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Identification of novel sphingolipid-binding motifs in mammalian membrane proteins
Abstract
Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.
... For some modifications, like isoprenylation, consensus motifs have been identified while for others, like S-palmitoylation, trained algorithms are available (31,38). Such trained algorithms have also been used for noncovalent interaction sites, like the CCM or sphingolipid interaction motif (45,68). As methods to define non-covalent lipid interaction sites in proteins are especially laborious, also molecular dynamics simulation have got more and more attention to confirm or also predict novel sites (69). ...
... In this case, the simulations, along with a point mutation screening of the p24 transmembrane helix, revealed the putative sphingolipid interaction motif N-V-(X) 2 -TL-(X) 2 IY-C. In a subsequent screening for similar (linear) interaction motifs a number of GPCRs were found (68). Among the found GPCRs, there were also members of the mGluR family, which had been associated with specific cholesterol binding earlier by structural and biochemical means (55,56,71). ...
... In a collaborative work with the group of Prof. Irmgard Sinning, mGluR2 has been found to be regulated by the cholesterol content in the membrane and might also harbor a sphingolipid binding site (55,68). The goal of my PhD thesis was to identify specific lipid binding sites in mGluR2 which may modulate its function. ...
The Metabotropic glutamate receptor 2 (mGluR2) belongs to the family of G-protein coupled receptors, a specific class of transmembrane proteins involved in cellular signaling. The functionality of such transmembrane proteins has been identified to largely depend on their microenvironment, namely the lipid bilayer surrounding them. However, the regulation of the receptors by their lipid microenvironment remains poorly understood. In particular, it remains unclear how specific protein-lipid interactions may modulate the function of mGluR2. In the last years, general motifs for non-covalent cholesterol and sphingolipid interaction within helical domains of transmembrane proteins have been described. In these motifs, both tryptophan and tyrosine residues have been found to play a decisive role. For that reason, an alanine mutagenesis screening, targeting tryptophan and tyrosine residues at the transmembrane surface, was carried out in the search of specific sphingolipid or cholesterol interaction sites for mGluR2. For the different Y→A and W→A variants, surface biotinylation and co-immunoprecipitation showed that neither trafficking nor dimerization were disturbed by substitution of these aromatic residues. In contrast, cellular photo-crosslinking assays demonstrated that cholesterol binding was compromised if one tyrosine residue located at the helix five or another at the helix six was replaced. Thus, these experiments suggested these two helices to contain specific cholesterol binding sites. To get a better molecular insight into these specific protein-lipid interactions, lipid binding to the transmembrane domain of mGluR2 was investigated in molecular dynamics (MD) simulation. The molecular dynamics simulations in GROMACS were performed in collaboration with the Max Planck tandem group of Dr. Camilo Aponte-Santamaría. All-atom and coarse-grained MD simulations of the mGluR2 transmembrane domain confirmed the experimental observation, by revealing a highly-localized density of cholesterol near these residues in helices five and six, which smeared out when they were changed to alanine in silico. The simulations also revealed flexibility of the protein structure at the exoplasmic end of helix six which changed upon introduction of point mutations. Overall, the work combining functional assays and MD simulations demonstrated the existence of specific cholesterol binding sites in mGluR2. It will be highly interesting to investigate the functional implications of this newly-found specific protein–cholesterol interaction on the activity and conformation of the receptor.
... Another example is the necessity of cardiolipin for the supercomplex formation between the respiratory chain complexes III and IV in mitochondria [13,14] . The study of the interaction between sphingomyelin species 18 (SM18) and = 8 = the transmembrane domain of the COPI machinery protein p24 revealed a conserved signature sequence (VXXTLXXIY) in a number of mammalian transmembrane proteins [15,16] . UniProt analysis using this sphingolipid-binding motif resulted in a list containing 615 novel candidates for sphingolipid-protein interactions [16] . ...
... The study of the interaction between sphingomyelin species 18 (SM18) and = 8 = the transmembrane domain of the COPI machinery protein p24 revealed a conserved signature sequence (VXXTLXXIY) in a number of mammalian transmembrane proteins [15,16] . UniProt analysis using this sphingolipid-binding motif resulted in a list containing 615 novel candidates for sphingolipid-protein interactions [16] . Two of these proteins are the endoplasmic reticulum (ER) localised transmembrane proteins glycosylphosphatidylinositol anchor (GPI) attachment 1 protein (GPAA1) and GPI inositol-deacylase (PGAP1). ...
... To [FWY]) for the sphingolipid binding motif was retrieved from [15], [16] . All sequences that did correspond to one of the binding motifs (see table 13) were considered a hit, when at least one amino acid was located inside a transmembrane domain (TMD). ...
The glycosylphosphatidylinositol (GPI) anchor is a lipid moiety attached to over 150 human proteins and plays a crucial role in cell surface display at the plasma membrane. The transamidase complex, consisting of the subunits PIGK/S/T/U and GPAA1, is localised to the endoplasmic reticulum (ER) and is responsible for attachment of the GPI-anchor to receiving proteins. Previously, interactions between the transamidase complex subunit GPAA1 and a sphingolipid have been shown. The aim of this thesis was to establish an experimental setup to analyse protein lipid interactions of the transamidase complex. These interactions were assessed via in-gel fluorescence and western blot quantification through click chemistry with bifunctional sphingolipids combined with transient expression of transamidase subunits. Thin layer chromatography was used to verify the metabolic status of exogenously added photoactivatable and clickable sphingosine (pacSph) at given time points. Results confirmed the previous reported interaction of GPAA1. The validity of this outcome was limited to GPAA1 only due to uncertainty with regards to the transient expression, such as affecting the stoichiometry of the transamidase complex and mislocalisation of the transiently overexpressed GPAA1. To circumvent these limitations, CRISPR/Cas9-based gene editing was performed to insert a C-terminal tag in the genomic region of GPAA1. This allowed to establish a cellular model to study sphingolipid and cholesterol interactions with the endogenous transamidase complex. Gene edited tagging of GPAA1 did not interfere with synthesis and trafficking of GPIanchor proteins to the cell surface. The analysis of the composition of the transamidase complex in this cell line by co-immunoprecipitation showed only PIGK/T/S to interact with GPAA1, but not PIGU. When styrene maleic anhydride (SMA) co-polymer was used to extract the transamidase complex and its native lipid environment, even PIGU was detected, suggesting that PIGU might be only loosely attached to the transamidase complex. Furthermore, first studies on mass spectrometric analysis of lipids extracted from an SMA-immunoprecipitation suggested that this approach allows to determine lipids in direct proximity of the transamaidase complex. Finally, GPAA1-pacSph metabolite interaction could be inhibited through the ceramide synthase inhibitor FB1, while the glucosylceramide synthase inhibitor PPMP did not interfere with the interaction. This suggested that ceramide or a metabolite upstream of this lipid is the interaction partner of GPAA1. In summary, the established cellular model verified previously published results in an endogenous setting. For the future, this model lays the foundation for quantitative determination of the lipid environment of the transamidase complex. Furthermore, this system might also provide structural information via single particle cryo-electron microscopy of affinity purified GPAA1 complexes.
... T1Rs belong to Class C of the GPCR family represented by the well-characterized metabotropic glutamate receptors; while T2Rs were grouped with either Class F or T represented by the frizzled receptors [6]. GPCRs contain a number of highly conserved motifs, such as D/ERY, NPxxY that are well studied, and non-conserved motifs like cholesterolbinding motifs (CRAC) and sphingolipid binding motifs (SBMs) that are poorly characterized [7][8][9][10][11]. Nevertheless, from a structural standpoint, the presence of these lipidbinding motifs across GPCR family underscores the importance of membrane lipids in the organization and function of GPCRs. ...
... Furthermore, it has been proposed that consensus sphingolipid binding signature sequences in Sertonin 1A receptors may be involved in forming interactions with sphingomyelin; however, the motifs were not functionally characterized [26,27]. In metabotropic glutamate receptor 2 (GRM2), a specific interaction between sphingomyelin and putative sphingolipid binding motif of GRM2 has been functionally characterized [11]. The role of sphingolipids in taste receptor (T1R and T2R) signaling has not been elucidated thus far. ...
... Prediction of putative SBMs in taste receptors (T1Rs and T2Rs) and other select GPCRs was pursued as described in the methods. The putative SBM which is purported to be predominantly present in the TM6 region of GPCRs [11] was not conserved in T2Rs (Fig. 1a). Only 3 out of the 25 human T2Rs, namely T2R9, T2R14, and T2R16 had the SBM in TM6 region (Fig. 1a). ...
Membrane lipids regulate the structure and function of G protein-coupled receptors (GPCRs). Previously we have shown that membrane cholesterol regulates the signaling of two human bitter taste receptors (T2Rs), T2R4 and T2R14. Another major plasma membrane lipid known to influence the function of membrane proteins including GPCRs is sphingomyelin. The role of sphingomyelin in T2R function is unexplored thus far. In this work, we examined the significance of sphingomyelin in T2R14 signaling. Results suggest that unavailability of membrane sphingomyelin did not affect the agonist-promoted T2R14 Ca²⁺ signaling in heterologous expression system and also in primary airway smooth muscle cells (HASM cells). In addition, T2R14 mediated downstream AMPK activation was also unaffected in sphingomyelin-depleted condition; however, cholesterol depletion impaired the T2R14-mediated AMPK activation. Angiotensin II type1A receptor (AT1R) expressed in HASM cells and signals through Ca²⁺ and AMPK was used as a control. Results suggest that similar to T2R14, membrane sphingomyelin depletion did not affect AT1R signaling. However, membrane cholesterol depletion impaired AT1R mediated Ca²⁺ signaling and AMPK activation. Interestingly, amino acid sequence analysis revealed the presence of putative sphingolipid binding motif in both T2R14 and AT1R suggesting that the presence of a motif alone might not be suggestive of sphingomyelin sensitivity. In conclusion, these results demonstrate that in contrast to membrane cholesterol, sphingomyelin does not affect the agonist-induced T2R14 signaling, however it may play a role in other aspects of T2R14 function.
... Starting from this sequence, a bioinformatics analysis identified a number of protein candidates with signatures homologous to this highly specific sphingolipid-recognition motif (Bjorkholm et al. 2014). Most of these proteins localize to the plasma membrane and to organelles of the secretory pathway and include the alpha-1 chain of the major histocompatibility complex class II (MHC II). ...
... Most of these proteins localize to the plasma membrane and to organelles of the secretory pathway and include the alpha-1 chain of the major histocompatibility complex class II (MHC II). The transmembrane domain of the MHC class II DQ alpha 1 chain (DQA1) assembles with the DQ beta 1 chain (DQB1) as a heterodimer through GXXXG-mediated protein-protein contacts (Dixon et al. 2014;King and Dixon 2010;Travers et al. 1984) and the protein's sphingomyelin-C18 interaction motif (Bjorkholm et al. 2014). Indeed, using 3 H-Derythro-photo-sphingosine, a photo-activatable radioactive sphingolipid precursor, it was shown that DQA1 indeed interacts with a sphingolipid (Bjorkholm et al. 2014). ...
