Partition Assay Research Articles

Topology and functional characterization of major outer membrane proteins of Treponema maltophilum and Treponema lecithinolyticum.

Numerous Treponema species are prevalent in the dysbiotic subgingival microbial community during periodontitis. The major outer sheath protein is a highly expressed virulence factor of the well-characterized species Treponema denticola. Msp forms an oligomeric membrane protein complex with adhesin and porin properties and contributes to host-microbial interaction. Treponema maltophilum and Treponema lecithinolyticum species are also prominent during periodontitis but are relatively understudied. Msp-like membrane surface proteins exist in T. maltophilum (MspA) and T. lecithinolyticum (MspTL), but limited information exists regarding their structural features or functionality. Protein profiling reveals numerous differences between these species, but minimal differences between strains of the same species. Using protein modeling tools, we predict MspA and MspTL monomeric forms to be large β-barrel structures composed of 20 all-next-neighbor antiparallel β strands which most likely adopt a homotrimer formation. Using cell fractionation, Triton X-114 phase partitioning, heat modifiability, and chemical and detergent release assays, we found evidence of amphiphilic integral membrane-associated oligomerization for both native MspA and MspTL in intact spirochetes. Proteinase K accessibility and immunofluorescence assays demonstrate surface exposure of MspA and MspTL. Functionally, purified recombinant MspA or MspTL monomer proteins can impair neutrophil chemotaxis. Expressions of MspA or MspTL with a PelB leader sequence in Escherichia coli also demonstrate surface exposure and can impair neutrophil chemotaxis in an in vivo air pouch model of inflammation. Collectively, our data demonstrate that MspA and MspTL membrane proteins can contribute to pathogenesis of these understudied oral spirochete species.

Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Differential Effects of Rhodococcus equi Virulence-Associated Proteins on Macrophages and Artificial Lipid Membranes.

Virulence-associated protein A (VapA) of Rhodococcus equi is a pathogenicity factor required for the multiplication of virulent R. equi strains within spacious macrophage vacuoles. The production of VapA is characteristic for R. equi isolates from pneumonic foals. VapB and VapN proteins in R. equi isolates from infected pig (VapB) and cattle (VapN) have amino acid sequences very similar to VapA and consequently have been assumed to be its functional correlates. Using model membrane experiments, phagosome pH acidification analysis, lysosome size measurements, protein partitioning, and degradation assays, we provide support for the view that VapA and VapN promote intracellular multiplication of R. equi by neutralizing the pH of the R. equi-containing vacuole. VapB does not neutralize vacuole pH, is not as membrane active as VapA, and does not support intracellular multiplication. This study also shows that the size of the sometimes enormous R. equi-containing vacuoles or the partitioning of purified Vaps into organic phases are not features that have predictive value for virulence of R. equi, whereas the ability of Vaps to increase phagosome pH is coupled to virulence. IMPORTANCE Rhodococcus equi is a major cause of life-threatening pneumonia in foals and occasionally in immunocompromised persons. Virulence-associated protein A (VapA) promotes R. equi multiplication in lung macrophages, which are the major host cells during foal infection. In this study, we compare cellular, biochemical, and biophysical phenotypes associated with VapA to those of VapB (typically produced by isolates from pigs) or VapN (isolates from cattle). Our data support the hypothesis that only some Vaps support multiplication in macrophages by pH neutralization of the phagosomes that R. equi inhabit.

Elucidating the Kinetic Mechanism of Human METTL16.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

Energetic analysis of the liquid–liquid equilibrium of systems containing polyethylene glycol (4000 g.mol−1 or 6000 g.mol−1) and salts (Na2SO4 or Na3C6H5O7) at different temperatures and their application in the bovine serum albumin and α-lactalbumin partitioning

