Showing papers in "Molecular Membrane Biology in 2013"
TL;DR: This review will discuss examples of vertebrate membrane protein overexpression in mammalian cells using a variety of viral, constitutive or inducible expression systems.
Abstract: The number of structures of integral membrane proteins from higher eukaryotes is steadily increasing due to a number of innovative protein engineering and crystallization strategies devised over the last few years. However, it is sobering to reflect that these structures represent only a tiny proportion of the total number of membrane proteins encoded by a mammalian genome. In addition, the structures determined to date are of the most tractable membrane proteins, i.e., those that are expressed functionally and to high levels in yeast or in insect cells using the baculovirus expression system. However, some membrane proteins that are expressed inefficiently in these systems can be produced at sufficiently high levels in mammalian cells to allow structure determination. Mammalian expression systems are an under-used resource in structural biology and represent an effective way to produce fully functional membrane proteins for structural studies. This review will discuss examples of vertebrate membrane protein overexpression in mammalian cells using a variety of viral, constitutive or inducible expression systems.
113 citations
TL;DR: The current state-of-the-art of this technology is compiled and parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes are summarized.
Abstract: Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the co-translat...
54 citations
TL;DR: The substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches and the molecular basis of this specificity was understood by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity.
Abstract: The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [32P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [14C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificit...
45 citations
TL;DR: The answer to the question on the origin of membrane materials for autophagosome biogenesis awaits further reconciliation of these different findings.
Abstract: Recent advances have revealed much about the signaling events and molecular components associated with autophagy. Although it is clear that there are multiple points of intersection and connection between autophagy and known vesicular membrane transport processes between membrane compartments, autophagy is modulated by a distinct set of molecular components, and the autophagosome has a unique membrane composition. A key question that has yet to be resolved with regards to autophagosome formation is its membrane source. Various evidences have indicated that membranes from the endoplasmic reticulum (ER), mitochondria, Golgi, endosomes and the plasma membrane could all potentially act as a source of autophagosomal membrane in non-specialized macroautophagy. Recent investigations have generated advances in terms of the mitochondria's involvement in starvation-induced autophagy, and refined localization of autophagosome formation to ER-mitochondria contact sites. On the other hand, data accumulates on the delivery of membrane sources to the pre-autophagosome structure by Atg9-containing mobile carriers, which likely originated from the Golgi-endosome system. The answer to the question on the origin of membrane materials for autophagosome biogenesis awaits further reconciliation of these different findings.
44 citations
TL;DR: This review examines the molecular components that may enable the dynamic regulation of sub-cellular AQP translocation in response to a specific trigger in the context of the regulation of cellular water flow and includes cytoskeletal proteins, kinases, calcium and retention or localization signals.
Abstract: Water passes through cell membranes relatively slowly by diffusion. In order to maintain water homeostasis, the rapid and specific regulation of cellular water flow is mediated by the aquaporin (AQP) family of membrane protein water channels. The wide range of tissues that are known to express AQPs is reflected by their involvement in many physiological processes and diseases; thirteen human AQPs have been identified to date and the majority are highly specific for water while others show selectivity for water, glycerol and other small solutes. Receptor mediated translocation, via hormone activation, is an established method of AQP regulation, especially for AQP2. There is now an emerging consensus that the rapid and reversible translocation of other AQPs from intracellular vesicles to the plasma membrane, triggered by a range of stimuli, confers altered membrane permeability thereby acting as a regulatory mechanism. This review examines the molecular components that may enable such AQP regulation; these include cytoskeletal proteins, kinases, calcium and retention or localization signals. Current knowledge on the dynamic regulation of sub-cellular AQP translocation in response to a specific trigger is explored in the context of the regulation of cellular water flow.
43 citations
TL;DR: Novel Klotho-dependent mechanisms that are clinically relevant in aging and age-related diseases are discussed that include regulatory effects on general metabolism and a more specific effect on mineral metabolism that correlates with its effect on aging.
Abstract: The Klotho gene was identified as an 'aging suppressor' in mice. Overexpression of the Klotho gene extends lifespan and defective Klotho results in rapid aging and early death. Both the membrane and secreted forms of Klotho have biological activity that include regulatory effects on general metabolism and a more specific effect on mineral metabolism that correlates with its effect on aging. Klotho serves as a co-receptor for fibroblast growth factor (FGF), but it also functions as a humoral factor that regulates cell survival and proliferation, vitamin D metabolism, and calcium and phosphate homeostasis and may serve as a potential tumor suppressor. Moreover, Klotho protects against several pathogenic processes in a FGF23-independent manner. These processes include cancer metastasis, vascular calcification, and renal fibrosis. This review covers the recent advances in Klotho research and discusses novel Klotho-dependent mechanisms that are clinically relevant in aging and age-related diseases.