... The transmembrane domain of the MHC class II DQ alpha 1 chain (DQA1) assembles with the DQ beta 1 chain (DQB1) as a heterodimer through GXXXG-mediated protein-protein contacts (Dixon et al. 2014;King and Dixon 2010;Travers et al. 1984) and the protein's sphingomyelin-C18 interaction motif (Bjorkholm et al. 2014). Indeed, using 3 H-Derythro-photo-sphingosine, a photo-activatable radioactive sphingolipid precursor, it was shown that DQA1 indeed interacts with a sphingolipid (Bjorkholm et al. 2014). ...
The major histocompatibility complex class II (MHC II) membrane proteins are key players in the adaptive immune response. An aberrant function of these molecules is associated with a large number of autoimmune diseases such as diabetes type I and chronic inflammatory diseases. The MHC class II is assembled from DQ alpha 1 and DQ beta 1 which come together as a heterodimer through GXXXG-mediated protein–protein interactions and a highly specific protein-sphingomyelin-C18 interaction motif located on DQA1. This association can have important consequences in regulating the function of these membrane proteins. Here, we investigated the structure and topology of the DQA1 and DQB1 transmembrane helical domains by CD-, oriented ²H and ¹⁵N solid-state NMR spectroscopies. The spectra at peptide-to-lipid ratios of 0.5 to 2 mol% are indicative of a topological equilibrium involving a helix crossing the membrane with a tilt angle of about 20° and another transmembrane topology with around 30° tilt. The latter is probably representing a dimer. Furthermore, at the lowest peptide-to-lipid ratio, a third polypeptide population becomes obvious. Interestingly, the DQB1 and to a lesser extent the DQA1 transmembrane helical domains exhibit a strong fatty acyl chain disordering effect on the inner segments of the ²H-labelled palmitoyl chain of POPC bilayers. This phosphatidylcholine disordering requires the presence of sphingomyelin-C18 suggesting that the ensemble of transmembrane polypeptide and sphingolipid exerts positive curvature strain.
... The functional significance of interactions between sphingolipids and TM domains was corroborated by the discovery of a specific C18-sphingomyelin interaction motif (VXXTLXXIY) in the single membrane-spanning vesicular transport protein p24. 12 Thereafter, additional putative sphingolipid-binding motifs conforming to the relaxed motif ( 13 However, for these proteins, nothing is known regarding their regulation by sphingolipids. Over 10 proteins that contain sphingolipid interaction motifs localize to endosomal/lysosomal compartments 12,13 that represent central organelles in sphingolipid degradation. ...
... TMHMM/). Further examination revealed that LAPTM4B contains two amino acid stretches that match the postulated relaxed sphingolipid-binding motif ( Figure 1A), 12,13 one located in TM3 and the other one partially overlapping with the predicted TM1. To assess whether these TM domains play a role in the interaction between ceramide and LAPTM4B, we established an in vitro lipid transfer assay that measures the association of a fluorescent ceramide probe with large unilamellar vesicles (LUVs) containing synthetic LAPTM4B-derived TM peptides ( Figure 1B, Figure S1A,B). ...
... Acidic amino acids are underrepresented within TM helices, with an estimated 1.4% incidence in the membrane core of human proteins. 41 Remarkably, the originally identified sphingolipid-binding motif in p24 12 is accompanied by an adjacent acidic residue, and the GPI-attachment protein 1 (GAA1) that displayed the highest interaction with a crosslinkable sphingolipid probe in the follow-up study 13 also contains an acidic glutamate residue near its sphingolipid-binding motif. Moreover, examination of the identified four membranespanning proteins expressing relaxed sphingolipid-binding motifs 13 reveals that 42% (13 of 31) of them are associated with TM-embedded acidic amino acids. ...
Membrane proteins are functionally regulated by the composition of the surrounding lipid bilayer. The late endosomal compartment is a central site for the generation of ceramide, a bioactive sphingolipid, which regulates responses to cell stress. The molecular interactions between ceramide and late endosomal transmembrane proteins are unknown. Here, we uncover in atomistic detail the ceramide interaction of Lysosome Associated Protein Transmembrane 4B (LAPTM4B), implicated in ceramide-dependent cell death and autophagy, and its functional relevance in lysosomal nutrient signaling. The ceramide-mediated regulation of LAPTM4B depends on a sphingolipid interaction motif and an adjacent aspartate residue in the protein’s third transmembrane (TM3) helix. The interaction motif provides the preferred contact points for ceramide while the neighboring membrane-embedded acidic residue confers flexibility that is subject to ceramide-induced conformational changes, reducing TM3 bending. This facilitates the interaction between LAPTM4B and the amino acid transporter heavy chain 4F2hc, thereby controlling mTORC signaling. These findings provide mechanistic insights into how transmembrane proteins sense and respond to ceramide.
... In follow-up studies a number of protein candidates with signatures homologous to this highly specific sphingolipidrecognition motif were identified by a bioinformatics approach (Bjorkholm et al., 2014). The majority of these proteins are associated with organelles of the secretory pathway and the plasma membrane, including membrane anchor domains of the major histocompatibility complex class II (MHC II). ...
... The majority of these proteins are associated with organelles of the secretory pathway and the plasma membrane, including membrane anchor domains of the major histocompatibility complex class II (MHC II). The transmembrane domain of the MHC class II DQ alpha 1 chain (DQA1) assembles with the DQ beta 1 chain (DQB1) as a heterodimer through GXXXG-mediated protein-protein interactions (Russ and Engelman, 2000;Dixon and Roy, 2019) where DQA1 carries the protein's sphingomyelin-C18 interaction motif (Bjorkholm et al., 2014). A cholesterol-recognition motif has also been identified within the C-terminal regions of the TMDs of DQA1 and DQB1 (Roy et al., 2013(Roy et al., , 2016. ...
MHC class II receptors carry important function in adaptive immunity and their malfunctioning is associated with diabetes type I, chronic inflammatory diseases and other autoimmune diseases. The protein assembles from the DQ alpha-1 and DQ beta-1 subunits where the transmembrane domains of these type I membrane proteins have been shown to be involved in homo- and heterodimer formation. Furthermore, the DQ alpha 1 chain carries a sequence motif that has been first identified in the context of p24, a protein involved in the formation of COPI vesicles of the intracellular transport machinery, to specifically interact with sphingomyelin-C18 (SM-C18). Here we investigated the membrane interactions and dynamics of DQ beta-1 in liquid crystalline POPC phospholipid bilayers by oriented 15N solid-state NMR spectroscopy. The 15N resonances are indicative of a helical tilt angle of the membrane anchor sequence around 20°. Two populations can be distinguished by their differential dynamics probably corresponding the DQ beta-1 mono- and homodimer. Whereas, this equilibrium is hardly affected by the addition of 5 mole% SM-C18 a single population is visible in DMPC lipid bilayers suggesting that the lipid saturation is an important parameter. Furthermore, the DQ alpha-1, DQ beta-1 and p24 transmembrane helical domains were reconstituted into POPC or POPC/SM-C18 lipid bilayers where the fatty acyl chain of either the phosphatidylcholine or of the sphingolipid have been deuterated. Interestingly in the presence of both sphingolipid and polypeptide a strong decrease in the innermost membrane order of the POPC palmitoyl chain is observed, an effect that is strongest for DQ beta-1. In contrast, for the first time the polypeptide interactions were monitored by deuteration of the stearoyl chain of SM-C18. The resulting 2H solid-state NMR spectra show an increase in order for p24 and DQ alpha-1 which both carry the SM recognition motif. Thereby the data are suggestive that SM-C18 together with the transmembrane domains form structures imposing positive curvature strain on the surrounding POPC lipids. This effect is attenuated when SM-C18 is recognized by the protein.
... Interestingly, the transmembrane domain of p24 (p24β1) has been shown to interact specifically with one single sphingomyelin species, SM18 (Contreras et al. 2012). This interaction depends on a sphingolipid-binding motif, which is also present in several G-protein-coupled receptors (Bjorkholm et al. 2014), putative cargoes of p24 proteins (see BTransport of cargo^), and has been postulated to modulate the equilibrium between monomeric and oligomeric states of p24 proteins (Contreras et al. 2012). ...
... p24A (p24β1) has been shown to bind several other GPCRs, including PAR-1, but also several nucleotide receptors as well as a μ-opiod receptor (Luo et al. 2011). Interestingly, both p24 (p24β1) and several 7TM GPCRs contain sphingolipid-binding motifs suggesting a regulation of these proteins by sphingolipids (Bjorkholm et al. 2014). ...
p24 family proteins have been known for a long time, but their functions have remained elusive. However, they are emerging as essential regulators of protein trafficking along the secretory pathway, influencing the composition, structure, and function of different organelles in the pathway, especially the ER and the Golgi apparatus. In addition, they appear to modulate the transport of specific cargos, including GPI-anchored proteins, G-protein-coupled receptors, or K/HDEL ligands. As a consequence, they have been shown to play specific roles in signaling, development, insulin secretion, and the pathogenesis of Alzheimer's disease. The search of new putative ligands may open the way to discover new functions for this fascinating family of proteins.
... Both motifs can be present in a single TMD (Fantini et al. 2019). The SM recognition sequence (VXXTLXXIY) was identified about 10 yr ago (Contreras et al. 2012 (Björkholm et al. 2014). In addition, some more universal features of the TMDs have been used to correlate the raft association, including the length, surface areas, and palmitoylation (Lorent et al. 2017). ...
Transmembrane signaling is essential for complex life forms. Communication across a bilayer lipid barrier is elaborately organized to convey precision and to fine-tune strength. Looking back, the steps that it has taken to enable this seemingly mundane errand are breathtaking, and with our survivorship bias, Darwinian. While this review is to discuss eukaryotic membranes in biological functions for coherence and theoretical footing, we are obliged to follow the evolution of the biological membrane through time. Such a visit is necessary for our hypothesis that constraints posited on cellular functions are mainly via the biomembrane, and relaxation thereof in favor of a coordinating membrane environment is the molecular basis for the development of highly specialized cellular activities, among them transmembrane signaling. We discuss the obligatory paths that have led to eukaryotic membrane formation, its intrinsic ability to signal, and how it set up the platform for later integration of protein-based receptor activation.
... It has been proposed that several transmembrane protein motifs are responsible for specific interactions with different lipid species. For instance, Pleckstrin homology (PH) domains and Phox homology (PX) domains are predominantly responsible for recognizing phospholipids; the cholesterol consensus motifs (characterized by L/V(X)(1-5)Y(X)(1-5)R/K) are responsible for cholesterol recognition; whereas V(XX)TL(XX)IY is one of the best characterized sphingolipid-binding motifs (Björkholm et al, 2014). Nevertheless, a universal rule for lipid-peptide interaction is still lacking because of insufficient information by far. ...
