
Methods Mol Biol ; 1860: 53-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317498

RESUMO

Dynamic light scattering (DLS) spectroscopy provides rapid information on the size distribution of a large number of particles in a mixture. Vesicle sizes change during the merger of lipid bilayers, and DLS analysis can provide rapid, accurate, and non-perturbative quantification of the size distribution of proteoliposomes in SNARE-dependent membrane fusion. In this chapter, we describe the methodologies and reagents used for DLS spectroscopy in a biochemical and biophysical study of SNARE-mediated membrane fusion.


Assuntos
Difusão Dinâmica da Luz/métodos , Fusão de Membrana , Proteínas SNARE/metabolismo , Difusão Dinâmica da Luz/instrumentação , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas SNARE/química , Proteínas SNARE/isolamento & purificação , Software
18.
Methods Mol Biol ; 1860: 145-159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317502

RESUMO

Force spectroscopy allows the manipulation of single molecules and the characterization of their properties and interactions thereby rendering it a powerful tool for biological sciences. Force spectroscopy at the level of individual molecules requires force resolution in the piconewton regime as achieved by optical tweezers (OT), magnetic tweezers (MT), and atomic force microscopy (AFM) with AFM providing the largest force range from tenth of piconewton to several micronewton. In membrane probe spectroscopy the commonly used sharp cantilever tip is replaced by a lipid-coated glass sphere. This technique expands the scope of force spectroscopy to processes at and between lipid bilayers, like the formation of coiled coils between SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins as well as subsequent membrane fusion. To this end, two solid-supported membranes equipped with SNARE proteins or fusion peptides are separately deposited on a flat glassy surface and on a micrometer glass sphere attached to the end of a tipless AFM cantilever. These two membranes are rapidly brought into contact until a defined force is reached. The AFM deflection readout is used to monitor the distance between the two bilayers, which allows to observe and identify fusion processes of the two lipid membranes, while the forces needed to separate the two surfaces give insights into the formation of SNARE complexes. By changing the contact pressure one can access fusion kinetics and to some extent reconstruct the energy landscape of membrane fusion. In this chapter we describe the preparation of membrane-coated colloidal probes attached to AFM cantilevers, experimental procedures, and necessary data analysis to perform membrane probe spectroscopy in the presence of fusogenic peptides or proteins.


Assuntos
Bicamadas Lipídicas/metabolismo , Fusão de Membrana , Proteínas SNARE/metabolismo , Análise Espectral/métodos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Pinças Ópticas , Domínios Proteicos , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas SNARE/química , Análise Espectral/instrumentação
19.
Methods Mol Biol ; 1860: 303-322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317514

RESUMO

Membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-family proteins is an essential process for intracellular membrane trafficking in all eukaryotic cells, which delivers proteins and lipids to their appropriate subcellular membrane compartments such as organelles and plasma membrane. The molecular basis of SNARE-mediated membrane fusion has been revealed by studying fusion of reconstituted proteoliposomes bearing purified SNARE-family proteins and chemically defined lipid species. This chapter describes the detailed experimental protocols for (1) purification of recombinant SNARE-family and SM (Sec1/Munc18-family) proteins in the yeast Saccharomyces cerevisiae; (2) preparation of reconstituted proteoliposomes bearing purified yeast SNARE proteins; and (3) developing an assay to monitor lipid mixing between reconstituted SNARE-bearing proteoliposomes. Lipid mixing assays for reconstituted SNARE-bearing proteoliposomes are useful for evaluating the intrinsic capacity of SNARE-family proteins to directly catalyze membrane fusion and to determine the specificity of membrane fusion.


Assuntos
Proteolipídeos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Corantes Fluorescentes/química , Lipossomos/química , Lipossomos/metabolismo , Fusão de Membrana , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ligação Proteica , Proteolipídeos/química , Proteolipídeos/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas SNARE/química , Proteínas SNARE/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
20.
J Gene Med ; 20(12): e3060, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30393908

RESUMO

BACKGROUND: Cardiac gene therapy using the adeno-associated virus serotype 9 vector is widely used because of its efficient transduction. However, the promoters used to drive expression often cause off-target localization. To overcome this, studies have applied cardiac-specific promoters, although expression is debilitated compared to that of ubiquitous promoters. To address these issues in the context of atrial-specific gene expression, an enhancer calsequestrin cis-regulatory module 4 (CRM4) and the highly atrial-specific promoter sarcolipin were combined to enhance expression and minimize off tissue expression. METHODS: To observe expression and bio-distribution, constructs were generated using two different reporter genes: luciferase and enhanced green fluorescent protein (EGFP). The ubiquitous cytomegalovirus (CMV), sarcolipin (SLN) and CRM4 combined with sarcolipin (CRM4.SLN) were compared and analyzed using the luciferase assay, western blotting, a quantitative polymerase chain reaction and fluorescence imaging. RESULTS: The CMV promoter containing vectors showed the strongest expression in vitro and in vivo. However, the module SLN combination showed enhanced atrial expression and a minimized off-target effect even when compared with the individual SLN promoter. CONCLUSIONS: For gene therapy involving atrial gene transfer, the CRM4.SLN combination is a promising alternative to the use of the CMV promoter. CRM4.SLN had significant atrial expression and minimized extra-atrial expression.


Assuntos
Calsequestrina/genética , Regulação da Expressão Gênica , Átrios do Coração/metabolismo , Proteínas Musculares/genética , Regiões Promotoras Genéticas/genética , Proteolipídeos/genética , Animais , Calsequestrina/metabolismo , Citomegalovirus/genética , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Proteínas Musculares/metabolismo , Proteolipídeos/metabolismo , Transfecção
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