43 citations
TL;DR: The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system and binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner.
Abstract: The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.
36 citations
TL;DR: A successful strategy for AQP small molecule inhibitor identification is established and AQP inhibitors may be relevant as experimental tools, to enhance the understanding of AQP function, and in the treatment of various diseases.
Abstract: Background: The involvement of aquaporin (AQP) water and small solute channels in the etiology of several diseases, including cancer, neuromyelitis optica and body fluid imbalance disorders, has been suggested previously. Furthermore, results obtained in a mouse model suggested that AQP9 function contributes to hyperglycemia in type-2 diabetes. In addition, the physiological role of several AQP family members remains poorly understood. Small molecule inhibitors of AQPs are therefore desirable to further study AQP physiological and pathophysiological functions. Methods: The binding of recently established AQP9 inhibitors to a homology model of AQP9 was investigated by molecular dynamics simulations and molecular docking. Putative inhibitor binding sites identified with this procedure were modified by site-directed mutagenesis. Active compounds were measured in a mammalian cell water permeability assay of mutated AQP9 isoforms and tested for changes in inhibitory effects. Controls: Three independent cell lines were established for each mutated AQP9 isoform and functionality of mutant isoforms was established. Principal findings: We have identified putative binding sites of recently established AQP9 inhibitors. This information facilitated successful identification of novel AQP9 inhibitors with low micromolar IC50 values in a cell based assay by in silico screening of a compound library targeting specifically this binding site. Significance: We have established a successful strategy for AQP small molecule inhibitor identification. AQP inhibitors may be relevant as experimental tools, to enhance our understanding of AQP function, and in the treatment of various diseases.
36 citations
TL;DR: This article interlink membrane protein production primarily through wheat-germ cell-free protein synthesis with the growing repertoire of membrane mimicking environments in the form of lipids, surfactants, amphipathic surfactant polymers, liposomes and nanodiscs that keep membrane proteins soluble.
Abstract: Membrane proteins control fundamental processes that are inherent to nearly all forms of life such as transport of molecules, catalysis, signaling, vesicle fusion, sensing of chemical and physical stimuli from the environment, and cell-cell interactions. Membrane proteins are harbored within a non-equilibrium fluid-like environment of biological membranes that separate cellular and non-cellular environments, as well as in compartmentalized cellular organelles. One of the classes of membrane proteins that will be specifically treated in this article are transport proteins of plant origin, that facilitate material and energy transfer at the membrane boundaries. These proteins import essential nutrients, export cellular metabolites, maintain ionic and osmotic equilibriums and mediate signal transduction. The aim of this article is to report on the progress of membrane protein functional and structural relationships, with a focus on producing stable and functional proteins suitable for structural and biophysical studies. We interlink membrane protein production primarily through wheat-germ cell-free protein synthesis (WG-CFPS) with the growing repertoire of membrane mimicking environments in the form of lipids, surfactants, amphipathic surfactant polymers, liposomes and nanodiscs that keep membrane proteins soluble. It is hoped that the advancements in these fields could increase the number of elucidated structures, in particular those of plant membrane proteins, and contribute to bridging of the gap between structures of soluble and membrane proteins, the latter being comparatively low.
29 citations
TL;DR: Insight into factors directing high production of eukaryotic membrane proteins is presented together with suggestions on ways to optimize the recombinant production in the yeast Pichia pastoris to explore the underlying bottleneck to a successful membrane protein production experiment.
Abstract: Aquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism - including humans. These proteins are vital molecules and their malfunction can lead to several severe disorders and diseases. Hence, an increased understanding of their structure, function and regulation is of the utmost importance for developing current and future drugs. Heading towards this goal, the first problem to overcome is to acquire the proteins in sufficient amounts to enable functional and structural characterization. Using a suitable host organism, large amounts of target molecules can possibly be produced, but for membrane proteins limitations are frequently encountered. In the work described here, we have produced the 13 human aquaporins (hAQPs) in one of the most successful hosts for recombinant overproduction of eukaryotic proteins; the yeast Pichia pastoris, in order to explore the underlying bottleneck to a successful membrane protein production experiment. Here we present exceptional yield of hAQP1, whereas some other hAQPs were below the threshold needed for scaled up production. In the overproduction process, we have established methods for efficient production screening as well as for accurate determination of the initial production yield. Furthermore, we have optimized the yield of low producing targets, enabling studies of proteins previously out of reach, exemplified with hAQP4 as well as the homologue PfAQP. Taken together, our results. present insight into factors directing high production of eukaryotic membrane proteins together with suggestions on ways to optimize the recombinant production in the yeast P. pastoris.
