An Original Approach to Measure Ligand/receptor Binding Affinity in Non-purified Samples

Several biochemical and biophysical methods are available to determine dissociation constants between a biological target and its ligands. Most of them require purification, labelling or surface immobilisation. However, these measurements remain challenging concerning membrane proteins because purification requires their extraction from the native lipid environment using different approaches, a process that may impact receptor conformation and functionality. We have developed a novel experimental procedure to determine binding affinities of a ligand to a membrane protein, the dopamine D2 receptor (D2R), directly from cell membrane fragments, using microscale thermophoresis (MST). Two main challenges had to be overcome: to determine the concentration of dopamine D2R in the crude sample; to find ways to minimize or account for non-specific binding of the ligand to cell fragments. Using MST, we were able to determine the D2R concentration in cell membrane fragments to be about 36.8 ± 2.6 pmol/mg. Then titration curves allowed the determination of a KD about 5.3 ± 1.7 nM, that is very close to the reported value. Important details of the experimental procedure are detailed to allow the transposition of this novel method to various membrane proteins.

Structures of the human peroxisomal fatty acid transporter ABCD1 in a lipid environment

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to fatty acid catabolism and lipid biosynthesis. Its dysfunction underlies toxic cytosolic accumulation of VLCFAs, progressive demyelination, and neurological impairments including X-linked adrenoleukodystrophy (X-ALD). We present cryo-EM structures of ABCD1 in phospholipid nanodiscs in a nucleotide bound conformation open to the peroxisomal lumen and an inward facing conformation open to the cytosol at up to 3.5 Å resolution, revealing details of its transmembrane cavity and ATP dependent conformational spectrum. We identify features distinguishing ABCD1 from its closest homologs and show that coenzyme A (CoA) esters of VLCFAs modulate ABCD1 activity in a species dependent manner. Our data suggest a transport mechanism where the CoA moieties of VLCFA-CoAs enter the hydrophilic transmembrane domain while the acyl chains extend out into the surrounding membrane bilayer. The structures help rationalize disease causing mutations and may aid ABCD1 targeted structure-based drug design.

Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme's catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.

Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.

OSBP-mediated cholesterol transfer determines epithelial polarity and associated cargo secretion

Golgi lipid environment regulates sorting and cargo secretion. However, the mechanisms that spatiotemporally control the lipid composition of the secretory membranes to drive cargo trafficking are poorly understood. Lipid transfer proteins regulate the concentration of specific lipids at membrane contact sites. We hypothesised that by catalysing cholesterol/PI(4)P exchange at ER-trans-Golgi membrane contact sites the lipid transfer protein oxysterol binding protein (OSBP) affects the secretion of a subset of cargoes. Here, we report that OSBP is a major epithelial protein as its inhibition leads to complete loss of apico-basal polarity. By mapping the OSBP proximity proteome with the biotin ligase TurboID, we found that OSBP controls the secretion of multiple membrane associated proteins, including key polarity determinants such as E-cadherin. Mechanistically, we established that OSBP contributes to E-cadherin secretion by supplying cholesterol to post-Golgi membranes. Importantly, when cells downregulate cell-cell junctions upon epithelial-to-mesenchymal transition, they re-wire their lipid homeostasis and downregulate OSBP as well, thus altering the trafficking of the OSBP-dependent secretory cargoes.

Revisiting the contribution of mitochondrial biology to the pathophysiology of skeletal muscle insulin resistance

While the etiology of type 2 diabetes is multifaceted, the induction of insulin resistance in skeletal muscle is a key phenomenon, and impairments in insulin signaling in this tissue directly contribute to hyperglycemia. Despite the lack of clarity regarding the specific mechanisms whereby insulin signaling is impaired, the key role of a high lipid environment within skeletal muscle has been recognized for decades. Many of the proposed mechanisms leading to the attenuation of insulin signaling — namely the accumulation of reactive lipids and the pathological production of reactive oxygen species (ROS), appear to rely on this high lipid environment. Mitochondrial biology is a central component to these processes, as these organelles are almost exclusively responsible for the oxidation and metabolism of lipids within skeletal muscle and are a primary source of ROS production. Classic studies have suggested that reductions in skeletal muscle mitochondrial content and/or function contribute to lipid-induced insulin resistance; however, in recent years the role of mitochondria in the pathophysiology of insulin resistance has been gradually re-evaluated to consider the biological effects of alterations in mitochondrial content. In this respect, while reductions in mitochondrial content are not required for the induction of insulin resistance, mechanisms that increase mitochondrial content are thought to enhance mitochondrial substrate sensitivity and submaximal adenosine diphosphate (ADP) kinetics. Thus, this review will describe the central role of a high lipid environment in the pathophysiology of insulin resistance, and present both classic and contemporary views of how mitochondrial biology contributes to insulin resistance in skeletal muscle.

Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers

Ion channels are membrane proteins that play important roles in a wide range of fundamental cellular processes. Studying membrane proteins at a molecular level becomes challenging in complex cellular environments. Instead, many studies focus on the isolation and reconstitution of the membrane proteins into model lipid membranes. Such simpler, in vitro, systems offer the advantage of control over the membrane and protein composition and the lipid environment. Rhodopsin and rhodopsin-like ion channels are widely studied due to their light-interacting properties and are a natural candidate for investigation with fluorescence methods. Here we review techniques for synthesizing liposomes and for reconstituting membrane proteins into lipid bilayers. We then summarize fluorescence assays which can be used to verify the functionality of reconstituted membrane proteins in synthetic liposomes.

Hepatocyte cholesterol content modulates glucagon receptor signalling

Glucagon decreases liver fat, and non-alcoholic fatty liver disease (NAFLD) is associated with hepatic glucagon resistance. Increasingly it is recognised that the function of G protein-coupled receptors can be regulated by their local plasma membrane lipid environment. The aim of this study was to evaluate the effects of experimentally modulating hepatocyte cholesterol content on the function of the glucagon receptor (GCGR). We found that glucagon-mediated cAMP production is inversely proportional to cholesterol content of human hepatoma and primary mouse hepatocytes after treatment with cholesterol-depleting and loading agents, with ligand internalisation showing the opposite trend. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Molecular dynamics simulations identified potential membrane-exposed cholesterol binding sites on the GCGR. Overall, our data suggest that increased hepatocyte membrane cholesterol could directly contribute to glucagon resistance in NAFLD.

Cryo-EM Structure of Mechanosensitive Channel YnaI Using SMA2000: Challenges and Opportunities

Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, the complete structure of the transmembrane domain of YnaI was not resolved. This reveals a significant limitation of SMA2000 or similar membrane-active copolymers. This limitation may come from the heterogeneity of the polymers and nonspecific interactions between the polymers and the relatively large hydrophobic pockets within the transmembrane domain of YnaI. However, this limitation offers development opportunities for detergent-free technology for challenging membrane proteins.