This work aims to determine the interaction parameters and the energetic analysis of the aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG 4000 g.mol−1 or PEG 6000 g.mol−1), salt (Na2SO4 or Na3C6H5O7) and water at different temperatures T = (293.2, 298.2, 303.2 and 308.2) K. Partition assays of bovine serum albumin (BSA) and α-lactalbumin in the different systems studied were also performed in order to demonstrate the application of the two-phase aqueous systems studied. The temperature had no effect on the ATPS phase equilibrium compositions while the polymer molar mass had an influence, presenting a greater biphasic region in the systems formed by PEG 6000. The systems composed by Na2SO4 showed greater biphasic region. The salting-out capacity and the effective excluded volume (EEV) of the systems were also determined. It was observed that the salting-out effect was greater in the systems composed of Na2SO4. The EEV was higher in the systems formed by PEG 6000 for both salts. In addition, the equilibrium data of the systems were thermodynamically modelled using UNIFAC model and it was found that the water-salt interaction was greater than the water-PEG interaction, thus proving the higher water concentration in the systems bottom phase. It was also verified that the UNIFAC model presented low values of the global deviations (2.970%), being considered adequate to predict the new parameters of interaction between the contribution groups of the studied systems. This study shows that the values found in thermodynamic modelling can be used to predict other systems that have these same contribution groups evaluated. It was also found that α-lactalbumin and BSA migrated preferentially to the bottom phase in most systems. It was observed lower kp in the systems formed by Na2SO4. Such behavior may be related due to the greater interaction of α-lactalbumin and BSA with the bottom phase of the system formed by Na2SO4. For the systems formed by sodium citrate, the partition coefficient for α-lactalbumin and BSA increased with the increase in the molar mass of the polymer (4000 to 6000). The salt type effect had more effect on the partition than the polymer molar mass, but both influenced.

Patterns of physiological parameters and nitrogen partitioning in flag leaf explain differential grain protein content in rice

The grain protein content (GPC) in rice is low, and more efforts with agronomic and molecular approaches were performed to increase them. However, the rice research focusing on the plant physiological behaviour that modulates the phenomenon of grain protein filling is very scarce. This work contains physiological parameters related to photosynthetic activity in the flag leaf in the grain filling period and N partitioning assays of high (Nutriar) and traditional (Camba) GPC cultivars. Results indicated a higher photosynthetic capacity, a better capacity to provide CO2 to the chloroplast and a healthier PSII structure in Camba relative to Nutriar. Chlorophyll fluorescence parameters decreased more steeply over time in the high protein variety, and a strong negative correlation was observed between GPC and PSII structure parameters. N content in the flag leaf at anthesis showed lower values and higher remobilisation during the grain filling period in Nutriar compared to Camba. The results of this work suggested that the inactivation of some PSII structures in higher GPC cultivars is associated with N remobilisation and would contribute to an increase in the free N available to be translocated to the grain.

Estimation of internal human daily intakes of organophosphate esters using one-compartment toxicokinetic model in the whole blood from Hebei Province, China

To evaluate the human health risks attributed by organophosphate ester (OPE) exposure, it is very important to estimate the daily intakes (DIs) of OPEs in human. In this study, the DIs of OPEs were estimated using a simplified one-compartment toxicokinetic model based on their total clearance rates in human and their whole blood concentrations. Thirty paired human whole blood and plasma samples were collected from participants in Hengshui, Hebei Province, China. The detection frequencies of most OPEs in the whole blood were lower than 50.0%. Thus, the OPE levels in whole blood were converted from the corresponding plasma levels using the fractions of OPEs in plasma (Fp), which were estimated from an in vitro partition assay and the values were in the range of 0.52–0.98. The measured whole blood concentrations of triphenyl phosphate (TPHP) and tris(chloroethyl) phosphate (TCEP) were comparable to those converted from the plasma concentrations, suggesting that the conversion method was reliable. The estimated total DIs of TPHP, TCEP, and tris(2-chloroisopropyl) phosphate were 1–30 times of those derived by the external exposure method, which usually excluded many exposure sources. The estimated human health risks based on the DIs indicated that the carcinogenic and non-carcinogenic effects of OPEs for the participants in Hengshui, Hebei Province, China, were negligible. This study recommended a more reliable and simpler method to estimate the human health risks attributed to the exposure of OPEs.

