Palmitoylation Levels Research Articles

Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway

BackgroundH-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.ResultsOur results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.ConclusionsWe made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.

Distinct Palmitoylation Events at the Amino-terminal Conserved Cysteines of Env7 Direct Its Stability, Localization, and Vacuolar Fusion Regulation in S. cerevisiae

Palmitoylation at cysteine residues is the only known reversible form of lipidation and has been implicated in protein membrane association as well as function. Many palmitoylated proteins have regulatory roles in dynamic cellular processes, including membrane fusion. Recently, we identified Env7 as a conserved and palmitoylated protein kinase involved in negative regulation of membrane fusion at the lysosomal vacuole. Env7 contains a palmitoylation consensus sequence, and substitution of its three consecutive cysteines (Cys(13)-Cys(15)) results in a non-palmitoylated and cytoplasmic Env7. In this study, we further dissect and define the role(s) of individual cysteines of the consensus sequence in various properties of Env7 in vivo. Our results indicate that more than one of the cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane localization, and phosphorylation. Furthermore, individually, each cysteine can serve as a minimum requirement for distinct aspects of Env7 behavior and function in cells. Cys(13) is sufficient for membrane association, Cys(15) is essential for the fusion regulatory function of membrane-bound Env7, and Cys(14) and Cys(15) are redundantly essential for protection of membrane-bound Env7 from proteasomal degradation. A role for Cys(14) and Cys(15) in correct sorting at the membrane is also discussed. Thus, palmitoylation at the N-terminal cysteines of Env7 directs not only its membrane association but also its stability, phosphorylation, and cellular function.

N‐terminal conserved cysteines of Env7 are palmitoylated and direct its stability, localization, and vacuolar fusion regulation in S. cerevisiae (739.12)

Palmitoylation at cysteine residues is the only known reversible lipid modification and has been implicated in protein membrane association as well as function. There is growing evidence that many palmitoylated proteins have regulatory roles in dynamic cellular functions including membrane fusion. Recently, we identified Env7 as a palmitoylated vacuolar membrane protein‐kinase involved in negative regulation of vacuolar fusion. Our phylogenetic analyses have established Env7 as an ortholog of human STK16, an endomembrane kinase with no known function. Env7 contains a conserved palmitoylation consensus sequence and substitution of the three consecutive cysteines (Cys13‐15) within that domain renders the protein non‐palmitoylated and cytoplasmic. In this study, we further dissect and define the role of individual cysteines of the consensus sequence in vivo. Our results indicate that all three cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane association, phosphorylation, and fusion regulatory function. Furthermore, individually, each can serve as a minimum requirement for distinct properties of Env7 in cells. Cys13 is sufficient for membrane association, Cys15 is essential for the fusion/fission regulatory function, and Cys14 or Cys15 is essential for protection of membrane bounded Env7 from proteasomal degradation. A role for Cys14 and Cys15 in correct sorting at the membrane is discussed. Thus, Env7 palmitoylation at the N‐terminal triple cysteines directs not only its membrane association, but also its stability, phosphorylation, and cellular function.Grant Funding Source: Supported by National Science Foundation

