Palmitoylation State Research Articles

Thermodynamics and S-Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes.

Aquaporin-4 (AQP4) is a water channel protein found primarily in the central nervous system (CNS) that helps to regulate water-ion homeostasis. AQP4 exists in two major isoforms: M1 and M23. While both isoforms have a homotetrameric quaternary structure and are functionally identical when transporting water, the M23 isoform forms large protein aggregates known as orthogonal arrays of particles (OAPs). In contrast, the M1 isoform creates a peripheral layer around the outside of these OAPs, suggesting a thermodynamically stable interaction between the two. Structurally, the M1 isoform has an N-terminal tail that is 22 amino acids longer than the M23 isoform and contains two solvent-accessible cysteines available for S-palmitoylation at cysteine-13 (Cys-13) and cysteine-17 (Cys-17) in the amino acid sequence. Earlier work suggests that the palmitoylation of these cysteines might aid in regulating AQP4 assemblies. This work discusses the thermodynamic driving forces for M1 protein-protein interactions and how the palmitoylation state of M1 affects them. Using temperature-dependent single-particle tracking, the standard state free energies, enthalpies, and entropies were measured for these interactions. Furthermore, we present a binding model based on measured thermodynamics and a structural modeling study. The results of this study demonstrate that the M1 isoform will associate with itself according to the following expressions: 2[AQP4-M1]4 ↔ [[AQP4-M1]4]2 when palmitoylated and 3[AQP4-M1]4 ↔ [AQP4-M1]4 + [[AQP4-M1]4]2 ↔ [[AQP4-M1]4]3 when depalmitoylated. This is primarily due to a conformational change induced by adding the palmitic acid groups at Cys-13 and Cys-17 in the N-terminal tails of the homotetramers. In addition, a statistical mechanical model was developed to estimate the Gibbs free energy, enthalpy, and entropy for forming dimers and trimers. These results were in good agreement with experimental values.

S-palmitoylation dependent self assembly of human aquaporin-4 orthogonal arrays of particles in biomimetic membranes.

Aquaporin-4 (AQP4) is a water channel protein located in the plasma membrane of astrocytes in the central nervous system (CNS) and helps to regulate water-ion homeostasis. Isoforms of AQP4 further order themselves into aggregates known as orthogonal arrays of particles (OAPs). AQP4 exists in two main isoforms, M1 and M23, with M23 being the isoform that favors stabilization by aggregation into OAPs. It has been hypothesized that the aggregation size is regulated by the amount of M1 isoform present in the membrane. In turn, the palmitoylation state of two cysteine residues on the N-terminal tail of the M1 isoform greatly affects its mobility and ability to inhibit OAP formation. In this study, M1 was chemically cleaved and palmitoylated in vitro, and inserted in biomimetic lipid bilayers to study kinetics and aggregation behavior using single-protein fluorescence tracking. This study showed that when M1 was palmitoylated, it displays fast diffusion through the membrane with short lived protein-protein interactions and no evidence of aggregation. The depalmitoylated form of M1 showed evidence of extensive aggregation and long-lived protein-protein interactions. This study is the first of its kind to investigate human AQP4 in vitro and to extract the kinetic rate constants for protein-protein interactions as well as estimates of their equilibrium constants.

Effect of SP-C and its palmitoylation state on membrane fragmentation and vesicle uptake

One of the largest surfaces of the human body in contact with the environment is the respiratory epithelium, constituted by different specialized cells. Alveolar type I cells are involved in gas exchange whereas alveolar type II cells prevent the alveoli from collapsing due to the synthesis and secretion of lung surfactant (LS). This lipid-protein complex covers the alveolar surface and reduces surface tension at the air-liquid interface. LS, the first element in contact with inhaled air, is also involved in innate defense mechanisms.