Mathematical Modeling of In Vitro‐In Vivo Atypical Kinetics of Diazepam

Cytochrome P450 (CYP) and uridine 5'‐diphospho‐glucuronosyltransferase (UGT) enzymes are responsible for metabolism of most marketed drugs. Due to the large binding sites of certain CYPs and UGTs, CYP and UGT substrates tend to show non ‐ Michaelis‐Menten kinetics (atypical kinetics). Although several in vitro data provide evidence for the existence of atypical kinetics, this characteristic is seldom observed with in vivo data. The purpose of this project is to characterize the atypical kinetics of diazepam and its sequential metabolism in in vitro and in vivo systems. Numerical methods will be used to obtain kinetic parameters, which will be used for in vitro‐in vivo correlations (IVIVC).Diazepam, which exhibits atypical kinetics in vitro, and its primary metabolites (temazepam, nordiazepam), and secondary metabolite (oxazepam) were characterized with in vitro studies with pooled male Sprague Dawley rat liver microsome (MRLM), pooled female Sprague Dawley (FRLM), and purified rat cyp isoforms (cyp3a1, cyp3a2, and cyp2c11) separately. Rat microsomal partition and rat plasma protein binding assays were conducted via equilibrium dialysis to determine the free fraction of the drug. Two substrate binding (ESS) models provided better fits than one substrate (ES) models, as determined from the resulting AICc values. Preliminary in vitro data from MRLM and cyp3a1 showed obvious sigmoidal saturation curves for temazepam formation. Only cyp3a1 show significant sigmoidal curves for nordiazepam formation. For cyp3a1, Km and Vmax values for temazepam and nordiazepam calculated with the EES model are significantly different from those with the ES model. The intrinsic clearance (CLint) values calculated from the MRLM and cyp3a1 ESS models is not constant within the observed range of diazepam plasma levels of rats. No obvious sigmoidicity of diazepam saturation curves was observed for cyp3a2 (male‐specific) and cyp2c11 (male‐specific) isoforms, which are other cyp isoforms responsible for diazepam metabolism in male rats. In order to characterize in vitro‐in vivo correlations for diazepam, in vivo PK studies with female and male rats are currently underway.Support or Funding InformationThis research was supported by NIGMS grants: R01GM114369 and R01GM104178.

A proteome‐wide assay for protein mistargeting in living cells

Nascent chains of secretory proteins are targeted to the endoplasmic reticulum (ER) to start their journey in the secretory pathway. Yet mistargeting may not be rare, especially during stress. Mistargeted nascent proteins then accumulate in the cytosol, which can be a potential threat to the proteostasis of the cell. A comprehensive understanding of how protein gets mistargeted, which kinds of proteins are more prone to mistargeting and which factors govern the regulation of mistargeting will be important. However, those questions are hard to answer due to the lack of methodologies: the gold standard relying on in vitro transcription/translation is quantitative but tedious, low through‐put and incompatible with living cells, while the naïve proxy of measuring ER maturation events is facile but not necessarily accurate. Here, we developed a protein partitioning assay by using a cytosol‐localized engineered ascorbate peroxidase (cytAPEX) or an ER lumen‐retained horseradish peroxidase (ERHRP) to proximity‐label cytosolic and ER luminal proteomes respectively. The compartmental selectivity of this assay was validated with exogenous and endogenous proteins. We showed protein localization can be independent of maturation events, like signal sequence cleavage. We measured how an ER stressor impacts the accumulation of ER proteins in the cytosol. We also characterized differential protein targeting under the condition of translocon inhibition. Using quantitative proteomics, we calculated the basal import efficiency of some ER‐directed proteins. Interestingly, these data have good agreement with the in vitro method. With this assay, we will be able to estimate the basal import efficiency or measure mistargeting at a proteome‐wide scale in living cells. This assay will also make it possible to investigate the mechanism behind mistargeting in response to ER stress.Support or Funding InformationUniversity of California, Riverside

P143 - Evaluation of single-use rapid equilibrium dialysis [red] device for automated octanol-water partitioning (LOGD7.4) assay to assess the lipophilicity of drug compounds

P143 - Evaluation of single-use rapid equilibrium dialysis [red] device for automated octanol-water partitioning (LOGD7.4) assay to assess the lipophilicity of drug compounds

Preclinical evaluation of cationic DOTA-triarginine-lipid conjugates for theranostic liquid brachytherapy.