Palmitoylation: A New Regulatory Role in Meiosis

The addition of lipid moieties to a protein can alter its localization, activity, and ability to associate with other proteins within the cell. There are several types of lipid modifications cells can employ on their proteins, but only one of these, palmitoylation (the addition of a palmitate chain), is reversible. This fact has led scientists to conjecture that cells may be able to fine tune the palmitoylation of their proteins in the same way that they can protein phosphorylation. If this were so, it would mean that cells have an entire regulatory scheme that is currently poorly understood. But, to what extent such regulation occurs and how it takes place is currently unknown, so Mingzi Zhang, Howard Hang, and colleagues decided to take a closer look at protein palmitoylation in their article published in PLOS Biology. The addition of palmitic acid onto a small GTPase (Rho3) is tuned by precise levels of the Erf2 DHHC-palmitoyltransferase, which helps fission yeast cells undergo sexual differentiation (meiosis). Zhang et al. studied protein palmitoylation in the fission yeast, Schizosaccharomyces pombe. Fission yeast cells possess three chromosomes and normally undergo what's called vegetative growth. In vegetative growth, yeast cells undergo mitosis; each chromosome is copied once and the genetic material is then divvied up evenly between two daughter cells. But, when starved of nutrients, fission yeast can enter a different growth program wherein two cells combine to generate “diploid cells with six chromosomes. After copying their chromosomes once, diploids undergo two rounds of meiotic divisions to generate four daughter cells—each with three chromosomes but a different genetic makeup and different shot at survival than either parent cell. Several proteins must cooperate to trigger and control this alternative growth program, and the authors wondered whether protein palmitoylation patterns might change during meiotic division. To detect palmitoylated proteins, the group employed a fatty acid reporter that can be installed onto target proteins by endogenous enzymes in living cells and sequentially visualized using bioorthogonal chemical methods. By applying this technology to cultures of a mutant yeast strain in which meiotic division can be synchronously induced in all cells, the researchers demonstrated that different proteins are palmitoylated during meiosis than in vegetative growth. Protein palmitoylation is carried out by a family of enzymes called palmitoyltransferases, and Zhang et al. observed that expression of one particular palmitoyltransferase, Erf2, increases during meiosis. So, they postulated that Erf2 might be involved in creating the meiotic palmitoylation pattern. Indeed, they observed that protein palmitoylation is drastically altered and meiosis is delayed in meiotic cells lacking the genes for either Erf2 or its enzymatic cofactor Erf4. This was not the case in cells lacking other palmitoyltransferases the group tested. To find out which proteins Erf2 modifies during meiosis, the authors immunoprecipitated fatty acid reporter-tagged proteins and identified them by mass spectrometry. This analysis turned up three proteins that are prominently palmitoylated in meiotic cells: Ras1, Isp3, and Rho3. The researchers decided to focus on Ras1 and Rho3, as these small GTPases are often important for cell signaling. Ras1 and Rho3 are expressed in both vegetative and meiotic cells. But, Zhang and colleagues showed that Ras1 is palmitoylated to the same extent in meiotic cells as it is in vegetative cells, whereas Rho3 is only palmitoylated in meiotic cells by Erf2. This suggests that acute palmitoylation of Rho3 but not of Ras1 is important for meiosis. Zhang et al. then demonstrate that simply changing the expression levels of the palmitoyltransferase Erf2 is sufficient to increase the palmitoylation levels and subsequent behavior of specific substrates such Rho3. This in turn can affect major cell transitions such as meiosis. To what extent other layers of regulation—such as changes to palmitoyltransferase localization or activity—affect cellular palmitoylation patterns remains to be seen, but this work gives us a glimpse of the possibilities. Zhang MM, Wu P-YJ, Kelly FD, Nurse P, Hang HC (2013) Quantitative Control of Protein S-Palmitoylation Regulates Meiotic Entry in Fission Yeast. doi:10.1371/journal.pbio.1001597

Palmitoylation of Superoxide Dismutase 1 (SOD1) Is Increased for Familial Amyotrophic Lateral Sclerosis-linked SOD1 Mutants

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons. Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment. Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Freely turning over palmitate in erythrocyte membrane proteins is not responsible for the anchoring of lipid rafts to the spectrin skeleton: A study with bio-orthogonal chemical probes

Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183–190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a “click chemistry” reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton.

