Palmitoylation regulates glutamate receptor distributions in postsynaptic densities through control of PSD95 conformation and orientation.

Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and EM, and examined how conformation regulated interactions with AMPA-type and NMDA-type glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration.

Altered palmitoylation and neuropathological deficits in mice lacking HIP14.

Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD).

Palmitoylation of nicotinic acetylcholine receptors.

It is well established that nicotinic acetylcholine receptors (nAChRs) undergo a number of different posttranslational modifications, such as disulfide bond formation, glycosylation, and phosphorylation. Recently, our laboratory has developed more sensitive assays of protein palmitoylation that have allowed us and others to detect the palmitoylation of relatively low abundant proteins such as ligand-gated ion channels. Here, we present evidence that palmitoylation is prevalent on many subunits of different nAChR subtypes, both muscle-type nAChRs and the neuronal "alpha(4)beta(2)" and "alpha(7)" subtypes most abundant in brain.

Palmitoylation of ATP-binding cassette transporter A1 is essential for its trafficking and function.

ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains.

Synaptic SAP97 isoforms regulate AMPA receptor dynamics and access to presynaptic glutamate.

The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane.

Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.

Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here a global characterization of rat neural palmitoyl-proteomes identifies most of the known neural palmitoyl proteins-68 in total, plus more than 200 new palmitoyl-protein candidates, with further testing confirming palmitoylation for 21 of these candidates. The new palmitoyl proteins include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins.

S-palmitoylation of gamma-secretase subunits nicastrin and APH-1.

Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM.

High affinity binding of epibatidine to serotonin type 3 receptors.

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of (125)I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT(3)Rs) block the remaining 25%. (125)I-Epibatidine binds with a high affinity to native 5-HT(3)Rs of N1E-115 cells and to receptors composed of only 5-HT(3A) subunits expressed in HEK cells.

Assays of protein palmitoylation.

Protein palmitoylation plays an important role in the structure and function of a wide array of proteins. Unlike other lipid modifications, protein palmitoylation is highly dynamic and cycles of palmitoylation and depalmitoylation can regulate protein function and localization. The dynamic nature of palmitoylation is poorly resolved because of limitations in assay methods.

Palmitoylation of huntingtin by HIP14 is essential for its trafficking and function.

Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14).

The role of palmitoylation in functional expression of nicotinic alpha7 receptors.

Neuronal alpha-bungarotoxin receptors (BgtRs) are nicotinic receptors that require as yet unidentified post-translational modifications to achieve functional expression. In this study, we examined the role of protein palmitoylation in BgtR expression. BgtR alpha7 subunits are highly palmitoylated in neurons from brain and other cells capable of BgtR expression, such as pheochromocytoma 12 (PC12) cells.

Labeling and quantifying sites of protein palmitoylation.

As a reversible posttranslational modification, protein palmitoylation has the potential to regulate the trafficking and function of a variety of proteins. However, the extent, function, and dynamic nature of palmitoylation are poorly resolved because of limitations in assay methods. Here, we introduce methods where hydroxylamine-mediated cleavage of the palmitoyl-thioester bond generates a free sulfhydryl, which can then be specifically labeled with sulfhydryl-reactive reagents.

Neuronal alpha-bungarotoxin receptors are alpha7 subunit homomers.

Nicotinic acetylcholine receptors in the nervous system are heterogeneous with distinct pharmacological and functional properties resulting from differences in post-translational processing and subunit composition. Because of nicotinic receptor diversity, receptor purification and biochemical characterization have been difficult, and the precise subunit composition of each receptor subtype is poorly characterized. Evidence is presented that alpha-bungarotoxin (Bgt)-binding nicotinic receptors found in pheochromocytoma 12 (PC12) cells are pentamers composed solely of alpha7 subunits.

alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits.

Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors.

We have characterized the alpha-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of alpha7-containing receptors that bind alpha-Bgt with high affinity (Kd = 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and alpha-Bgt block approximately 90% of the whole-cell current.

Purification and partial characterization of acrosome reaction inhibiting glycoprotein from human seminal plasma.

The human acrosome reaction (AR; sperm exocytosis) is absolutely required for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human seminal plasma was purified by differential centrifugation, carboxymethyl cellulose chromatography, chromatofocusing, and Sephacryl S300 gel filtration. A highly purified protein with a molecular weight of 74,000 was obtained as determined by gel filtration and SDS-PAGE.

Atrial natriuretic peptide (ANP) as a stimulus of the human acrosome reaction and a component of ovarian follicular fluid: correlation of follicular ANP content with in vitro fertilization outcome.

Atrial natriuretic peptides (ANPs) from several species induced the human acrosome reaction. The maximal response was highest for human ANP (18.6% above unstimulated or baseline values) and decreased progressively for peptides derived from animals lower on the phylogenetic scale.

Inhibition of platelet factor XIIIa-catalyzed reactions by calmodulin.

Calmodulin was found to exhibit an inhibitory effect on platelet factor XIIIa-catalyzed incorporation of pseudodonor amines into dimethylcasein, platelet actin and myosin. The inhibitory action of calmodulin on the calcium-dependent enzyme reactions was analogous to the effects of EGTA and parvalbumin on these reactions. The extent of inhibition of factor XIIIa activity was a function of calmodulin concentration when factor XIII and Ca2+ concentrations were held constant.

Rabbit myocardial lysophospholipase-transacylase. Purification, characterization, and inhibition by endogenous cardiac amphiphiles.

Rabbit myocardial lysophospholipase-transacylase was purified 69,000-fold to near homogeneity by ammonium sulfate precipitation, DEAE-Sephacel, hydroxylapatite chromatography, and high precision liquid chromatography. The purified protein was a single band (Mr = 63,000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It had a specific activity of 4 mumol/mg/min for fatty acid release and 2 mumol/mg/min for phosphatidylcholine synthesis.