Cytokine Profiling in Low- and High-Density Small Extracellular Vesicles from Epidermoid Carcinoma Cells

Exosomes or small extracellular vesicles (sEVs) are membrane-bound nanoparticles that carry various macromolecules and act as autocrine and paracrine signaling messengers. In this study, sEVs from epidermoid carcinoma cells influenced by membrane presentation of the glycoprotein desmoglein 2 and its palmitoylation state were investigated. In this study, sEVs were isolated by sequential ultracentrifugation followed by iodixanol density gradient separation. They were then subjected to multiplex profiling of cytokines associated with the surface of intact sEVs. The results revealed a previously undescribed active sorting of cytokines onto the surface of low-density and high-density sEV subpopulations. Specifically, an altered surface presentation of desmoglein 2 decreased FGF-2 and VEGF in low-density sEVs. In addition, in response to desmoglein 2, IL-8 and RANTES were increased in low-density sEVs but only slightly decreased in high-density sEVs. Finally, IL-6 and G-CSF were increased dramatically in high-density sEVs. This comprehensive analysis of the cytokine production profile by squamous cell carcinoma‒derived sEVs highlights their contribution to immune evasion, pro-oncogenic and proangiogenic activity, and the potential to identify diagnostic disease biomarkers.

LB719 Cytokine profiling in low- and high-density small extracellular vesicles from epidermoid carcinoma cells

Exosomes or small extracellular vesicles (sEVs) are membrane-bound, nanoparticles carrying various macromolecules, acting as autocrine and paracrine signaling messengers. Here, sEVs from epidermoid carcinoma cells influenced by membrane presentation of the glycoprotein desmoglein 2 and its palmitoylation state were investigated. sEVs were isolated by sequential ultracentrifugation followed by iodixanol density gradient separation and subjected to multiplex cytokine profiling. Subpopulations of different densities showed active sorting of surface-associated cytokines, chemokines and growth factors.

Wnt-controlled sphingolipids modulate Anthrax Toxin Receptor palmitoylation to regulate oriented mitosis in zebrafish

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis.

Palmitoylation of the KATP channel Kir6.2 subunit promotes channel opening by regulating PIP2 sensitivity

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Domains of the TF protein important in regulating its own palmitoylation

Sindbis virus particles contain the viral proteins capsid, E1 and E2, and low levels of a small membrane protein called TF. TF is produced during a (−1) programmed ribosomal frameshifting event during the translation of the structural polyprotein. TF from Sindbis virus-infected cells is present in two palmitoylated states, basal and maximal; unpalmitoylated TF is not detectable. Mutagenesis studies demonstrated that without palmitoylation, TF is not incorporated into released virions, suggesting palmitoylation of TF is a regulated step in virus assembly. In this work, we identified Domains within the TF protein that regulate its palmitoylation state. Mutations and insertions in Domain III, a region proposed to be in the cytoplasmic loop of TF, increase levels of unpalmitoylated TF found during an infection but still unpalmitoylated TF was not incorporated into virions. Mutations in Domain IV, the TF unique region, are likely to impact the balance between basal and maximal palmitoylation.

Mechanisms of CPT1C-Dependent AMPAR Trafficking Enhancement.

In neurons, AMPA receptor (AMPAR) function depends essentially on their constituent components:the ion channel forming subunits and ion channel associated proteins. On the other hand, AMPAR trafficking is tightly regulated by a vast number of intracellular neuronal proteins that bind to AMPAR subunits. It has been recently shown that the interaction between the GluA1 subunit of AMPARs and carnitine palmitoyltransferase 1C (CPT1C), a novel protein partner of AMPARs, is important in modulating surface expression of these ionotropic glutamate receptors. Indeed, synaptic transmission in CPT1C knockout (KO) mice is diminished supporting a positive trafficking role for that protein. However, the molecular mechanisms of such modulation remain unknown although a putative role of CPT1C in depalmitoylating GluA1 has been hypothesized. Here, we explore that possibility and show that CPT1C effect on AMPARs is likely due to changes in the palmitoylation state of GluA1. Based on in silico analysis, Ser 252, His 470 and Asp 474 are predicted to be the catalytic triad responsible for CPT1C palmitoyl thioesterase (PTE) activity. When these residues are mutated or when PTE activity is inhibited, the CPT1C effect on AMPAR trafficking is abolished, validating the CPT1C catalytic triad as being responsible for PTE activity on AMPAR. Moreover, the histidine residue (His 470) of CPT1C is crucial for the increase in GluA1 surface expression in neurons and the H470A mutation impairs the depalmitoylating catalytic activity of CPT1C. Finally, we show that CPT1C effect seems to be specific for this CPT1 isoform and it takes place solely at endoplasmic reticulum (ER). This work adds another facet to the impressive degree of molecular mechanisms regulating AMPAR physiology.