Liquid brachytherapy is an emerging technology for internal radiation therapy where liquids containing radionuclides are administered directly into solid tumors. These technologies are less invasive than conventional brachytherapy, and can potentially improve the dose coverage and homogeneity of the radioactivity distribution within the tumor. For this purpose, we have developed a novel cationic micelle system for delivery of a range of radionuclides. The system is applicable for emitters of alpha, beta or photon radiation, and enables dose-mapping via theranostic nuclear imaging.Methods: The cationic micelles were developed as linear surfactants comprising the chelator DOTA, a triarginine sequence and a palmitoyl or stearoyl fatty acid chain. The critical micelle concentration of the surfactants was determined, and the micelles were radiolabelled with 64Cu or 177Lu in high radiochemical purity (>95%). The tumor retention and biodistribution of the 64Cu-radiolabeled surfactants, administered as micelles or formulated in liposomes, were investigated in vivo by PET/CT in a tumor bearing mouse model.Results: The interaction of the micelles with anionic lipid membranes was demonstrated to be favourable, using a liposome partition assay. In vivo, the surfactants formulated both as cationic micelles and liposomes displayed the best intratumoral retention, with micelles providing more homogeneous activity distribution.Conclusion: A cationic, surfactant-based drug delivery system was developed and demonstrated promise as a vehicle for liquid brachytherapy when formulated as micelles or in liposomes. The system enables accurate dosimetry due to the flexible radiochemistry of DOTA.

Interactions between Sterols and Phospholipids with Different Headgroups - Influence on Lateral Segregation

Cholesterol is an important modulator of the structure and function of mammalian cell membranes. The effect of cholesterol is mediated through the interactions between the sterol and the phospholipid components of the membrane. Recently, we have reported a correlation between the relative affinity cholesterol have for different phospholipid components and the degree to which the sterol can facilitate lateral domain formation. In the current project, the aim is to determine the affinity of cholesterol and other sterols for phospholipids with different headgroups, and to analyze how the measured affinities relate to the formation of lateral membrane structure. The affinity of the sterols for phospholipid bilayers is measured using different fluorescence based partitioning assays, and the formation of lateral domains in the bilayers is determined by measuring the fluorescence lifetimes of trans-parinaric acid as a function of lipids composition. The results from these experiments offer insights into the mechanisms driving the formation of lateral domain formation that will help us understand the processes that occurs in cellular membranes.

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts.

Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [45Ca] and [54Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [45Ca] or [54Mn] during a light incubation step. After incubation, chloroplasts are either kept intact or osmotically/mechanically treated to release thylakoids. The amount of incorporated [45Ca] or [54Mn] can be determined by liquid scintillation counting and the relative distribution calculated.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.

Quantitative and functional analysis of CD69+ NKG2D+ T regulatory cells in healthy subjects

T regulatory (Treg) cells have a key role in immune homeostasis and the pathogenesis of chronic inflammatory and autoimmune diseases. CD69 is an early leukocyte activation molecule that under steady state conditions is detected in a small proportion of lymphocytes in peripheral blood and lymphoid tissues. Although it has been reported that a subset of CD69+ T cells behaves as Treg lymphocytes, the possible relationship between CD69+ Treg cells and CD4+NKG2D+ T lymphocytes, which also exert immunosuppressive activity, has not been explored. In this study, we analyzed the expression of CD69 and NKG2D by T lymphocytes from the peripheral blood of twenty-five healthy subjects by multi-parametric flow cytometry analysis, and their suppressive activity by an assay of inhibition of lymphocyte activation (CD40L expression) and proliferation (carboxyfluorescein partition assay). We found a very small percentage of CD4+CD69+NKG2D+ T cells (median 0.002%, Q1–Q3, 0.001–0.004%), which also expressed TGF-β (Latency Associated Peptide or LAP) and IL-10, in all samples analyzed. These cells exerted an important in vitro suppressive effect on both activation and proliferation of T effector cells. Our data suggest that at very small numbers, CD4+CD69+NKG2D+ lymphocytes seem to exert a relevant functional immune-regulatory role in healthy subjects.