Palmitoylation Controls Death Receptor Function in Chronic Lymphocytic Leukemia

Abstract 2829 Introduction:Post-translational modifications are important fine-tuning elements for controlling protein activity and signaling. Palmitoylation is a common post-translational modification and defined as the addition of palmitic acid to internal cysteins. Interestingly, in contrast to other lipid modifications, it is reversible. Control over the palmitoylation cycle therefore provides indirect control over protein localization and function. While a number of proteins with palmitoyl transferase activity are known, LYPLA1 (lysophospholipase 1) is the only enzyme known to be responsible for the process of depalmitoylation. CLL cells are known to be resistant to TRAIL-mediated apoptosis. While TRAIL-R1 is reported to be palmitoylated, TRAIL-R2 seems to contain a region with basic amino acids in its membrane-proximal cytoplasmatic domain. Some studies showed that palmitoylation is crucial for several steps of death receptor signaling. Therefore, regulation of depalmitoylation by LYPLA1 seems to be an important tool for the regulation of death receptor function.Methods and Results: Global palmitoylation in CLL cells was investigated by screening for all palmitoylated proteins via a click chemistry assay. There, cells were metabolically labeled, coupled to a specific reporter group and then analyzed by in-gel fluorescence. Comparison of healthy B cells, healthy PBMCs and CLL cells revealed a significant difference in global palmitoylation (+38.5 % in B cells, n=6, p<0.001; +57.8 % in healthy PBMCs, n=6, p<0.001 compared to CLL cells, n=10). We identified LYPLA1 as overexpressed in CLL compared to healthy controls on both protein and mRNA level. We generated a potent LYPLA1 inhibitor. We could show, that inhibition of LYPLA1 led to a significant increase of the overall protein palmitoylation level in CLL cells (+24.7 % n=6, p=0.0118). Ours and other groups have shown, that treatment of cancer cells with TRAIL and X-linked inhibitor of apoptosis protein (XIAP)-inhibition lead to apoptosis in otherwise TRAIL resistant CLL cells. Since death receptors might be palmitoylated, we extended these studies. Treatment of CLL cells with TRAIL, XIAP- and LYPLA1-inhibition led to significantly increased apoptosis compared to TRAIL treatment and XIAP-inhibition alone (+43.2 %, n=12, p=0.0089). Palmitoylation of death receptors was investigated with the help of acyl-biotin exchange chemistry. We could show that palmitoylation of TRAIL-R1 was significantly increased after LYPLA1-inhibiton (+58.7 %, n=3, p=0.0169). It could be demonstrated, that inhibition of LYPLA1 in combination with death receptor stimulation increased the amount of activated caspase-8 in comparison to solely TRAIL and DMSO treated cells (+41.8 %, n=3, p=0.0199), indicating that palmitoylation plays a crucial role in apoptotic signaling far from XIAP. In addition to that, we could show that inhibition of depalmitoylation of TRAIL-R1 led to more death receptor located to lipid rafts. To understand how LYPLA1 is regulated, we investigated two highly conserved miRNAs which were predicted as key regulators of LYPLA1 and which are significantly downregulated in CLL. Indeed, luciferase assays revealed that both miRNAs were able to downregulate LYPLA1 expression. Conclusion:We show for the first time, that LYPLA1 is a central enzyme which regulates the apoptotic signaling of TRAIL. Furthermore, we identified LYPLA1 to be regulated by miRNAs, which are deregulated in CLL. These novel findings allow speculation, that LYPLA1 inhibitors could be used for the treatment of CLL. Future experiments should therefore aim at investigating the LYPLA1 signaling pathway as a potential target for CLL/ cancer therapy.L.P.F. and V.F. contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

Conditioned Place Preference Associated with Level of Palmitoylation of PSD-95 in Rat Hippocampus and Nucleus Accumbens

Psychostimulant-mediated synaptic plasticity in the hippocampus and nucleus accumbens is one of the pathological features of addiction, a disease of learning and memory. Dynamic palmitoylation of PSD-95 modulates synaptic plasticity, but its role in addiction is not fully understood. Using a morphine-conditioned place preference (CPP) rat model and Acyl-biotin exchange (ABE) labeling we found a correlation between CPP and levels of palmitoylated PSD-95 in the hippocampus and nucleus accumbens. Rats that developed significant CPP had higher levels of palmitoylation of PSD-95 in the hippocampus and nucleus accumbens. Furthermore, palmitoylation of PSD-95 was significantly decreased in the hippocampus but increased in the nucleus accumbens during the beginning of withdrawal. With long-term withdrawal, palmitoylated PSD-95 in these regions recovered, while CPP waned and physical signs gradually disappeared. However, morphine reinjection restored strong CPP without producing any significant changes in palmitoylation of PSD-95. Our findings suggest that CPP is correlated with the dynamics of PSD-95 palmitoylation in rat hippocampus and nucleus accumbens, and could be one of the mechanisms for morphine-dependent synaptic plasticity.

Palmitoylation Controls Dopamine Transporter Kinetics, Degradation, and Protein Kinase C-dependent Regulation

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.

Reversible Palmitoylation Regulates Surface Stability of AMPA Receptors in the Nucleus Accumbens in Response to Cocaine In Vivo

Palmitoylation is emerging as one of the most important posttranslational modifications of excitatory synaptic proteins in mammalian brain cells. As a reversible and regulatable modification sensitive to changing synaptic inputs, palmitoylation of ionotropic glutamate receptors contributes not only to the modulation of normal receptor and synaptic activities but also to the pathogenesis of various neuropsychiatric disorders. Here we report that palmitoylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is regulated by the psychostimulant, cocaine, and such regulation is involved in cocaine action. We tested palmitoylation and surface expression of AMPA receptors in striatal neurons and psychomotor behavior in response to cocaine in rats. All four AMPA receptor subunits (GluA1-4 or GluR1-4) are palmitoylated in the nucleus accumbens (NAc) of adult rats. Among them, GluA1 and GluA3 are preferentially upregulated in their palmitoylation levels by a systemic injection of cocaine. The upregulated GluA1 and 3 palmitoylation is a transient and reversible event. Consequently, it increases the susceptibility of surface-expressed GluA1 and 3 to internalization trafficking, leading to a temporal loss of surface receptor expression. Blockade of the regulated GluA1/3 palmitoylation with a palmitoylation inhibitor in the local NAc reverses the loss of surface GluA1/3. The inhibition of palmitoylation concurrently sustains behavioral responsivity to cocaine as well. Our data identify a novel drug-palmitoylation coupling in the center of limbic reward circuits. Through palmitoylating selective AMPA receptor subunits, cocaine activity dependently regulates trafficking and subcellular localization of the receptor in NAc neurons and dynamically controls psychomotor sensitivity to the psychoactive drug in vivo.