14 A patient derived antibody targeting the tetraspanin CD9 inhibits tumour progression and metastasis

IntroductionImmune reactions against cancer cells can be induced by immunotherapy. We investigated the possibility that an antibody response has contributed to the success of the immunotherapy of a cancer patient.Material and methodsA patient with metastatic melanoma was successfully treated by adoptive transfer of ex vivo expanded autologous tumour reactive T cells. This patient is still tumour free 11 years after treatment and had developed both CD4 and CD8 T cells specific for neoantigens. We isolated B cells from peripheral blood and generated clones following immortalization by forced expression of Bcl-6 and Bcl-xL.Results and discussionsA B-cell clone producing antibody AT1412 recognising a novel cell surface epitope on the tetraspanin CD9 was isolated. CD9 is widely expressed and plays a role in many phases of disease progression. AT1412 showed significant stronger binding to melanoma cells than to melanocytes indicating that the AT1412 epitope is overexpressed on tumour cells. In addition, AT1412 also strongly reacted with many solid tumours types including colon, pancreas and, breast cancer, as well as liquid tumours such as AML, B-ALL and multiple myeloma. Further analysis revealed that AT1412 favours binding to a clustered form of CD9. These clusters are dependent on the palmitoylation state of CD9 and known to be enriched on the surface of tumorigenic cells.Using immunodeficient mice harbouring a human immune system (HIS mice) we observed that AT1412 blocks tumour progression as a single agent and more strongly in combination with an anti-PD1 antibody. Highly significant, AT1412 strongly stimulated the influx of macrophages and CD8 +T cells into the tumour providing an explanation for the synergistic effects of anti PD1 and AT1412.In sharp contrast to previously described anti-CD9 antibodies AT1412 does not induce aggregation of platelets. This is in line with an absence of antibody-related adverse events, including thrombosis and colitis, in the patient during and after treatment. Altogether this indicates that the antibody is safe for use in humans.ConclusionThese data suggest that antibody AT1412 contributed to the success of the immunotherapy in this patient. This antibody could provide help to tumor-reactive immune cells in the eradication of tumour masses and is as such an attractive candidate drug for cancer treatment.Our results show that the B-cell repertoire of a patient who is cured after immunotherapy provides a highly attractive source of antibodies with clinical potential.

Stochastic palmitoylation of accessible cysteines in membrane proteins revealed by native mass spectrometry

Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins. However, how specificity of palmitoylation and multiple palmitoylations in membrane proteins are determined is not well understood. Here, we profile palmitoylation states of three human claudins, human CD20 and cysteine-engineered prokaryotic KcsA and bacteriorhodopsin by native mass spectrometry. Cysteine scanning of claudin-3, KcsA, and bacteriorhodopsin shows that palmitoylation is independent of a sequence motif. Palmitoylations are observed for cysteines exposed on the protein surface and situated up to 8 Å into the inner leaflet of the membrane. Palmitoylation on multiple sites in claudin-3 and CD20 occurs stochastically, giving rise to a distribution of palmitoylated membrane-protein isoforms. Non-native sites in claudin-3 indicate that membrane-protein function imposed evolutionary restraints on native palmitoylation sites. These results suggest a generic, stochastic membrane-protein palmitoylation process that is determined by the accessibility of palmitoyl-acyl transferases to cysteines on membrane-embedded proteins, and not by a preferred substrate-sequence motif.