Monosodium uric acid (MSU) crystal, the etiological agent of gout, has been shown to trigger innate immune responses via multiple pathways. It is known that MSU-induced lipid sorting on plasma membrane promotes the phosphorylation of Syk and eventually leads to the activation of phagocytes. However, whether this membrane lipid-centric mechanism is regulated by other processes is unclear. Previous studies showed that Clec12a, a member of the C-type lectin receptor family, is reported to recognize MSU and suppresses this crystalline structure-induced immune activation. How this scenario is integrated into the lipid sorting-mediated inflammatory responses by MSU, and particularly, how Clec12a intercepts lipid raft-originated signaling cascade remains to be elucidated. Here, we found that the ITIM motif of Clec12a is dispensable for its inhibition of MSU-mediated signaling; instead, the transmembrane domain of Clec12a disrupts MSU-induced lipid raft recruitment and thus attenuates downstream signals. Single amino acid mutagenesis study showed the critical role of phenylalanine in the transmembrane region for the interactions between C-type lectin receptors and lipid rafts, which is critical for the regulation of MSU-mediated lipid sorting and phagocyte activation. Overall, our study provides new insights for the molecular mechanisms of solid particle-induced immune activation and may lead to new strategies in inflammation control.
... Our next step, in the development of a new version of ProRafts, will be to train the predictor with additional features, such as the sphingolipid-binding sequence homologous motifs, which were identified in the human immunodeficiency virus (HIV)-1 surface envelope glycoprotein gp120, the human prior proteins (PrP) and the Alzheimer -amyloid β peptide (Mahfoud et al., 2002;Fantini, 2003), and other mammalian membrane proteins (Björkholm et al., 2014). ...
Background: Protein raftophilicity refers to the affinity of proteins for cell biomembrane lipid domains, called rafts. Rafts are fluctuating nanoscale platforms that are enriched in cholesterol and sphingolipids, and that are considered relevant for cell signalling, viral function, and biomembrane trafficking. The dynamic partitioning of proteins into rafts depends on the physical and physico-chemical properties of the biomembranes where such proteins are embedded or attached; however it also depends on specific protein features, such as acylation, glypidation, specific amino acid sequence motifs, transmembrane hydrophobic length, and surface accessible area to solvent. In this paper we present a method, and the resulting ProRafts predictor, that can be used to predict if a given mammal protein may be raftophilic or non-raftophilic, without having an a priori knowledge of the physical and physico-chemical properties of the biomembranes where such protein is embedded or attached. ProRafts is based on a machine-learning algorithm, XGBoost, where data regarding the features of known raftophilic human-proteins fed the algorithm.
Results: ProRafts enabled to predict correctly more than 80% of human proteins that are a priori known to be raftophilic; this is a promising result considering the limited size of the training dataset that we could build with data retrieved from protein databases. In addition, although we used protein features of known human raftophilic proteins, it was possible to identify accurately raft-proteins from other mammals than humans, such as mouse and rats. This finding suggests that certain protein features are sufficient to predict raftophilicity of proteins from different species. Moreover, our results indicated that phosphorylation may play a more relevant role for protein raftophilicity than indicated by previous studies.
Conclusion: Raftophilic proteins can be used as biomarkers in medical research, or can serve as targeting sites for therapeutics. In this respect, the machine learning method presented in this paper is a useful tool to guide experimental validations of raftophilicity of proteins in biomembranes, and facilitate the choice of proteins that can be used for experiments on biomimetic membranes.
... Ceramides contribute mainly, but not exclusively, to the bilayers of MAM, ER, and Golgi [92]; for example, even in late phagosomes, glucosylceramides formed by glucosylceramide synthase (GCase) and UDP glucose are elevated for ceramide processing [93]. A generic signature binding sequence, VAMTLGQIYY, for sphingomyelin [94][95][96] (structure in Figure 2B), has been identified within the TM helix of the Golgi coat protein, COPI p24 [97]. Sphingomyelin/p24 interactions regulate a functionally key monomer to dimer equilibrium. ...
Both bioactive sphingolipids and Sigma-1 receptor (S1R) chaperones occur ubiquitously in mammalian cell membranes. Endogenous compounds that regulate the S1R are important for controlling S1R responses to cellular stress. Herein, we interrogated the S1R in intact Retinal Pigment Epithelial cells (ARPE-19) with the bioactive sphingoid base, sphingosine (SPH), or the pain-provoking dimethylated SPH derivative, N,N-dimethylsphingosine (DMS). As informed by a modified native gel approach, the basal and antagonist (BD-1047)-stabilized S1R oligomers dissociated to protomeric forms in the presence of SPH or DMS (PRE-084 as control). We, thus, posited that SPH and DMS are endogenous S1R agonists. Consistently, in silico docking of SPH and DMS to the S1R protomer showed strong associations with Asp126 and Glu172 in the cupin beta barrel and extensive van der Waals interactions of the C18 alkyl chains with the binding site including residues in helices 4 and 5. Mean docking free energies were 8.73–8.93 kcal/mol for SPH and 8.56–8.15 kcal/mol for DMS, and calculated binding constants were ~40 nM for SPH and ~120 nM for DMS. We hypothesize that SPH, DMS, and similar sphingoid bases access the S1R beta barrel via a membrane bilayer pathway. We further propose that the enzymatic control of ceramide concentrations in intracellular membranes as the primary sources of SPH dictates availability of endogenous SPH and DMS to the S1R and the subsequent control of S1R activity within the same cell and/or in cellular environments.
... Sphingolipids can bind to specific pockets in the TMDs of membrane proteins (25). VXXTLXXIY is the best-characterized sphingolipid-binding motif, and new motifs are constantly being found (9). Such sphingolipid-binding domains have been found in Alzheimer, prion, and HIV-1 proteins (85). ...
Lipid–protein interactions in cells are involved in various biological processes, including metabolism, trafficking, signaling, host–pathogen interactions, and transmembrane transport. At the plasma membrane, lipid–protein interactions play major roles in membrane organization and function. Several membrane proteins have motifs for specific lipid binding, which modulate protein conformation and consequent function. In addition to such specific lipid–protein interactions, protein function can be regulated by the dynamic, collective behavior of lipids in membranes. Emerging analytical, biochemical, and computational technologies allow us to study the influence of specific lipid–protein interactions, as well as the collective behavior of membranes on protein function. In this article, we review the recent literature on lipid–protein interactions with a specific focus on the current state-of-the-art technologies that enable novel insights into these interactions.
Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The lipid bilayer of cancer cells has less unsaturated fatty acids, preventing lipid peroxidation and increasing the fluidity of the PM [168], a biophysical change associated with resistance to chemotherapy. Dynamic destabilization of lipid rafts, the main lipid microdomain, has been related to several pathologies [169], particularly as these microdomains are enriched in Cho and sphingolipids that are essential for correct cell functioning [170,171]. ...
Membranes are mainly composed of a lipid bilayer and proteins, constituting a checkpoint for the entry and passage of signals and other molecules. Their composition can be modulated by diet, pathophysiological processes, and nutritional/pharmaceutical interventions. In addition to their use as an energy source, lipids have important structural and functional roles, e.g., fatty acyl moieties in phospholipids have distinct impacts on human health depending on their saturation, carbon length, and isometry. These and other membrane lipids have quite specific effects on the lipid bilayer structure, which regulates the interaction with signaling proteins. Alterations to lipids have been associated with important diseases, and, consequently, normalization of these alterations or regulatory interventions that control membrane lipid composition have therapeutic potential. This approach, termed membrane lipid therapy or membrane lipid replacement, has emerged as a novel technology platform for nutraceutical interventions and drug discovery. Several clinical trials and therapeutic products have validated this technology based on the understanding of membrane structure and function. The present review analyzes the molecular basis of this innovative approach, describing how membrane lipid composition and structure affects protein-lipid interactions, cell signaling, disease, and therapy (e.g., fatigue and cardiovascular, neurodegenerative, tumor, infectious diseases).
... Some proteins are also involved in signal transduction or play an enzymatic role (Lane 2015;Stellacci et al. 2009). In most cases, interactions of the lipids with cholesterol or sphingomyelin in rafts is crucial for their proper functioning, thus the destabilisation of the rafts, lead to severe clinical complications (Björkholm 2014;Weiser 2014). Cancer cell lipid composition differs from the nonmalignant cell profile, but it also varies between malignancy types (Bernardes and Fialho, 2018;Casares 2019;Pakiet et al., 2019). ...
Cancer cell possesses numerous adaptations to resist the immune system response and chemotherapy. One of the most significant properties of the neoplastic cells is the altered lipid metabolism, and consequently, the abnormal cell membrane composition. Like in the case of phosphatidylcholine, these changes result in the modulation of certain enzymes and accumulation of energetic material, which could be used for a higher proliferation rate. The changes are so prominent, that some lipids, such as phosphatidylserines, could even be considered as the cancer biomarkers. Additionally, some changes of biophysical properties of cell membranes lead to the higher resistance to chemotherapy, and finally to the disturbances in signalling pathways. Namely, the increased levels of certain lipids, like for instance phosphatidylserine, lead to the attenuation of the immune system response. Also, changes in lipid saturation prevent the cells from demanding conditions of the microenvironment. Particularly interesting is the significance of cell membrane cholesterol content in the modulation of metastasis. This review paper discusses the roles of each lipid type in cancer physiology. The review combined theoretical data with clinical studies to show novel therapeutic options concerning the modulation of cell membranes in oncology.
... Importantly, the DAG:C1-EGFP-NES affinities (1/K d ) of the two natural lipids SAG and SOG differed by one order of magnitude (in both models, Fig. 5Q and SI Appendix, Fig. S5-3L), indicating a clear side-chain specificity of the DAGbinding domain. This finding demonstrates that species-specific lipid-protein interactions can occur within biological membranes, a hypothesis that has been frequently put forward (12), but only rarely experimentally tested (35). ...
Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol–protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.
... Different studies reported the important role of SL in modulating the function of different membrane proteins, including GPCR. 55,56 Thus, our findings have additional clinical implications as imply that derangement in SL metabolism and levels might impact GPCR-mediated signaling and the effects elicited by drugs targeting GPCR. 56 Downregulation of SL de novo production impairs VEGFR2mediated signaling and vasodilation following VEGF stimulation. ...
Ceramides are sphingolipids that modulate a variety of cellular processes via 2 major mechanisms: functioning as second messengers and regulating membrane biophysical properties, particularly lipid rafts, important signaling platforms. Altered sphingolipid levels have been implicated in many cardiovascular diseases, including hypertension, atherosclerosis, and diabetes mellitus-related conditions; however, molecular mechanisms by which ceramides impact endothelial functions remain poorly understood. In this regard, we generated mice defective of endothelial sphingolipid de novo biosynthesis by deleting the Sptlc2 (long chain subunit 2 of serine palmitoyltransferase)-the first enzyme of the pathway. Our study demonstrated that endothelial sphingolipid de novo production is necessary to regulate (1) signal transduction in response to NO agonists and, mainly via ceramides, (2) resting eNOS (endothelial NO synthase) phosphorylation, and (3) blood pressure homeostasis. Specifically, our findings suggest a prevailing role of C16:0-Cer in preserving vasodilation induced by tyrosine kinase and GPCRs (G-protein coupled receptors), except for Gq-coupled receptors, while C24:0- and C24:1-Cer control flow-induced vasodilation. Replenishing C16:0-Cer in vitro and in vivo reinstates endothelial cell signaling and vascular tone regulation. This study reveals an important role of locally produced ceramides, particularly C16:0-, C24:0-, and C24:1-Cer in vascular and blood pressure homeostasis, and establishes the endothelium as a key source of plasma ceramides. Clinically, specific plasma ceramides ratios are independent predictors of major cardiovascular events. Our data also suggest that plasma ceramides might be indicative of the diseased state of the endothelium.