29 citations
TL;DR: LIMR was shown to be highly specific for Lcn1, binding the lipocalin with low micromolar to high nanomolar affinity, raising doubts about the physiological relevance of the reported binding of BLG and UG to the receptor.
Abstract: Human lipocalin-1 interacting membrane receptor (LIMR) was the first lipocalin receptor to be identified, as a specific receptor for lipocalin-1 (Lcn1). Subsequently LIMR has been reported to interact with other ligands as well, notably with the bovine lipocalin β-lactoglobulin (BLG) and with the unrelated secretoglobin uteroglobin (UG). To study the ligand-binding behaviour of this prototypic lipocalin receptor in more detail, a system was developed for the recombinant expression of LIMR in Drosophila Schneider 2 (S2) cells, and for the subsequent solubilization and purification of the protein. The receptor forms dimers or larger oligomers when solubilized in n-dodecyl β-D-maltoside (DDM). The full-length, functional receptor was captured onto a surface plasmon resonance (SPR) chip via an α-V5 antibody, and the binding of various potential ligands was followed in time. In this way, LIMR was shown to be highly specific for Lcn1, binding the lipocalin with low micromolar to high nanomolar affinity....
TL;DR: The analysis shows that the major contact points of the substrate binding site are indeed crucial for function and in defining the specificity, and the matrix salt bridge network is more critical for function than the cytoplasmic salt bridge networks in agreement with its central position.
Abstract: Mitochondrial carriers transport inorganic ions, nucleotides, amino acids, keto acids and cofactors across the mitochondrial inner membrane. Structurally they consist of three domains, each containing two transmembrane α-helices linked by a short α-helix and loop. The substrate binds to three major contact points in the central cavity. The class of substrate (e.g., adenine nucleotides) is determined by contact point II on transmembrane α-helix H4 and the type of substrate within the class (e.g., ADP, coenzyme A) by contact point I in H2, whereas contact point III on H6 is most usually a positively charged residue, irrespective of the type or class. Two salt bridge networks, consisting of conserved and symmetric residues, are located on the matrix and cytoplasmic side of the cavity. These residues are part of the gates that are involved in opening and closing of the carrier during the transport cycle, exposing the central substrate binding site to either side of the membrane in an alternating way. ...
TL;DR: A three-dimensional model is generated for each of the galanin receptors based on its homologs in the same family and finds significant differences in the organization of the binding pockets among the three types of receptors, which might be the key for specific molecular recognition of ligands.
Abstract: The galanin receptor family comprises of three members, GalR1, GalR2 and GalR3, all belonging to the G-protein-couple receptor superfamily. All three receptors bind the peptide hormone galanin, but show distinctly different binding properties to other molecules and effects on intracellular signaling. To gain insight on the molecular basis of receptor subtype specificity, we have generated a three-dimensional model for each of the galanin receptors based on its homologs in the same family. We found significant differences in the organization of the binding pockets among the three types of receptors, which might be the key for specific molecular recognition of ligands. Through docking of fragments of the galanin peptide and a number of ligands, we investigated the involvement of transmembrane and loop residues in ligand interaction.
TL;DR: How recent biochemical and biophysical studies are beginning to reveal how solute coupling is achieved is discussed.
Abstract: The availability of high-resolution atomic structures for transport proteins provides unprecedented opportunities for understanding their mechanism of action. The details of conformational change can be deduced from these structures, especially when multiple conformations are available. However, the singular ability of transporters to couple the movement of one solute to that of another requires even more information than what is supplied by a crystal structure. This short commentary discusses how recent biochemical and biophysical studies are beginning to reveal how solute coupling is achieved.
TL;DR: Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies, but showed that Q408E is unstable under these conditions.
Abstract: Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1 mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.
TL;DR: The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site.
Abstract: Na+/H+ antiporters play a primary role in Na+/H+ homeostasis in cells and many organelles and have long been drug targets. The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs (Trans membrane segments) are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site. Remarkably, inverted repeats containing interrupted helices with similar functional implications have since been observed in structures of other bacterial secondary transporters with almost no sequence homology. Finally, the structure reveals that NhaA is organized into two functional regions: a ‘pH sensor' – a cluster of amino acyl side chains that are involved in pH regulation; and a catalytic region that is 9 A removed from the pH sensor. Alternative access...