Delivery of Lung Surfactant SP-C Based Nanostructures to Respiratory Air-Liquid Interfacial Films

The breathing function is sustained by Lung Surfactant (LS), a macromolecular complex lining the alveolar air-liquid interface. In the context of the physiology of the LS system, the absence or dysfunction of surfactant protein SP-C is associated with several lung diseases. With the main goal of developing new therapeutic tools for the treatment of patients with respiratory dysfunction, we have evaluated different methodologies from the field of nanotechnology using scaffolding molecules that break membranes in nanoscale particles incorporating membrane proteins in their natural environment. We have explored the scaffolding reversible encapsulation of lipid bilayers incorporating either palmitoylated or a non-palmitoylated recombinant version of SP-C for the study of its structure-function relationships. In this work, SP-C has been solubilized using nanoparticles made with different lipid composition (POPC, POPC/POPG, POPC/POPG/DPPC or the Native Surfactant Lipid Fraction), assessing them as a potential native-like environment in order to preserve the tridimensional structure and functional properties of the protein. The nanoparticles behave themselves as a highly adsorptive material in the air-liquid interface, where they inhibited pulmonary surfactant adsorption. When SP-C is incorporated into the nanoparticles the interfacial adsorption capability of the complex increased slightly depending on the palmitoylation state of the protein. We have also analyzed the interaction of the complexes with surfactant monolayers and bilayers. Fluorescently labeled nanocomplexes seem to associate to both lipid structures with no apparent lipid transfer. These nanostructures could then constitute an efficient vehicle to direct molecules such as proteins or drugs to the respiratory air-liquid interface.

Identification of PSD-95 Depalmitoylating Enzymes.

Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. Protein palmitoylation, the most common lipid modification, dynamically regulates neuronal protein localization and function. Its unique reversibility is conferred by DHHC-type palmitoyl acyl transferases (palmitoylating enzymes) and still controversial palmitoyl-protein thioesterases (depalmitoylating enzymes). Here, we identified the membrane-anchored serine hydrolases, ABHD17A, 17B, and 17C, as the physiological PSD-95 depalmitoylating enzymes that regulate PSD-95 palmitoylation cycles in neurons. This study describes the first direct evidence for the neuronal depalmitoylating enzyme and provides a new aspect of the dynamic regulatory mechanisms of synaptic development and synaptic plasticity. In addition, our established APEGS assay, which provides unbiased and quantitative information about the palmitoylation state and dynamics, revealed the distinct regulatory mechanisms for synaptic palmitoylation.

AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors.

AMPARs mediate the vast majority of fast excitatory synaptic transmission in the brain and their biophysical and trafficking properties depend on their subunit composition and on several posttranscriptional and posttranslational modifications. Additionally, in the brain AMPARs associate with auxiliary subunits, which modify the properties of the receptors. Despite the abundance of AMPAR partners, recent proteomic studies have revealed even more interacting proteins that could potentially be involved in AMPAR regulation. Amongst these, carnitine palmitoyltransferase 1C (CPT1C) has been demonstrated to form an integral part of native AMPAR complexes in brain tissue extracts. Thus, we aimed to investigate whether CPT1C might be able to modulate AMPAR function. Firstly, we confirmed that CPT1C is an interacting protein of AMPARs in heterologous expression systems. Secondly, CPT1C enhanced whole-cell currents of GluA1 homomeric and GluA1/GluA2 heteromeric receptors. However, CPT1C does not alter the biophysical properties of AMPARs and co-localization experiments revealed that AMPARs and CPT1C are not associated at the plasma membrane despite a strong level of co-localization at the intracellular level. We established that increased surface GluA1 receptor number was responsible for the enhanced AMPAR mediated currents in the presence of CPT1C. Additionally, we revealed that the palmitoylable residue C585 of GluA1 is important in the enhancement of AMPAR trafficking to the cell surface by CPT1C. Nevertheless, despite its potential as a depalmitoylating enzyme, CPT1C does not affect the palmitoylation state of GluA1. To sum up, this work suggests that CPT1C plays a role as a novel regulator of AMPAR surface expression in neurons. Fine modulation of AMPAR membrane trafficking is fundamental in normal synaptic activity and in plasticity processes and CPT1C is therefore a putative candidate to regulate neuronal AMPAR physiology.