Palmitoylation and Membrane Interactions of the Neuroprotective Chaperone Cysteine-string Protein

Cysteine-string protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localized palmitoyl transferases DHHC3, DHHC7, DHHC15, or DHHC17. Indeed, these DHHC proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localize to the endoplasmic reticulum (ER) and are physically separated from the Golgi-localized DHHC proteins. Palmitoylation of an ER-localized mutant could be rescued by brefeldin A treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following brefeldin A washout and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the protein to locate its partner Golgi-localized DHHC proteins directly by membrane "sampling." Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting.

Cysteine-string protein isoform beta (Cspβ) is targeted to the trans-Golgi network as a non-palmitoylated CSP in clonal β-cells

Cysteine string proteins (CSPs) belong to the DnaJ-like chaperone family and play an important role in regulated exocytosis in neurons and endocrine cells. The palmitoylation of several residues in a cysteine string domain may anchor CSPs to the exocytotic vesicle surface and in pancreatic β-cells, Cspα is localized on insulin containing large dense core vesicles (LDCVs). An isoform closely related to Cspα, Cspβ, has been obtained from testis cell cDNA libraries. To gain insights on this isoform and more generally on the properties of CSPs, we compared Cspα and Cspβ. In pull-down experiments, Cspβ was able to interact to the same extent with two of the known Cspα chaperone partners, Hsc70 and SGT. Upon transient overexpression in clonal β-cells, Cspβ but not Cspα was mainly produced as a non-palmitoylated protein and mutational analysis indicated that domains distinct from the cysteine string are responsible for this difference. As Cspβ remained tightly bound to membranes, intrinsic properties of CSPs are sufficient for interactions with membranes. Indeed, recombinant Cspα and Cspβ were capable to interact with membranes even in their non-palmitoylated forms. Furthermore, overexpressed Cspβ was not associated with LDCVs, but was localized at the trans-Golgi network. Our results suggest a possible correlation between the specific membrane targeting and the palmitoylation level of CSPs.

Searching for the Protein Acyltransferase of Gpa1

This project focuses on mechanisms that localize proteins to cell membranes. Specifically, we worked to identify the enzyme that catalyzes S-palmitoylation of Gpa1, a G-protein alpha subunit in yeast, which results in its localization to the plasma membrane. Enyzmes that catalyze palmitoylation are called protein acyltransferases (PATs). These proteins increase the membrane localization of their substrates through the addition of one or more lipids. Gpa1 is dually lipidated, undergoing palmitoylation by an unknown PAT but only after first being myristoylated by N-myristoyltransferase. Gpa1 resides in the inner leaflet of the plasma membrane via its N-terminal myristoyl and palmitoyl chains and functions in the yeast mating pheromone response cascade. Identified PATs have transmembrane domains, undergo autoacylation, and have a DHHC motif embedded in a cysteine-rich domain (CRD). The DHHC-CRD motif is essential for PAT activity. We produced a myristoylated Gpa1 fusion construct (Gpa1f) in E. coli and purified it by chromatography. Using myristoylated Gpa1f as substrate, we assayed five PAT candidates from the yeast DHHC-CRD family for in vitro PAT activity. According to these assays, a small level of palmitoylation of myristoylated Gpa1f occurs in the presence of Pfa3. This research was supported by the Washington University School of Medicine.

Wild-Type-Like Viral Replication Potential of Human Immunodeficiency Virus Type 1 Envelope Mutants Lacking Palmitoylation Signals

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.