TL;DR: A process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor, resulting in an intracellular loop 2 deletion mutant with N-terminal modifications is described, which is suitable for biophysical characterization and structural studies.
Abstract: In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a KD of 138 ± 55 nM, a ΔH of −9708 ± 1195 cal/mol and a ΔS of −1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a ...
TL;DR: In this paper, two membrane-bound pyrophosphatases (M-PPases) were crystallized recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme (PaPPase).
Abstract: Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 A. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, wa...
TL;DR: It is shown that DENV2C6 partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition.
Abstract: Dengue virus (DENV) C protein is essential for viral assembly. DENV C protein associates with intracellular membranes through a conserved hydrophobic domain and accumulates around endoplasmic reticulum-derived lipid droplets which could provide a platform for capsid formation during assembly. In a previous work we described a region in DENV C protein which induced a nearly complete membrane rupture of several membrane model systems, which was coincident with the theoretically predicted highly hydrophobic region of the protein. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to this DENV C region, DENV2C6. We show that DENV2C6 partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. These results identify an important region in the DENV C protein which might be directly implicated in the DENV life cycle t...
TL;DR: During fat absorption the permeability of the gut epithelium is increased mainly in the crypts, a possible explanation is that cell membranes of immature crypt cells, lacking detergent-resistant lipid raft microdomains, are less resistant to the deleterious effects of bile salts and free fatty acids.
Abstract: Absorption of dietary fat in the small intestine involves epithelial exposure to potentially harmful molecules such as bile salts and free fatty acids. We used organ culture of porcine jejunal explants incubated with a pre-digested mixture of fat (plant oil), bile and pancreatin to mimick the physiological process of dietary fat absorption, and short exposures to the fat mixture caused fat droplet accumulation within villus enterocytes. Lucifer yellow (LY), a fluorescent membrane-impermeable polar tracer was included to monitor epithelial integrity. Both in controls and during fat absorption LY penetrated the epithelium and accumulated in the basal lamina and the lamina propria. LY was also seen in the paracellular space, whereas villus enterocytes were generally only weakly labeled except for small amounts taken up by apical endocytosis. In the crypts, however, fat absorption induced cell permeabilization with LY accumulating in the cytosol and nucleus. Morphologically, both apical and basolateral membranes appeared intact, indicating that the leakiness was caused by minor lesions in the membrane. Albeit to a lesser extent, bile alone was capable of permeabilizing crypt cells, implying that the surfactant properties of bile salts are involved in the process. In addition to LY, crypt enterocytes also became permeable for albumin, ovalbumin and insulin. In conclusion, during fat absorption the permeability of the gut epithelium is increased mainly in the crypts. A possible explanation is that cell membranes of immature crypt cells, lacking detergent-resistant lipid raft microdomains, are less resistant to the deleterious effects of bile salts and free fatty acids.
TL;DR: All three proteins were successfully solubilized and purified in milligram quantities from inner membrane preparations and their suitability for inclusion in crystallization trials was assessed by analysis of structural integrity and thermal stability using circular dichroism spectroscopy followed by examination for monodispersity using gel filtration chromatography.
Abstract: A systematic approach was used for the cloning and amplified expression in Escherichia coli of the genes for each of three inositol transport proteins (IolF, IolT, YfiG) from Bacillus subtilis that are evolutionarily-related to human transporters. Inducible amplified expression of each was achieved to levels of ~ 10–15% of total protein in E. coli inner membrane preparations. The functional integrity of each heterologously-expressed protein was demonstrated by measuring the kinetics of 3 H-myo-inositol transport into energized whole cells; this confirmed that IolT is the major inositol transporter, IolF is an inefficient transporter of this substrate and demonstrated that YfiG is an inositol transport protein for the first time. Competition for 3 H-myo-inositol transport by 17 unlabelled compounds revealed all three proteins to be highly specific in recognizing inositols over sugars. IolT was confirmed to be highly specific for both myo- and D-chiro-inositol and IolF was confirmed to prefer D-chiro-inositol over myo-inositol. YfiG selectively recognized myo-inositol, D-chiro-inositol and, uniquely, L-chiro-inositol. All three proteins were successfully solubilized and purified in milligram quantities from inner membrane preparations and their suitability for inclusion in crystallization trials was assessed by analysis of structural integrity and thermal stability using circular dichroism spectroscopy followed by examination for monodispersity using gel filtration chromatography.