The differential palmitoylation states of N-Ras and H-Ras determine their distinct Golgi subcompartment localizations.

Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C-terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H-Ras and N-Ras) localize on the Golgi apparatus whereas K-Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub-organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live-cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H-Ras is distributed throughout the Golgi stacks, the singly palmitoylated N-Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants, we show that the different sub-Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane, but also their distribution within the Golgi stacks.

Quantitative Control of Protein S-Palmitoylation Regulates Meiotic Entry in Fission Yeast

Author SummaryPalmitoylation is a fatty acid modification, which increases the hydrophobicity of proteins, known to control the membrane trafficking and function of many proteins important for cellular homeostasis and disease. Being a reversible process, palmitoylation is proposed to be a major cellular regulator. While changes in global protein palmitoylation patterns are often associated with different physiological states, the mechanisms that regulate this posttranslational modification and their functional consequences have been elusive. Using improved methods for protein palmitoylation analysis in the genetically tractable fission yeast Schizosaccharomyces pombe, we discovered that the global protein palmitoylation pattern is significantly altered and correlates with entry into meiosis, a major developmental transition. We further showed that the precise level of a palmitoylating enzyme is important to shape the subset of lipid-modified proteins and influence the start of this cellular transition. As palmitoylated proteins are involved in eukaryotic cellular pathways, our results provide a crucial insight into how the quantitative control of palmitoylating enzymes may impact human physiology and disease.

The Palmitoylation State of PMP22 Modulates Epithelial Cell Morphology and Migration

PMP22 (peripheral myelin protein 22), also known as GAS 3 (growth-arrest-specific protein 3), is a disease-linked tetraspan glycoprotein of peripheral nerve myelin and constituent of intercellular junctions in epithelia. To date, our knowledge of the post-translational modification of PMP22 is limited. Using the CSS-Palm 2.0 software we predicted that C85 (cysteine 85), a highly conserved amino acid located between the second and third transmembrane domains, is a potential site for palmitoylation. To test this, we mutated C85S (C85 to serine) and established stable cells lines expressing the WT (wild-type) or the C85S-PMP22. In Schwann and MDCK (Madin–Darby canine kidney) cells mutating C85 blocked the palmitoylation of PMP22, which we monitored using 17-ODYA (17-octadecynoic acid). While palmitoylation was not necessary for processing the newly synthesized PMP22 through the secretory pathway, overexpression of C85S-PMP22 led to pronounced cell spreading and uneven monolayer thinning. To further investigate the functional significance of palmitoylated PMP22, we evaluated MDCK cell migration in a wound-healing assay. While WT-PMP22 expressing cells were resistant to migration, C85S cells displayed lamellipodial protrusions and migrated at a similar rate to vector control. These findings indicate that palmitoylation of PMP22 at C85 is critical for the role of the protein in modulating epithelial cell shape and motility.

Effects of palmitoylation of Cys(415) in helix 8 of the CB(1) cannabinoid receptor on membrane localization and signalling.

The CB(1) cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB(1) receptor by analysing the functional consequences of site-specific mutation of Cys(415) , the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling. The palmitoylation state of CB(1) receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys(415) was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [(35) S]GTPγS, cAMP and co-immunoprecipitation assays. Endogenous CB(1) receptors in rat brain were palmitoylated. Mutation of Cys(415) prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB(1) receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins. CB(1) receptors were post-translationally modified by palmitoylation. Mutation of Cys(415) provides a receptor that is functionally impaired in terms of membrane targeting and signalling. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Palmitoylation State Impacts Induction of Innate and Acquired Immunity by the Salmonella enterica Serovar Typhimurium msbB Mutant

Palmitoylation State Impacts Induction of Innate and Acquired Immunity by the Salmonella enterica Serovar Typhimurium <i>msbB</i> Mutant