... and at the end of this section. Interestingly, the 5-HT3R contains the sphingolipid binding motif at the extracellular part of the M1 helix (Björkholm et al., 2014). But more generally, amphipathic molecules can have a great impact on the structure and/or function of pLGIC. ...
Cys-loop receptors are pentameric ligand-gated ion channels (pLGIC), which play a crucial role in rapid neurotransmission. They are the targets of a legion of drugs (antiemetics, general anesthetics, benzodiazepines, smoke cessation drugs, etc.) and their physiological properties are intensively studied. When pLGICs bind neurotransmitters, they undergo conformational changes, from a resting closed-pore state to a transient open-pore state; they can also enter a ligand-bound, closed-pore, desensitized state. Moreover, the gating properties of pLGICs can be influenced by a variety of compounds (e.g. lipids, competitive inhibitors, allosteric modulators, ions such as Ca2+), which makes them flexible receptors capable of integrating different signals into conformational changes.In this thesis we focus on structural studies of the mouse serotonin type 3 receptor (m5-HT3R). The first structure of the m5-HT3R, obtained by X-ray crystallography using stabilizing nanobodies, was a closed-pore inhibited conformation {Hassaine:2014de}. As a follow-up, we aimed to obtain structures of the m5-HT3R in other conformations, in order to elucidate its gating mechanism. For this purpose we used both X-ray crystallography and cryo-electron microscopy and thus the whole thesis follows two story-lines.A general introduction of the pLGIC family is followed by a detailed structural description of the m5-HT3R. In the results section, we present the optimized protocol for the receptor purification, we report that limiting diffraction is a bottleneck in the crystallographic trials and we emphasize limits met using nanobodies for conformational stabilization of the receptor. In the electron microscopy results part we present the optimization of the sample and grid preparation that ultimately permitted data collection. We report four different structures representing distinct functional states of the m5-HT3R: an inhibited tropisetron-bound closed conformation, an open-pore state and a putative pre-active state obtained in the presence of serotonin, and finally a closely-related putative pre-active state in the presence of serotonin and of the allosteric modulator TMPPAA. We compare our data with structures of the same receptor obtained by other laboratory.It was shown for the first time in our work how the antagonist (tropisetron) and the neurotransmitter (serotonin) bind to the full-length m5-HT3R. And our structures deepen the knowledge of the receptor's gating mechanism.
... In addition, CD82 inhibition of tumour cell movement depends on glycosphingolipids [39,[47][48][49]. But CD82 unlikely binds glycosphingolipids directly, given that sphingolipid recognition motif [50] cannot be found in CD82. As glycosphingolipids are clustered with cholesterol in the outer leaflet [51], CD82 may interact glycosphingolipids indirectly through cholesterol. ...
Tumour metastasis suppressor KAI1/CD82 inhibits tumour cell movement. As a transmembrane protein, tetraspanin CD82 bridges the interactions between membrane microdomains of lipid rafts and tetraspanin-enriched microdomains (TEMs). In this study, we found that CD82 and other tetraspanins contain cholesterol recognition/interaction amino-acid consensus (CRAC) sequences in their transmembrane domains and revealed that cholesterol binding of CD82 determines its interaction with lipid rafts but not with TEMs. Functionally, CD82 needs cholesterol binding to inhibit solitary migration, collective migration, invasion and infiltrative outgrowth of tumour cells. Importantly, CD82–cholesterol/–lipid raft interaction not only promotes extracellular release of lipid raft components such as cholesterol and gangliosides but also facilitates extracellular vesicle (EV)–mediated release of ezrin–radixin–moesin (ERM) protein Ezrin. Since ERM proteins link actin cytoskeleton to the plasma membrane, we show for the first time that cell movement can be regulated by EV-mediated releases, which disengage the plasma membrane from cytoskeleton and then impair cell movement. Our findings also conceptualize that interactions between membrane domains, in this case converge of lipid rafts and TEMs by CD82, can change cell movement. Moreover, CD82 coalescences with both lipid rafts and TEMs are essential for its inhibition of tumour cell movement and for its enhancement of EV release. Finally, our study underpins that tetraspanins as a superfamily of functionally versatile molecules are cholesterol-binding proteins.
Abbreviations: Ab: antibody; CBM: cholesterol-binding motif; CCM: cholesterol consensus motif; CRAC/CARC: cholesterol recognition or interaction amino-acid consensus; CTxB: cholera toxin B subunit; ECM: extracellular matrix; ERM: ezrin, radixin and moesin; EV: extracellular vesicles; FBS: foetal bovine serum; mAb: monoclonal antibody; MST: microscale thermophoresis; pAb: polyclonal antibody; and TEM: tetraspanin-enriched microdomain
... More recently, the protein p24 was found to bind to sphingomyelin via a defined region [49]. Based on this finding, a set of sphingolipid-binding motifs, e.g., VX2V2X2LF, was elucidated [50]. However, to our knowledge, no interactions between sphingolipids and specific residues within these motifs were identified, nor is it clear where the required terminal aromatic residue of the motif should be positioned relative to the membrane in which the protein is embedded. ...
The accumulation of lipids in the late endosomes and lysosomes of Niemann–Pick type C disease (NPCD) cells is a consequence of the dysfunction of one protein (usually NPC1) but induces dysfunction in many proteins. We used molecular docking to propose (a) that NPC1 exports not just cholesterol, but also sphingosine, (b) that the cholesterol sensitivity of big potassium channel (BK) can be traced to a previously unappreciated site on the channel’s voltage sensor, (c) that transient receptor potential mucolipin 1 (TRPML1) inhibition by sphingomyelin is likely an indirect effect, and (d) that phosphoinositides are responsible for both the mislocalization of annexin A2 (AnxA2) and a soluble NSF (N-ethylmaleimide Sensitive Fusion) protein attachment receptor (SNARE) recycling defect. These results are set in the context of existing knowledge of NPCD to sketch an account of the endolysosomal pathology key to this disease.
... Lastly our work reveals established TMED2 functions in the secretory pathway to regulate transport of GPI-anchored protein to the apical membrane and secretion to the extracellular space are required in during labyrinth layer formation. Our work also suggests that less established function for TMED2 in quality control, to ensure that properly modified or folded proteins exit the secretory pathway, and in ensuring the proper lipid association in proteins exiting the secretory pathway (Björkholm et al., 2014;Contreras et al., 2012) may also be important for chorioallantoic attachment and fusion. ...
TMED2, a member of the transmembrane emp24 domain (TMED) family, is required for transport of cargo proteins between the ER and Golgi. TMED2 is also important for normal morphogenesis of mouse embryos and their associated placenta, and in fact Tmed2 homozygous mutant embryos arrest at mid-gestation due to a failure of placental labyrinth layer formation. Differentiation of the placental labyrinth layer depends on chorioallantoic attachment (contact between the chorion and allantois), and branching morphogenesis (mingling of cells from these two tissues). Since Tmed2 mRNA was found in both the chorion and allantois, and 50% of Tmed2 homozygous mutant embryos failed to undergo chorioallantoic attachment, the tissue-specific requirement of Tmed2 during placental labyrinth layer formation remained a mystery. Herein, we report differential localization of TMED2 protein in the chorion and allantois, abnormal ER retention of Fibronectin in Tmed2 homozygous mutant allantoises and cell-autonomous requirement for Tmed2 in the chorion for chorioallantoic attachment and fusion. Using an ex vivo model of explanted chorions and allantoises, we showed that chorioallantoic attachment failed to occur in 50% of samples when homozygous mutant chorions were recombined with wild type allantoises. Furthermore, though expression of genes associated with trophoblast differentiation was maintained in Tmed2 mutant chorions with chorioallantoic attachment, expression of these genes was attenuated. In addition, Tmed2 homozygous mutant allantoises could undergo branching morphogenesis, however the region of mixing between mutant and wild type cells was reduced, and expression of genes associated with trophoblast differentiation was also attenuated. Our data also suggest that Fibronectin is a cargo protein of TMED2 and indicates that Tmed2 is required cell-autonomously and non-autonomously in the chorion and the allantois for placental labyrinth layer formation.
... Additionally, the atypical and recently described deoxysphingolipids were reported to cause mitochondrial fragmentation and inhibit mitochondrial function [53]. As key players in membrane structure and topology, membrane sphingolipids may regulate numerous functions of membranebound proteins [54] as well as membrane-localized events that are sensitive to alterations in membrane biophysical properties, including signal transduction through membrane signaling platforms, or endocytosis. Thus, while few molecular mechanisms for ceramides in heart have been shown, it seems reasonable that mechanisms established to play roles in heart-relevant processes (e.g. ...
Obesity, type 2 diabetes, and metabolic syndrome induce dyslipidemia resulting in inundation of peripheral organs with fatty acids. These not only serve as substrates for energy production, but also contribute to aberrant production of bioactive lipids. Moreover, lipid metabolism is affected in many cardiac disorders including heart failure, ischemia reperfusion injury, and others. While lipids serve crucial homeostatic roles, perturbing biosynthesis of lipid mediators leads to aberrant cell signaling, which contributes to maladaptive cardiovascular programs. Bioactive sphingolipids, in particular, have been implicated in pathophysiology in the heart and vasculature by a variety of studies in cells, animal models, and humans. Because of the burgeoning interest in sphingolipid-driven biology in the cardiovascular system, it is necessary to discuss the experimental considerations for studying sphingolipid metabolism and signaling, emphasizing the caveats to some widely available experimental tools and approaches. Additionally, there is a growing appreciation for the diversity of ceramide structures generated via specific enzymes and bearing disparate cellular functions. While targeting these individual species and enzymes constitutes a major advance, studies show that sphingolipid synthesis readily adapts to compensate for experimental targeting of any individual pathway, thereby convoluting data interpretation. Furthermore, though some molecular mechanisms of sphingolipid action are known, signaling pathways impacted by sphingolipids remain incompletely understood. In this review, we discuss these issues and highlight recent studies as well as future directions that may extend our understanding of the metabolism and signaling actions of these enigmatic lipids in the cardiovascular context.
... We sought to determine the specific mechanism by which MHC II overcrowding alters the lipid raft or tetraspanin web and determine whether this alteration is responsible for poor engagement and activation of thymocytes by MHC II K-deficient DCs. It has been suggested that MHC II binds cholesterol and sphingolipid through its transmembrane domain (Roy et al., 2013;Björkholm et al., 2014). We postulated that cholesterol and sphingolipid in the lipid raft and tetraspanin web may be sequestered by MHC II because MHC II accumulates in these membrane domains. ...