TL;DR: This work investigated the cytotoxicity of lysophosphatidylcholine (lysoPC), a major phospholipid component generated upon LDL oxidation, on bone-forming MG-63 osteoblast-like cells and confirmed the involvement of TRPV2 activation in lysoPC-induced cell death.
Abstract: Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Accordingly, atherogenic determinants such as oxidized low density lipoprotein (OxLDL) particles have been shown to alter bone cell functions. In this work, we investigated the cytotoxicity of lysophosphatidylcholine (lysoPC), a major phospholipid component generated upon LDL oxidation, on bone-forming MG-63 osteoblast-like cells. Cell viability was reduced by lysoPC in a concentration-dependent manner with a LC50 of 18.7 ± 0.7 mM. LysoPC-induced cell death was attributed to induction of both apoptosis and necrosis. Since impairment of intracellular calcium homeostasis is often involved in mechanism of cell death, we determined the involvement of calcium in lysoPC-induced cytotoxicity. LysoPC promoted a rapid and transient increase in intracellular calcium attributed to mobilization from calcium stores, followed by a sustained influx. Intracellular calcium mobilization was associated to phospholipase C (PLC)-dependent mobilization of calcium from the endoplasmic reticulum since inhibition of PLC or calcium depletion of reticulum endoplasmic with thapsigargin prevented the calcium mobilization. The calcium influx induced by lysoPC was abolished by inhibition of transient receptor potential vanilloid (TRPV) channels with ruthenium red whereas gadolinium, which inhibits canonical TRP (TRPC) channels, was without effect. Accordingly, expression of TRPV2 and TRPV4 were shown in MG-63 cells. The addition of TRPV2 inhibitor Tranilast in the incubation medium prevent the calcium influx triggered by lysoPC and reduced lysoPC-induced cytotoxicity whereas TRPV4 inhibitor RN 1734 was without effect, which confirms the involvement of TRPV2 activation in lysoPCinduced cell death.
TL;DR: The utility of solid-state NMR combined with sample deuteration for mapping the binding interface of low affinity ligands with membrane proteins is demonstrated.
Abstract: Solid-state NMR combined with sample deuteration was used to probe the proximity of the low-affinity substrate D-glucose to its binding site within the Escherichia coli sugar transport protein GalP. Samples of E. coli inner membranes with amplified expression of GalP were incubated in D(2)O with D-[(13)C(6)]glucose and (13)C NMR signals from the substrate were assigned in two-dimensional dipolar-assisted rotational resonance (DARR) spectra. The signals were confirmed as representing D-glucose bound to GalP as the peaks were abolished after the substrate was displaced from the specific site with the inhibitor forskolin. The (13)C chemical shift values for D-[(13)C(6)]glucose in solution revealed some differences compared to those for ligand bound to GalP, the differences being most pronounced for positions C1 and C2, and especially for C1 in the α-anomer. (13)C cross-polarization build-up was measured for C1 and C2 of D-[(13)C(6)]glucose and D-[(2)H(7), (13)C(6)]glucose in GalP membranes suspended in D(2)O. The build-up curves for the deuterated substrate reflect intermolecular (1)H-(13)C interactions between the protein and the fully deuterated substrate; the signal build-up suggests that the α-anomer is situated closer to the protein binding site than is the β-anomer, consistent with its relatively high signal intensities and more pronounced chemical shift changes in the 2D-correlation spectra. These results demonstrate the utility of solid-state NMR combined with sample deuteration for mapping the binding interface of low affinity ligands with membrane proteins.
TL;DR: The capability of the Leishmania tarentolae cell-free system for the production of a huge variety of human solute carriers in the precipitate mode is demonstrated and purified SLC17A3 shows the formation of an oligomeric state.
Abstract: Cell-free protein production offers a versatile alternative to complement in vivo expression systems. However, usage of prokaryotic cell-free systems often leads to non-functional proteins. We modified a previously designed cell-free system based on the protozoan Leishmania tarentolae, a parasite of the Moorish gecko Tarentola mauritanica, together with a species-independent translational sequences-based plasmid to produce human membrane proteins in 2 hours reaction time. We successfully established all four commonly used expression modes for cell-free synthesis of membrane proteins with a human organic anion transporter, SLC17A3, as a model membrane protein: (i) As precipitates without the addition of any hydrophobic environment, (ii) in the presence of detergents, (iii) with the addition of liposomes, and (iv) supplemented with nanodiscs. We utilized this adapted system to synthesize 22 human solute carriers from 20 different families. Our results demonstrate the capability of the Leishmania tar...