Mass Spectrometric Analysis of Agonist Effects on Posttranslational Modifications of the β-2 Adrenoceptor in Mammalian Cells

Posttranslational modifications (PTMs) of the beta-2 adrenoceptor (B2AR) play a fundamental role in receptor regulation by agonists. We have examined the effects of several agonists on net levels of B2AR palmitoylation and phosphorylation using epitope tagging in stably transfected human embryonal kidney (HEK) 293 cells, immunoaffinity purification, and mass spectrometry combined with the method of stable isotope labeling by amino acids in cell culture (SILAC). Palmitoylation of Cys341 was confirmed and did not change detectably after 30 min exposure of cells to saturating concentrations of dopamine, epinephrine, or isoproterenol. However, all of these agonists produced a marked increase in net phosphorylation. Phosphorylation of the third cytoplasmic loop was increased to a similar degree by all three agonists, whereas differences between agonists were observed in net phosphorylation of the carboxyl-terminal cytoplasmic domain (isoproterenol approximately epinephrine >> dopamine). Interestingly, agonist-induced phosphorylation of the carboxyl-terminal cytoplasmic domain was observed exclusively in a proximal portion (between residues 339-369). None of the agonists produced detectable phosphorylation in a distal portion of the cytoplasmic tail, which contains all sites of agonist-induced phosphorylation identified previously by in vitro reconstitution. These results provide insight to agonist-dependent regulation of the B2AR in intact cells, suggest the existence of significant differences in regulatory phosphorylation events occurring between in vitro and in vivo conditions, and outline a general analytical approach to investigate regulated PTM of receptors in mammalian cells.

The juxtamembrane lysine and arginine residues of surfactant protein C precursor influence palmitoylation via effects on trafficking.

Surfactant protein (SP)-C propeptide (proSP-C) becomes palmitoylated on cysteines 5 and 6 before mature SP-C is formed by several proteolytic steps. To study the structural requirements for the palmitoylation of proSP-C, his-tagged human proSP-C (his-proSP-C) and his-proSP-C mutants were expressed in Chinese hamster ovary cells and analyzed by metabolic labeling with [(3)H]palmitate and immunocytochemistry. Substitution of cysteines 5 and 6 by serines showed that these were the only two cysteine residues palmitoylated in his-proSP-C. Substitution of the juxtamembrane basic residues lysine and arginine by uncharged glutamines led to a large decrease in palmitoylation level of proSP-C. The addition of brefeldin A nearly abolished this decrease for the lysine and double mutant; the palmitoylation of the arginine mutant increased also, but not to wild-type (WT) levels. Fluorescence immunocytochemistry showed that WT proSP-C was localized in punctate vesicles throughout the cell, whereas the mutant lacking the juxtamembrane positive charges was found more perinuclear, probably in the endoplasmic reticulum (ER). This indicates that the two basic juxtamembrane residues influence palmitoylation of proSP-C by preventing the transport of proSP-C out of the ER, implying that proSP-C becomes palmitoylated normally in a compartment distal to the ER.

Antisense palmitoyl protein thioesterase 1 (PPT1) treatment inhibits PPT1 activity and increases cell death in LA-N-5 neuroblastoma cells.

Infantile neuronal ceroid lipofuscinosis (INCL) is a childhood neurodegenerative disease caused by the selective death of cortical neurons and retinal degeneration, as the result of a palmitoyl protein thioesterase 1 (PPT1) deficiency. Recently, we showed that overexpression of PPT1 protects LA-N-5 human neuroblastoma cells against apoptotic death (Cho and Dawson [2000a] J. Neurochem. 74:1478-1488) and we now show that inhibition of PPT1 increases the susceptibility of these cells to apoptotic cell death. Transient transfection of LA-N-5 neuroblastoma cells with PPT1-FLAG resulted in a strong expression of PPT-FLAG-tagged protein as evidenced by Western blot analysis and immunofluorescence. Co-transfection of a reverse-oriented (antisense) PPT1 (AS-PPT1) decreased the expression of PPT-FLAG to almost zero, reduced PPT1 enzyme activity (as measured by an in vitro assay) and increased the susceptibility to apoptosis induced by C(2) ceramide. Similarly, inhibition of PPT1 with a synthetic inhibitor (AcG-palmitoyl diaminoproprionate-VKIKK) (DAP1) (100 microM) increased the susceptibility of the cells to apoptosis induced by either C(2)-ceramide or etoposide, a common chemotherapeutic agent used in the treatment of neuroblastoma. Cells stably overexpressing PPT1 were resistant to apoptosis induced by DAP1 suggesting that the inhibitor has a specific action and confirming that low levels of protein palmitoylation block the death pathway. Drugs that raise the level of protein palmitoylation are pro-apoptotic and PPT1 inhibition may enhance the killing efficacy of chemotherapeutic agents used to kill neuroblastoma-derived cells.