Dendritic cells (DCs) produce major histocompatibility complex II (MHCII) in large amounts to function as professional antigen presenting cells. Paradoxically, DCs also ubiquitinate and degrade MHCII in a constitutive manner. Mice deficient in the MHCII-ubiquitinating enzyme membrane-anchored RING-CH1, or the ubiquitin-acceptor lysine of MHCII, exhibit a substantial reduction in the number of regulatory T (Treg) cells, but the underlying mechanism was unclear. Here we report that ubiquitin-dependent MHCII turnover is critical to maintain homeostasis of lipid rafts and the tetraspanin web in DCs. Lack of MHCII ubiquitination results in the accumulation of excessive quantities of MHCII in the plasma membrane, and the resulting disruption to lipid rafts and the tetraspanin web leads to significant impairment in the ability of DCs to engage and activate thymocytes for Treg cell differentiation. Thus, ubiquitin-dependent MHCII turnover represents a novel quality-control mechanism by which DCs maintain homeostasis of membrane domains that support DC’s Treg cell–selecting function.
... ENG did not associate with any particular SM in this compartment ( Supplementary Fig. 2D), suggesting that not all ENG in the trans-Golgi was sequestered in the SM-18:0 enriched lipid compartment. In silico analysis did not reveal any known sphingolipid-binding motif in ENG and MMP14 46 . ...
Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women.
... The composition of biological membranes varies as particularly the pool of phosphoinositides (PIs) fluctuates and in this way modulates the activity of proteins. PIs can bind to regions of disorder, whereas cholesterol and sphingolipids typically bind to the structured transmembrane domains [150][151][152]. PI typically, but not always binds within the JM region and may further interact with residues from transmembrane helices. ...
Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane proteins, and address why the flexibility afforded by disorder is mechanistically important. Intrinsically disordered regions are present in many common classes of membrane proteins including ion channels and transporters; G-protein coupled receptors (GPCRs), receptor tyrosine kinases and cytokine receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding protein disorder.
... Several cholesterol and sphingolipid binding motifs for partitioning various proteins in lipid rafts have been described in the literature and include aliphatic (L, V), aromatic (Y, F) and positively charged (K and R) amino acid residues (reviewed in Epand et al., 2010;Björkholm et al., 2014). The most popular are the Cholesterol Recognition/interaction Amino acid Consensus sequence (CRAC domain) (L/V)-X 1−5 -(Y)-X 1−5 -(K/R) and the CARC domain (K/R)-X 1−5 -(Y/F)-X 1−5 -(L/V), which exhibits the opposite orientation along the polypeptide chain. ...
... The sequence analysis of GPCRs shows that sphingolipids binding motifs are often presented in its sixth helix. This helix is involved in the large-scale relocation required for binding the G protein during receptor activation [66,67]. We found that in the D 1 receptor there are more cholesterol recognition/interaction aminoacid consensus CRAC and CARC motifs than the CBM binding motifs (Table S1). ...
... Similarly the b2-adrenergic receptor (b2R) was shown to require cholesterol at the dimer interface to preserve its native structure [14,15]. Interestingly, a recent survey of over 100 high resolution structures of membrane proteins with bound lipids did not arrive at any common molecular determinants for lipid binding sites, precluding the reliable prediction of selective lipid interactions in all but a few instances [16,17]. Therefore, insights into the connection between structural and functional implications of lipid binding are for the most part limited to proteinspecific, highly selective interactions that can be maintained even under delipidating conditions. ...
Biological membranes form barriers that are essential for cellular integrity and compartmentalisation. Proteins in the membrane have co-evolved with their hydrophobic lipid environment, which serves as a solvent for proteins with very diverse requirements. As a result, their interactions range from non-selective to highly discriminating. Mass spectrometry enables us to monitor how lipids interact with membrane proteins and assess their effects on structure and dynamics. Recent studies illustrate the ability to differentiate specific lipid binding, preferential interactions with lipid subsets, and nonselective annular contacts. Here, we consider the biological implications of different lipid-binding scenarios and propose that binding occurs on a sliding selectivity scale, in line with the view of biological membranes as facilitators of dynamic protein and lipid organization.
... In addition, a sphingolipid binding motif (VXXTLXXIY) has been identified in the transmembrane domain of p24, a component of the COP-I machinery, which results in a highly specific interaction of p24 with sphingomyelin (55). Recent bioinformatic analysis of mammalian proteomes has uncovered multiple membrane proteins containing predicted sphingolipid binding motifs, including many G-protein-coupled receptors (56). These findings suggest that there are many more sphingolipid binding proteins yet to be discovered. ...
... Binding of raft lipids like podoplanin might be one strategy to integrate into lipid rafts. INTRODUCTION 17 [163] and can be located in single and in multiple spanning proteins like GPCRs (Gprotein coupled receptors) [164]. ...
Das Hämagglutinin (HA) der Influenzaviren wird während der Assemblierung in Cholesterin- und Sphingolipid-reiche Domänen (Rafts) der Plasmamembran rekrutiert. Vorangehende Studien konnten mittels Fluoreszenzresonanzenergietransfer eine Raft-Integration nachweisen, die von zwei Raft-Zielsignalen abhängig war; zum einen von drei S-acylierten Cysteinen in der zytoplasmatischen Domäne und zum anderen von hydrophoben Aminosäuren (VIL) am Beginn der Transmembrandomäne (TMD). Zudem zeigte sich ein möglicher Einfluss des VIL-Motives auf den intrazellulären Proteintransport. Um diese Annahme zu bestätigen, wurden HA Mutanten in Zellen exprimiert und ihre Ankunft im medialen und trans-Golgi verfolgt. In dieser Arbeit konnte eine Beteiligung des VIL-Motives am Transport bestätigt werden, jedoch nicht der S-Acylierungen. Zudem wurde eine generelle Abhängigkeit des Transportes von der Sphingolipidsynthese beobachtet. Da sowohl die Cholesterinsynthese als auch die Sphingolipidsynthese für den Transport von HA benötigt werden, habe ich die Hypothese aufgestellt, dass das VIL-Motiv in der Lage sein könnte, mit Raft Lipiden zu interagieren. Ein Sequenzvergleich ergab, dass kein Sphingolipid-Bindemotiv vorhanden ist, jedoch ein potenzielles Cholesterin-Consensus-Motiv (CCM, W/Y-I/V/L-K/R). Dieses Motiv wurde nur in der Sequenz von Gruppe 1 jedoch nicht Gruppe 2 HAs gefunden und umfasst das Leucin des VIL Motives. Tatsächlich ist die Mutation des Leucins aber nicht des vorangehenden Isoleucins für den verzögerten Transport verantwortlich. Untersuchungen weiter Einzel- und Mehrfachmutanten konnten eine Abhängigkeit des intrazellulären Transportes von einer möglichen Cholesterinbindung verifizieren. Zudem konnte auch ein zunehmender Effekt auf die Kinetiken vom medialen Golgi zum TGN beobachtet werden, welcher auch die Oberflächenexpression negativ beeinflusste. FLIM-FRET Analysen zeigten zusätzlich eine reduzierte Raft Assoziation der CCMMutanten mit Rafts an der Plasmamembran. Daher kann man spekulieren, dass HA mit Cholesterin interagiert, wodurch sein intrazellulärer Transport durch den Golgi und die Assoziation mit Rafts gewährleistet wird.
... As a negative control for protein-sphingolipid interactions, we used ASGR1, which is lacking the sphingolipid-binding motif of p24 and GPAA1, but is exposed to the bulk 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 13 pool of cellular sphingolipids at the plasma membrane. 33 No crosslinked product of ASGR1 with sphingolipids was observed. Interestingly, we observed a slower migration behavior of nearly all membrane spanning proteins in pacFA/+UV treated cells, which was not observed in pacSph/+UV-treated cells. ...
Sphingolipids are essential structural components of cellular membranes and are crucial regulators of cellular processes. While current high-throughput approaches allow for the systematic mapping of interactions of soluble proteins with their lipid-binding partners, photo-crosslinking is the only technique that enables for the proteome-wide mapping of integral membrane proteins with their direct lipid environment. Here we report the synthesis of a photoactivatable and clickable analog of sphingosine. When administered to sphingosine-1-phosphate lyase deficient cells, pacSph allows its metabolic fate and the subcellular flux of de novo synthesized sphingolipids to be followed in a time resolved manner. The chemoproteomic profiling yielded over 180 novel sphingolipid-binding proteins, of which we validated a number, demonstrating the unique value of this technique as a discovery tool. This work provides an important resource for the understanding of the global cellular interplay between sphingolipids and their interacting proteins.
... Of note, LAPTM proteins have been implicated in facilitating transmembrane movement of small amphiphilic molecules 23,[26][27][28][29] . Interestingly, the predicted third transmembrane domain of LAPTM4B contains the motif LVAITVLIY, matching the relaxed sphingolipid binding motif described 45,46 . However, when we introduced the interfering T212F mutation 45 in this motif, the protein remained functional as judged by its ability to rescue LE ceramide-BODIPY accumulation in LAPTM4B-depleted cells (data not shown). ...
Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase-dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.
... Recently, a sphingolipid motif was found in the transmembrane protein p24, which is involved in vesicular transport in the early secretory pathway (Contreras et al., 2012). Later a number of membrane proteins with a similar motif have been found (Bjorkholm et al., 2014), indicating that such lipid binding may be a relatively common event. The binding of lipids to proteins may regulate their function, as for example in the case of p24, but possibly lipids bound to the surface of transmembrane proteins could influence how they partition between different lipid environments in much the same way as palmitoylation has been reported to do. ...
Natural liquid crystalline membranes are made up of many different lipids carrying a mixture of saturated and unsaturated fatty acyl chains. Whereas in the past considerable attention has been paid to cholesterol content, the phospholipid head groups and the membrane surface charge the detailed fatty acyl composition was often considered less important. However, recent investigations indicate that the detailed fatty acyl chain composition has pronounced effects on the oligomerization of the transmembrane helical anchoring domains of the MHC II receptor or the membrane alignment of the cationic antimicrobial peptide PGLa. In contrast the antimicrobial peptides magainin 2 and alamethicin are less susceptible to lipid saturation. Using histidine-rich LAH4 designer peptides the high energetic contributions of lipid saturation in stabilizing transmembrane helical alignments are quantitatively evaluated. These observations can have important implications for the biological regulation of membrane proteins and should be taken into considerations during biophysical or structural experiments.
The cytokine interferon‐gamma (IFN‐γ) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN‐γ exerts it signaling action by binding to a specific cell surface receptor, the IFN‐γ receptor (IFN‐γR), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN‐γR signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN‐γR2 chains into plasma membrane lipid nanodomains, orchestrating IFN‐γR oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol‐binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN‐γR transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol‐IFNγR2 interaction may represent a potential therapeutic strategy for various IFN‐γ‐dependent diseases. This work uncovers the role of cholesterol as the molecular determinant controlling interferon‐gamma receptor (IFN‐γR) activity. Through a multidisciplinary approach the structural signature within the IFN‐γR required for cholesterol binding is identified. Binding of cholesterol is critical for receptor plasma membrane compartmentalization, heterodimerization, and signal transduction. This work identifies a therapeutic target for prevention and treatment of IFN‐γ dependent diseases.