TL;DR: Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.
Abstract: Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.
TL;DR: A fluorescence-based assay is presented to investigate Rab GTPase membrane extraction and delivery by RabGDI and showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane.
Abstract: Rab proteins are a large family of GTP-binding proteins that regulate cellular membrane traffic and organelle identity. Rab proteins cycle between association with membranes and binding to RabGDI. Bound on membranes, each Rab has a very specific cellular location and it is this remarkable degree of specificity with which Rab GTPases recognize distinct subsets of intracellular membranes that forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the correct membrane at the correct time. The molecular mechanisms controlling Rab localization remain poorly understood. Here, we present a fluorescence-based assay to investigate Rab GTPase membrane extraction and delivery by RabGDI. Using EGFP-Rab fusion proteins the amount of Rab:GDI complex obtained by GDI extraction of Rab proteins from HEK293 membranes could be determined, enabling control of complex concentration. Subsequent partitioning of the Rab GTPases into vesicles made up of artificial binary lipid mixtures showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane.
TL;DR: Light is shed on the molecular components of this diffusion barrier at its base, and intriguing functional similarities with other better characterized cellular barriers are revealed.
Abstract: The primary cilium that protrudes from the plasma membrane of many eukaryotic cell types is very much a cellular organelle in its own right. Its unique membrane and luminal composition is effectively compartmentalized by diffusion barrier at its base, known as the transition zone. Recent works have now shed light on the molecular components of this diffusion barrier, and revealed intriguing functional similarities with other better characterized cellular barriers.
TL;DR: The structural requirements for binding to a pocket that accommodates the two C-terminal residues, YY, in the crystal structures of the sodium pump are examined and a significant correlation between the docking scores from two different methods and the experimentally determined sodium affinities are shown, which strengthens the previous hypothesis that sodium binding is coupled to docking of the C- terminus.
Abstract: The Na,K-ATPase is essential to all animals, since it maintains the electrochemical gradients that energize the plasma membrane Naturally occurring inhibitors of the pump from plants have been used pharmaceutically in cardiac treatment for centuries The inhibitors block the pump by binding on its extracellular side and thereby locking it To explore the possibilities for designing an alternative way of targeting the pump function, we have examined the structural requirements for binding to a pocket that accommodates the two C-terminal residues, YY, in the crystal structures of the pump To cover the sample space of two residues, we first performed docking studies with the 400 possible dipeptides For validation of the in silico predictions, pumps with 13 dipeptide sequences replacing the C-terminal YY were expressed in Xenopus laevis oocytes and examined with electrophysiology Our data show a significant correlation between the docking scores from two different methods and the experimentally determined sodium affinities, which strengthens the previous hypothesis that sodium binding is coupled to docking of the C-terminus From the dipeptides that dock the best and better than wild-type YY, it may therefore be possible to develop specific drugs targeting a previously unexplored binding pocket in the sodium pump
TL;DR: New potent inhibitors for the ABC-type MDR efflux pumps studied are revealed with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria.
Abstract: The increasing number of multidrug-resistant pathogenic microorganisms is a serious public health issue Among the multitude of mechanisms that lead to multidrug resistance, the active extrusion of toxic compounds, mediated by MDR efflux pumps, plays an important role In our study we analyzed the inhibitory capability of 26 synthesized zosuquidar derivatives on three ABC-type MDR efflux pumps, namely Saccharomyces cerevisiae Pdr5 as well as Lactococcus lactis LmrA and LmrCD For Pdr5, five compounds could be identified that inhibited rhodamine 6G transport more efficiently than zosuquidar One of these is a compound with a new catechol acetal structure that might represent a new lead compound Furthermore, the determination of IC(50) values for rhodamine 6G transport of Pdr5 with representative compounds reveals values between 03 and 09 μM Thus the identified compounds are among the most potent inhibitors known for Pdr5 For the ABC-type efflux pumps LmrA and LmrCD from L lactis, seven and three compounds, which inhibit the transport activity more than the lead compound zosuquidar, were found Interestingly, transport inhibition for LmrCD was very specific, with a drastic reduction by one compound while its diastereomers showed hardly an effect Thus, the present study reveals new potent inhibitors for the ABC-type MDR efflux pumps studied with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria
TL;DR: The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.
Abstract: The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein 'u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.