Endogenous fatty acids in olfactory hairs influence pheromone binding protein structure and function in Lymantria dispar

The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs). We have extracted and identified free fatty acids from lymph of sensory hairs, and we demonstrate that these function as endogenous ligands for gypsy moth PBP1 and PBP2. Homology modeling of both PBPs, and docking of fatty acids reveal multiple binding sites: one internal, the others external. Pheromone binding assays suggest that these fatty acids increase PBP-pheromone binding affinity. We show that fatty acid binding causes an increase in α-helix content in the N-terminal domain, but not in the C-terminal peptide of both proteins. The C-terminal peptide was shown to form a α-helix in a hydrophobic, homogeneous environment, but not in the presence of fatty acid micelles. Through partition assays we show that the fatty acids prevent adsorption of the pheromone on hydrophobic surfaces and facilitate pheromone partition into an aqueous phase. We propose that lymph is an emulsion of fatty acids and PBP that influence each other and thereby control the partition equilibria of hydrophobic odorants.

Optimized extraction of a single-chain variable fragment of antibody by using aqueous micellar two-phase systems

In this work, the purification of a single-chain variable fragment (scFv) of an antibody by using liquid–liquid extraction in aqueous micellar two-phase systems was optimized by means of central composite design. Protein partitioning assays were performed by using the selected system composition in previous works: Triton X-114 at 4% wt/wt, yeast fermentation supernatant at 60% wt/wt, McIlvaine buffer pH 7.00. The other system component concentrations, Cibacron Blue F3GA (CB), Fabsorbent™ F1P HF (HF) and NaCl, were selected as independent variables. ScFv recovery percentage (%R) and purification factor (PF) were selected as the responses. According to the optimization process both, scFv recovery percentage and purification factor were favored with the addition of HF and NaCl in a range of concentrations around the central point of the second central composite design (HF 0.0120% w/w, CB 0.0200% w/w, NaCl 0.200% w/w). These experimental conditions allowed the concentration and pre-purification of scFv in the micelle-rich bottom phase of the systems with a recovery percentage superior to 88% and a purification factor of approximately 3.5. These results improved the previously presented works and demonstrated the convenience of using aqueous micellar two-phase systems as a first step in the purification of scFv molecules.

Structural Basis of Dynamic Membrane Recognition by trans-Golgi Network Specific FAPP Proteins

Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) domains of FAPP proteins to the trans-Golgi network. The mechanism involved is unclear but requires recognition of phosphatidylinositol-4-phosphate (PI4P) within the Golgi membrane. We investigated the molecular basis of FAPP1-PH domain interactions with PI4P bilayers in liposome sedimentation and membrane partitioning assays. Our data reveals a mechanism in which FAPP-PH proteins preferentially target PI4P-containing liquid disordered membranes, while liquid ordered membranes were disfavored. Additionally, NMR spectroscopy was used to identify the binding determinants responsible for recognizing trans-Golgi network-like bicelles including phosphoinositide and neighboring lipid molecules. Membrane penetration by the FAPP1-PH domain was mediated by an exposed, conserved hydrophobic wedge next to the PI4P recognition site and ringed by a network of complementary polar residues and basic charges. Our data illuminates how insertion of a structured loop provides selectivity for sensing membrane fluidity and targeting to defined membrane zones and organelles. The determinants of this membrane sensing process are conserved across the CERT, OSBP and FAPP family. Hence, lipid gradients not only result in differential membrane ordering along the secretory pathway but also specifically localize diverse proteins through recognition of ensembles of lipid ligands in dynamic and deformable bilayers in order to promote anterograde trafficking.

Identification of Nucleic Acid High Affinity Binding Sequences of Proteins by SELEX.

A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein-nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound by the protein of interest. The cloning and sequencing of these sequences finally completes their identification.