Membrane proteins often cluster in nanoscale membrane domains (lipid rafts) that coalesce into ceramide-rich platforms during cell stress, however the clustering mechanisms remain uncertain. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis (CF), forms clusters that are cholesterol dependent and become incorporated into long-lived platforms during hormonal stimulation. We report here that clustering does not involve known tethering interactions of CFTR with PDZ domain proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are abolished by the disease mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate CF therapeutics that correct mutant protein folding restore both trafficking and normal lipid interactions in the plasma membrane.
This article has an associated First Person interview with the first author of the paper.
Membranes are complex structures composed primarily of proteins and lipids stabilised by dynamic cooperative non-covalent interactions. During the course of evolution, proteins and lipids have co-evolved following a set of interdependent rules based on their respective physical and chemical properties that govern the biogenesis, organisation and function of membrane proteins. Our understanding of the function of membrane lipids has progressed from a static canonical role as a cell barrier matrix supporting membrane proteins to dynamic roles as molecular chaperones, topological determinants, allosteric ligands and organisers of complex biological machines. Combined molecular genetic and biochemical approaches have uncovered a picture of biological membranes in which lipids and proteins interact dynamically, reversibly, temporally and spatially depending on different cellular circumstances and demands. Membrane proteins can undergo conformational changes and even transmembrane re-organisation in response to variations in the lipid environment or posttranslational modification without a severe thermodynamic cost. Lipids can contribute to execution of genetic code by decoding protein sequences subject to a variable lipid composition.
Structural lipids in membranes are composed of three general types of membrane lipids: (glycero)phospholipids, sphingolipids, and sterols. Sphingolipids are the derivatives of sphingosine and sites of biological recognition. Sphingolipid does not contain glycerol, different from (glycero)phospholipid. Sphingolipid contains a long-chain fatty acid and sphingosine (4-sphingenine)-based long-chain amino alcohol, and in certain case, phosphoric acid is diester-linked to the polar head group. Sphingolipid has a three subclasses of ceramide derivatives, including sphingomyelins, neural (uncharged) glycolipid, and ganglioside, depending on head groups. Sphingomyelin has phosphocholine or phosphoethanol-amine as a polar head and thus these can belong to a phospholipid. Neutral glycolipid and ganglioside have one or more sugar residues in their head groups and linked C-1 OH of ceramide without phosphate. These sugar-containing sphingolipids are also called GSL.
Eukaryotic membranes can be partitioned into lipid-driven, ordered membrane microdomains called lipid rafts, which function to laterally sort membrane lipids and proteins. As protein selectivity underlies the functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of protein affinity for ordered membrane domains. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multi-pass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored. Here, we used cell-derived Giant Plasma Membrane Vesicles (GPMVs) to systematically explore multi-pass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multi-pass TMPs, the large majority (92%) had minimal raft affinity. The only exceptions were two myelin-associated four pass TMPs, Myelin And Lymphocyte protein (MAL) and ProteoLipid Protein (PLP). We characterized the mechanisms for their exceptional raft affinity and observed that PLP requires cholesterol and sphingolipids for optimal ordered domain association and that PLP and MAL appear to compete for cholesterol-mediated raft affinity. These observations suggest broad conclusions about the composition of ordered membrane domains in cells and point to previously unrecognized drivers of raft affinity for multi-pass transmembrane proteins.
Plant plasmodesmata (PDs) are specialized channels that enable communication between neighboring cells. The intercellular permeability of PDs affecting plant development, defense and responses to stimulus must be tightly regulated. Analysis of specific PD membrane lipid composition and their impact on PD permeability will provide new insights into PD regulatory mechanism. Herein, we report that the Arabidopsis sld1 sld2 double mutant, lacking sphingolipid long-chain base 8 desaturases 1 and 2, displayed decreased PD permeability due to a significant increase in callose accumulation. Plasmodesmata-located protein 5 (PDLP5) was significantly enriched in the leaf epidermal cells of sld1 sld2 and showed specific binding affinity to phytosphinganine (t18:0), suggesting that the enrichment of t18:0-based sphingolipids in sld1 sld2 PDs might facilitate the recruitment of more PDLP5. The double mutants showed enhanced resistance to the fungal-wilt pathogen Verticillium dahlia or the bacterium Peudomonas syringae pv tomato DC3000. This phenotype was fully restored in sld1 sld2 pdlp5. Thus, we proposed that phytosphinganine might regulate PDs functions and cell-to-cell communication by modifying the level of PDLP5 in PD membranes.
Every cell produces thousands of distinct lipid species, but methodology for studying the biological roles of individual lipids is insufficient. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We developed novel photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modelling enabled the determination of binding constants for diacylglycerol-protein interactions and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol structural diversity. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and thus differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales.
The p24 proteins play an important role in the secretory pathway where they selectively connect various cargo to other proteins thereby being involved in the controlled assembly and disassembly of the coat protein complexes and lipid sorting. Recently, a highly selective lipid interaction motif has been identified within the p24 transmembrane domain (TMD) which recognizes the combination of sphingomyelin head group and the exact length of the C18 fatty acyl chain (SM-C18). Here we present investigations of the structure, dynamics and sphingomyelin interactions of the p24 transmembrane region using CD-, tryptophan fluorescence- and solid-state NMR spectroscopies of the polypeptides and the surrounding lipids. Membrane insertion and/or conformation of the TMD are strongly dependent on the membrane lipid composition where the transmembrane helical insertion is strongest in the presence of POPC and SM-C18. By analyzing solid-state NMR angular restraints from a large number of labelled sites a tilt angle of 19 degrees has been found for the transmembrane helical domain at a peptide-to-lipid ratio of 1 mole%. Only minor changes in the solid-state NMR spectra are observed due to the presence of SM-C18, the only visible alterations are associated with the SM-C18 recognition motif close to the carboxyterminal part of the hydrophobic transmembrane region in proximity of the SM head group. Finally, the deuterium order parameters of POPC-d31 are nearly unaffected by the presence of SM-C18 or the polypeptide alone, but decreases noticeably when the sphingomyelin and the polypeptide were added in combination.
Recent years have witnessed the evolution of the cell biology of lipids into an extremely active area of investigation. Deciphering the involvement of lipid metabolism and lipid signaling in membrane trafficking pathways defines a major nexus of contemporary experimental activity on this front. Significant effort in that direction is invested in understanding the trans-Golgi network/endosomal system where unambiguous connections between membrane trafficking and inositol lipid and phosphatidylcholine metabolism were first discovered. However, powered by new advances in contemporary cell biology, the march of science is rapidly expanding that window of inquiry to include ever more diverse arms of the lipid metabolome, and to include other compartments of the secretory pathway as well.
The membrane topology of the peptide 18A, a derivative of apolipoprotein A-I, is investigated in structural detail. Apolipoprotein A-I is the dominant protein component of high density lipoproteins with important functions in cholesterol metabolism. 18A (Ac-DWLKA FYDKV AEKLK EAF- NH 2 ) was designed to mimic the structure of tandem domains of class A amphipathic helices and has served as a lead peptide for biomedical applications. At low peptide-to-lipid ratios 18A partitions into phosphatidylcholine membranes with helix topologies parallel to the membrane surface, an alignment that is maintained when disc-like bicelles form at higher peptide-to-lipid ratios. Notably, the bicelles interact cooperatively with the magnetic field of the NMR spectrometer, thus the bilayer normal is oriented perpendicular to the magnetic field direction. A set of peptides that totals four ¹⁵ N or ² H labelled positions of 18A allowed the accurate analysis of tilt and azimuthal angles relative to the membrane surface under different conditions. The topology agrees with a double belt arrangement forming a rim that covers the hydrophobic fatty acyl chains of the bicelles. In another set of experiments, it was shown that POPC nanodiscs prepared in the presence of diisobutylene/maleic acid (DIBMA) polymers can also be made to align in the magnetic field. Finally, the transmembrane domains of the DQ alpha-1 and DQ beta-1 subunits of the major histocomptability complex (MHC) class II have been prepared and reconstituted into magnetically oriented bicelles for NMR structural analysis.
Sphingolipids correspond to a major class of lipids which serve as indispensable structural components of membranes and play an important role in various cellular functions. They constitute ~10–20% of total membrane lipids and are known to form segregated domains in biological membranes. Sphingolipids have been shown to play a vital role in the function of various G protein-coupled receptors (GPCRs). We report here the presence of sphingolipid-binding motif (SBM) in representative GPCRs such as cholecystokinin, oxytocin and secretin receptors, and subtypes of human serotonin receptors. We previously reported the importance of sphingolipids in the function of the serotonin1A receptor, a representative member of the GPCR superfamily, involved in behavioral, cognitive, and developmental functions. In this work, we show that the serotonin1A receptor contains a putative SBM, corresponding to amino acids 205 to 213. In addition, our analysis shows that SBM is an intrinsic characteristic feature of the serotonin1A receptor and is conserved throughout the course of natural evolution. Our results represent the first report on the presence of SBM in serotonin1A receptors and provide novel insight on the molecular mechanism of GPCR-sphingolipid interaction.
A molecular-level insight into the lipid binding to membrane proteins and how it induces a protein’s conformational change.
Sphingolipids are highly enriched in the nervous system where they are pivotal constituents of the plasma membranes and are important for proper brain development and functions. Sphingolipids are not merely structural elements, but are also recognized as regulators of cellular events by their ability to form microdomains in the plasma membrane. The significance of such compartmentalization spans broadly from being involved in differentiation of neurons and synaptic transmission to neuronal–glial interactions and myelin stability. Thus, perturbations of the sphingolipid metabolism can lead to rearrangements in the plasma membrane, which has been linked to the development of various neurological diseases. Studying microdomains and their functions has for a long time been synonymous with studying the role of cholesterol. However, it is becoming increasingly clear that microdomains are very heterogeneous, which among others can be ascribed to the vast number of sphingolipids. In this review, we discuss the importance of microdomains with emphasis on sphingolipids in brain development and function as well as how disruption of the sphingolipid metabolism (and hence microdomains) contributes to the pathogenesis of several neurological diseases.
Sphingolipids in general and ceramides in particular, contribute to pathophysiological mechanisms by modifying signalling and metabolic pathways. Here, we present the available evidence for a bidirectional homeostatic crosstalk between sphingolipids and glycerophospholipids, whose dysregulation contributes to lipotoxicity induced metabolic stress. The initial evidence for this crosstalk originates from simulated models designed to investigate the biophysical properties of sphingolipids in plasma membrane representations. In this review, we reinterpret some of the original findings and conceptualise them as a sort of “ying/yang” interaction model of opposed/complementary forces, which is consistent with the current knowledge of lipid homeostasis and pathophysiology. We also propose that the dysregulation of the balance between sphingolipids and glycerophospholipids results in a lipotoxic insult relevant in the pathophysiology of common metabolic diseases, typically characterised by their increased ceramide/sphingosine pools.
To evaluate the precise role of sphingomyelin synthase 2 (SMS2) in sphingomyelin (SM) metabolism and their anti-inflammatory properties, we analyzed species of major SM and ceramide (Cer) (18:1, 18:0 sphingoid backbone, C14 - C26 N-acyl part) in SMS2 knockout and wild-type mouse plasma and liver using HPLC-MS. SMS2 deficiency significantly decreased very long chain SM (SM (d18:1/22:0) and SM (d18:1/24:0 or d18:0/24:1)) and increased very long chain Cer (Cer (d18:1/24:0 or d18:0/24:1) and Cer (d18:1/24:1)), but not long chain SM (SM (d18:1/16:0), SM (d18:1/18:0 or d18:0/18:1) and SM (d18:1/18:1)) in plasma.
To examine the effects of SM on inflammation, we studied the role of very long chain SM in macrophage activation. Addition of SM (d18:1/24:0) strongly upregulated several macrophage activation markers, SM (d18:1/6:0) and Cer (d18:1/24:0) however, did not.
It was suggested that very long chain SM but not long chain SM were decreased in SMS2-deficient mice liver and plasma. And the exogenously added very long chain SM (d18:1/24:0) could activate macrophages directly, suggesting a novel role of plasma very long chain SM in modulating macrophage activation and resulting inflammation.
The plasma membrane is an essential cellular structure that separates the cell interior from the extracellular environment, while allowing for the exchange of signals and materials that are essential for cell survival and function. The complexity of the lipids in the plasma membrane has been long appreciated, but recent developments in lipidomics and imaging technologies have improved our understanding of plasma membrane lipid dynamics. New studies have started to unveil important functions for plasma membrane lipids in regulating T cell signalling. Importantly, it has been shown that the modulation of membrane lipids can be used to harness T cell activity to treat cancer and autoimmunity. Therefore, lipid-based immunotherapy might be a promising new clinical strategy.
The authors Rabie Saidi and Tunca Dogan were omitted from the list of the UniProt consortium in the acknowledgements section of this paper. The corrected consortium list is provided below.
The UniProt Consortium
UniProt has been prepared by Rolf Apweiler, Alex Bateman, Maria Jesus Martin, Claire O'Donovan, Michele Magrane, Yasmin Alam–Faruque, Emanuele Alpi, Ricardo Antunes, Joanna Arganiska, Elisabet Barrera Casanova, Benoit Bely, Mark Bingley, Carlos Bonilla, Ramona Britto, Borisas Bursteinas, Wei Mun Chan, Gayatri Chavali, Elena Cibrian–Uhalte, Alan Da Silva, Maurizio De Giorgi, Tunca Dogan, Francesco Fazzini, Paul Gane, Leyla Garcia Castro, Penelope Garmiri, Emma Hatton–Ellis, Reija Hieta, Rachael Huntley, Duncan Legge, Wudong Liu, Jie Luo, Alistair MacDougall, Prudence Mutowo, Andrew Nightingale, Sandra Orchard, Klemens Pichler, Diego Poggioli, Sangya Pundir, Luis Pureza, Guoying Qi, Steven Rosanoff, Rabie Saidi, Tony Sawford, Aleksandra Shypitsyna, Edward Turner, Vladimir Volynkin, Tony Wardell, Xavier Watkins, Hermann Zellner, Matt Corbett, Mike Donnelly, Pieter van Rensburg, Mickael Goujon, Hamish McWilliam and Rodrigo Lopez at the European Bioinformatics Institute (EMBL–EBI); Ioannis Xenarios, Lydie Bougueleret, Alan Bridge, Sylvain Poux, Nicole Redaschi, Lucila Aimo, Andrea Auchincloss, Kristian Axelsen, Parit Bansal, Delphine Baratin, Pierre–Alain Binz, Marie–Claude Blatter, Brigitte Boeckmann, Jerven Bolleman, Emmanuel Boutet, Lionel Breuza, Cristina Casal–Casas, Edouard de Castro, Lorenzo Cerutti, Elisabeth Coudert, Beatrice Cuche, Mikael Doche, Dolnide Dornevil, Severine Duvaud, Anne Estreicher, Livia Famiglietti, Marc Feuermann, Elisabeth Gasteiger, Sebastien Gehant, Vivienne Gerritsen, Arnaud Gos, Nadine Gruaz–Gumowski, Ursula Hinz, Chantal Hulo, Janet James, Florence Jungo, Guillaume Keller, Vicente Lara, Philippe Lemercier, Jocelyne Lew, Damien Lieberherr, Thierry Lombardot, Xavier Martin, Patrick Masson, Anne Morgat, Teresa Neto, Salvo Paesano, Ivo Pedruzzi, Sandrine Pilbout, Monica Pozzato, Manuela Pruess, Catherine Rivoire, Bernd Roechert, Michel Schneider, Christian Sigrist, Karin Sonesson, Sylvie Staehli, Andre Stutz, Shyamala Sundaram, Michael Tognolli, Laure Verbregue and Anne–Lise Veuthey at the SIB Swiss Institute of Bioinformatics (SIB); Cathy H. Wu, Cecilia N. Arighi, Leslie Arminski, Chuming Chen, Yongxing Chen, John S. Garavelli, Hongzhan Huang, Kati Laiho, Peter McGarvey, Darren A. Natale, Baris E. Suzek, C. R. Vinayaka, Qinghua Wang, Yuqi Wang, Lai–Su Yeh, Meher Shruti Yerramalla and Jian Zhang at the Protein Information Resource (PIR).
The mission of the Universal Protein Resource (UniProt) (http://www.uniprot.org) is to support biological research by providing a freely accessible, stable, comprehensive, fully classified, richly and
accurately annotated protein sequence knowledgebase. It integrates, interprets and standardizes data from numerous resources
to achieve the most comprehensive catalogue of protein sequences and functional annotation. UniProt comprises four major components,
each optimized for different uses, the UniProt Archive, the UniProt Knowledgebase, the UniProt Reference Clusters and the
UniProt Metagenomic and Environmental Sequence Database. UniProt is produced by the UniProt Consortium, which consists of
groups from the European Bioinformatics Institute (EBI), the SIB Swiss Institute of Bioinformatics (SIB) and the Protein Information
Resource (PIR). UniProt is updated and distributed every 4 weeks and can be accessed online for searches or downloads.
Metabotropic glutamate receptor (mGluR), a prototypical family 3 G protein-coupled receptor (GPCR), has served as a model for studying GPCR dimerization, and growing evidence has revealed that a glutamate-induced dimeric rearrangement promotes activation of the receptor. However, structural information of the seven-transmembrane domain (TMD) is severely limited, in contrast to the well-studied family 1 GPCRs including rhodopsins and adrenergic receptors. Homology modeling of mGluR8 TMD with rhodopsin as a template suggested the presence of a conserved water-mediated hydrogen-bonding network (HBN) between helices VI and VII, which presumably constrains the receptor in an inactive conformation. We therefore conducted a mutational analysis to assess structural similarities between mGluR and family 1 GPCRs. Mutational experiments confirmed that the disruption of the HBN by T789Y(6.43) mutation induced high constitutive activity. Unexpectedly, this high constitutive activity was suppressed by glutamate, the natural agonist ligand, indicating that glutamate acts as a partial inverse agonist to this mutant. Fluorescence energy transfer analysis of T789Y(6.43) suggested that the glutamate-induced reduction of the activity originated not from the dimeric rearrangement but from conformational changes within each protomer. Double mutational analysis showed that the specific interaction between Y789(6.43) and G831(7.45) in T789Y(6.43) mutant was important for this phenotype. Therefore, the present study is consistent with the notion that mGluR shares a common activation mechanism with family 1 GPCRs, where rearrangement between helices VI and VII causes the active state formation.
PROSITE (http://prosite.expasy.org/) consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns
and profiles to identify them. It is complemented by ProRule a collection of rules, which increases the discriminatory power
of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids.
PROSITE signatures, together with ProRule, are used for the annotation of domains and features of UniProtKB/Swiss-Prot entries.
Here, we describe recent developments that allow users to perform whole-proteome annotation as well as a number of filtering
options that can be combined to perform powerful targeted searches for biological discovery. The latest version of PROSITE
(release 20.85, of 30 August 2012) contains 1308 patterns, 1039 profiles and 1041 ProRules.
Summary: CD-HIT is a widely used program for clustering biological sequences to reduce sequence redundancy and improve the performance of other sequence analyses. In response to the rapid increase in the amount of sequencing data produced by the next-generation sequencing technologies, we have developed a new CD-HIT program accelerated with a novel parallelization strategy and some other techniques to allow efficient clustering of such datasets. Our tests demonstrated very good speedup derived from the parallelization for up to ∼24 cores and a quasi-linear speedup for up to ∼8 cores. The enhanced CD-HIT is capable of handling very large datasets in much shorter time than previous versions.Availability:
http://cd-hit.org.Contact:
liwz@sdsc.eduSupplementary information:
Supplementary data are available at Bioinformatics online.
The PRINTS database, now in its 21st year, houses a collection of diagnostic protein family ‘fingerprints’. Fingerprints are groups of conserved motifs, evident in multiple sequence alignments, whose unique inter-relationships provide distinctive signatures for particular protein families and structural/functional domains. As such, they may be used to assign uncharacterized sequences to known families, and hence to infer tentative functional, structural and/or evolutionary relationships. The February 2012 release (version 42.0) includes 2156 fingerprints, encoding 12 444 individual motifs, covering a range of globular and membrane proteins, modular polypeptides and so on. Here, we report the current status of the database, and introduce a number of recent developments that help both to render a variety of our annotation and analysis tools easier to use and to make them more widely available.Database URL:
www.bioinf.manchester.ac.uk/dbbrowser/PRINTS/
Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.
The Golgi serves as a hub for intracellular membrane traffic in the eukaryotic cell. Transport within the early secretory pathway, that is within the Golgi and from the Golgi to the endoplasmic reticulum, is mediated by COPI-coated vesicles. The COPI coat shares structural features with the clathrin coat, but differs in the mechanisms of cargo sorting and vesicle formation. The small GTPase Arf1 initiates coating on activation and recruits en bloc the stable heptameric protein complex coatomer that resembles the inner and the outer shells of clathrin-coated vesicles. Different binding sites exist in coatomer for membrane machinery and for the sorting of various classes of cargo proteins. During the budding of a COPI vesicle, lipids are sorted to give a liquid-disordered phase composition. For the release of a COPI-coated vesicle, coatomer and Arf cooperate to mediate membrane separation.
HSV-1 viral capsid maturation and egress from the nucleus constitutes a self-controlled process of interactions between host cytoplasmic membrane proteins and viral capsid proteins. In this study, a member of the tetraspanin superfamily, CTMP-7, was shown to physically interact with HSV-1 protein VP26, and the VP26-CTMP-7 complex was detected both in vivo and in vitro. The interaction of VP26 with CTMP-7 plays an essential role in normal HSV-1 replication. Additionally, analysis of a recombinant virus HSV-1-UG showed that mutating VP26 resulted in a decreased viral replication rate and in aggregation of viral mutant capsids in the nucleus. Together, our data support the notion that biological events mediated by a VP26 - CTMP-7 interaction aid in viral capsid enveloping and egress from the cell during the HSV-1 infectious process.
CD-HIT is a widely used program for clustering and comparing large biological sequence datasets. In order to further assist the CD-HIT users, we significantly improved this program with more functions and better accuracy, scalability and flexibility. Most importantly, we developed a new web server, CD-HIT Suite, for clustering a user-uploaded sequence dataset or comparing it to another dataset at different identity levels. Users can now interactively explore the clusters within web browsers. We also provide downloadable clusters for several public databases (NCBI NR, Swissprot and PDB) at different identity levels.
Availability: Free access at http://cd-hit.org
Contact: liwz@sdsc.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Membrane lipids and proteins are non-randomly distributed and are unable to diffuse freely in the plane of the membrane. This is because of multiple constraints imposed both by the cortical cytoskeleton and by the preference of lipids and proteins to cluster into diverse and specialized membrane domains, including tetraspanin-enriched microdomains, glycosylphosphatidyl inositol-linked proteins nanodomains and caveolae, among others. Recent biophysical characterization of tetraspanin-enriched microdomains suggests that they might be specially suited for the regulation of avidity of adhesion receptors and the compartmentalization of enzymatic activities. Moreover, modulation by tetraspanins of the function of adhesion receptors involved in inflammation, lymphocyte activation, cancer and pathogen infection suggests potential as therapeutic targets. This review explores this emerging picture of tetraspanin microdomains and discusses the implications for cell adhesion, proteolysis and pathogenesis.
A wealth of protein and DNA sequence data is being generated by genome projects and other sequencing efforts. A crucial barrier
to deciphering these sequences and understanding the relations among them is the difficulty of detecting subtle local residue
patterns common to multiple sequences. Such patterns frequently reflect similar molecular structures and biological properties.
A mathematical definition of this "local multiple alignment" problem suitable for full computer automation has been used to
develop a new and sensitive algorithm, based on the statistical method of iterative sampling. This algorithm finds an optimized
local alignment model for N sequences in N-linear time, requiring only seconds on current workstations, and allows the simultaneous
detection and optimization of multiple patterns and pattern repeats. The method is illustrated as applied to helix-turn-helix
proteins, lipocalins, and prenyltransferases.
Molecular biologists frequently can obtain interesting insight by aligning a set of related DNA, RNA or protein sequences. Such alignments can be used to determine either evolutionary or functional relationships. Our interest is in identifying functional relationships. Unless the sequences are very similar, it is necessary to have a specific strategy for measuring-or scoring-the relatedness of the aligned sequences. If the alignment is not known, one can be determined by finding an alignment that optimizes the scoring scheme.
We describe four components to our approach for determining alignments of multiple sequences. First, we review a log-likelihood scoring scheme we call information content. Second, we describe two methods for estimating the P value of an individual information content score: (i) a method that combines a technique from large-deviation statistics with numerical calculations; (ii) a method that is exclusively numerical. Third, we describe how we count the number of possible alignments given the overall amount of sequence data. This count is multiplied by the P value to determine the expected frequency of an information content score and, thus, the statistical significance of the corresponding alignment. Statistical significance can be used to compare alignments having differing widths and containing differing numbers of sequences. Fourth, we describe a greedy algorithm for determining alignments of functionally related sequences. Finally, we test the accuracy of our P value calculations, and give an example of using our algorithm to identify binding sites for the Escherichia coli CRP protein.
Programs were developed under the UNIX operating system and are available by anonymous ftp from ftp://beagle.colorado.edu/pub/consensus.
Each intracellular organelle critically depends on maintaining its specific lipid composition that in turn contributes to the biophysical properties of the membrane. With our knowledge increasing about the organization of membranes with defined microdomains of different lipid compositions, questions arise regarding the molecular mechanisms that underlie the targeting to/segregation from microdomains of a given protein. In addition to specific lipid-transmembrane segment interactions as a basis for partitioning, the presence in a given microdomain may alter the conformation of proteins and, thus, the activity and availability for regulatory modifications. However, for most proteins, the specific lipid environment of transmembrane segments as well as its relevance to protein function and overall membrane organization are largely unknown. To help fill this gap, we have synthesized a novel photoactive sphingolipid precursor that, together with a precursor for phosphoglycerolipids and with photo-cholesterol, was investigated in vivo with regard to specific protein transmembrane span-lipid interactions. As a proof of principle, we show specific labeling of the ceramide transporter with the sphingolipid probe and describe specific in vivo interactions of lipids with caveolin-1, phosphatidylinositol transfer protein beta, and the mature form of nicastrin. This novel photolabile sphingolipid probe allows the detection of protein-sphingolipid interactions within the membrane bilayer of living cells.
Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.
The current best membrane-protein topology-prediction methods are typically based on sequence statistics and contain hundreds of parameters that are optimized on known topologies of membrane proteins. However, because the insertion of transmembrane helices into the membrane is the outcome of molecular interactions among protein, lipids and water, it should be possible to predict topology by methods based directly on physical data, as proposed >20 years ago by Kyte and Doolittle. Here, we present two simple topology-prediction methods using a recently published experimental scale of position-specific amino acid contributions to the free energy of membrane insertion that perform on a par with the current best statistics-based topology predictors. This result suggests that prediction of membrane-protein topology and structure directly from first principles is an attainable goal, given the recently improved understanding of peptide recognition by the translocon.
• bioinformatics
• membrane insertion
• topology prediction
• translocon
• biological hydrophobicity scale
Empty-gut disease is a common disease of Chinese oak silkworm, Antheraea pernyi (Tussah). The study on the pathogen of the disease is of great help in prevention and cure of the disease. Previous studies showed that the pathogen was a new species in the genus Streptococcus, which was therefore named as Streptococcus pernyi sp.nov. In this study, with the purified pathogen, we performed some physiological and biochemical experiments, and also cloned and sequenced 16S rRNA gene of the pathogen. Subsequently, we analyzed its similarity and genetic distance with other related bacteria by using DNAstar software, and a NJ tree was then constructed for phylogenic analysis by MEGA 3.0. Unexpectedly, the results showed that the pathogen causing empty-gut disease of Tussah belongs to the genus Enterococcus with 99% bootstrap support. Therefore, Streptococcus pernyi sp.nov should be renamed as Enterococcus pernyi. Reclassification of the pathogen should keep pace with the development of modern taxonomy. In doing so, it will better our understanding of the pathogen and contribute to further study of the pathogen.
G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine.
During the past few years, crystallography of G protein-coupled receptors (GPCRs) has experienced exponential growth, resulting in the determination of the structures of more than 16 distinct receptors-9 of them in the first half of 2012 alone. Including closely related subtype homology models, this coverage amounts to approximately 12% of the human GPCR superfamily. The adrenergic, rhodopsin, and adenosine receptor systems are also described by agonist-bound active-state structures, including a structure of the receptor-G protein complex for the β2-adrenergic receptor. Biochemical and biophysical techniques, such as nuclear magnetic resonance and hydrogen-deuterium exchange coupled with mass spectrometry, are providing complementary insights into ligand-dependent dynamic equilibrium between different functional states. Additional details revealed by high-resolution structures illustrate the receptors as allosteric machines that are controlled not only by ligands but also by sodium, lipids, cholesterol, and water. This wealth of data is helping redefine our knowledge of how GPCRs recognize such a diverse array of ligands and how they transmit signals 30 angstroms across the cell membrane; it also is shedding light on a structural basis of GPCR allosteric modulation and biased signaling. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 53 is January 06, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Although cell membranes are packed with proteins mingling with lipids, remarkably little is known about how proteins interact with lipids to carry out their function. Novel analytical tools are revealing the astounding diversity of lipids in membranes. The issue is now to understand the cellular functions of this complexity. In this Perspective, we focus on the interface of integral transmembrane proteins and membrane lipids in eukaryotic cells. Clarifying how proteins and lipids interact with each other will be important for unraveling membrane protein structure and function. Progress toward this goal will be promoted by increasing overlap between different fields that have so far operated without much crosstalk.
To find motifs that mediate helix-helix interactions in membrane proteins, we have analyzed frequently occurring combinations of residues in a database of transmembrane domains. Our analysis was performed with a novel formalism, which we call TMSTAT, for exactly calculating the expectancies of all pairs and triplets of residues in individual sequences, taking into account differential sequence composition and the substantial effect of finite length in short segments. We found that the number of significantly over and under-represented pairs and triplets was much greater than the random expectation. Isoleucine, glycine and valine were the most common residues in these extreme cases. The main theme observed is patterns of small residues (Gly, Ala and Ser) at i and i+4 found in association with large aliphatic residues (Ile, Val and Leu) at neighboring positions (i.e. i+/-1 and i+/-2). The most over-represented pair is formed by two glycine residues at i and i+4 (GxxxG, 31.6 % above expectation, p<1x10(-33)) and it is strongly associated with the neighboring beta-branched residues Ile and Val. In fact, the GxxxG pair has been described as part of the strong interaction motif in the glycophorin A transmembrane dimer, in which the pair is associated with two Val residues (GVxxGV). GxxxG is also the major motif identified using TOXCAT, an in vivo selection system for transmembrane oligomerization motifs. In conjunction with these experimental observations, our results highlight the importance of the GxxxG+beta-branched motif in transmembrane helix-helix interactions. In addition, the special role for the beta-branched residues Ile and Val suggested here is consistent with the hypothesis that residues with constrained rotameric freedom in helical conformation might reduce the entropic cost of folding in transmembrane proteins. Additional material is available at http://engelman.csb.yale. edu/tmstat and http://bioinfo.mbb.yale. edu/tmstat.
The endogenous lipid signaling agent oleoylethanolamide (OEA) has recently been described as a peripherally acting agent that reduces food intake and body weight gain in rat feeding models. This paper presents evidence that OEA is an endogenous ligand of the orphan receptor GPR119, a G protein-coupled receptor (GPCR) expressed predominantly in the human and rodent pancreas and gastrointestinal tract and also in rodent brain, suggesting that the reported effects of OEA on food intake may be mediated, at least in part, via the GPR119 receptor. Furthermore, we have used the recombinant receptor to discover novel selective small-molecule GPR119 agonists, typified by PSN632408, which suppress food intake in rats and reduce body weight gain and white adipose tissue deposition upon subchronic oral administration to high-fat-fed rats. GPR119 therefore represents a novel and attractive potential target for the therapy of obesity and related metabolic disorders.
- P Björkholm
P. Björkholm et al. / Biochimica et Biophysica Acta 1838 (2014) 2066–2070
- V Popoff
- F Adolf
- B Brugger
- F Wieland
V. Popoff, F. Adolf, B. Brugger, F. Wieland, COPI budding within the Golgi stack, Cold
Spring Harb. Perspect. Biol. 3 (2011) a005231.
Update on activities at the Universal Protein Resource (UniProt) in 2013
The UniProt Consortium, Update on activities at the Universal Protein Resource
(UniProt) in 2013, Nucleic Acids Res. 41 (2013) D43-D47.