Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.

Tissue development, differentiation, and physiology require specialized cellular adhesion and signal transduction at sites of cell-cell contact. Scaffolding proteins that tether adhesion molecules, receptors, and intracellular signaling enzymes organize macromolecular protein complexes at cellular junctions to integrate these functions. One family of such scaffolding proteins is the large group of membrane-associated guanylate kinases (MAGUKs). Genetic studies have highlighted critical roles for MAGUK proteins in the development and physiology of numerous tissues from a variety of metazoan organisms. Mutation of Drosophila discs large (dlg) disrupts epithelial septate junctions and causes overgrowth of imaginal discs. Similarly, mutation of lin-2, a related MAGUK in Caenorhabditis elegans, blocks vulval development, and mutation of the postsynaptic density protein PSD-95 impairs synaptic plasticity in mammalian brain. These diverse roles are explained by recent biochemical and structural analyses of MAGUKs, which demonstrate their capacity to assemble well--efined--yet adaptable--protein complexes at cellular junctions.

Membrane-associated guanylate kinases (MAGUKs), such as PSD-95, are modular scaffolds that organize signaling complexes at synapses and other cell junctions. MAGUKs contain PDZ domains, which recruit signaling proteins, as well as a Src homology 3 (SH3) and a guanylate kinase-like (GK) domain, implicated in scaffold oligomerization. The crystal structure of the SH3-GK module from PSD-95 reveals that these domains form an integrated unit: the SH3 fold comprises noncontiguous sequence elements divided by a hinge region and the GK domain. These elements compose two subdomains that can assemble in either an intra- or intermolecular fashion to complete the SH3 fold. We propose a model for MAGUK oligomerization in which complementary SH3 subdomains associate by 3D domain swapping. This model provides a possible mechanism for ligand regulation of oligomerization.

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Methylenecyclopropyl)formyl-CoA (MCPF-CoA), a toxic metabolite of methylenecyclopropylglycine (MCPG), is known to have hypoglycemic activity due to its ability to interrupt the β-oxidation pathway of fatty acid metabolism. Earlier experiments have shown that acetoacetyl-CoA thiolase, 3-ketoacyl-CoA thiolase, acyl-CoA dehydrogenases, and enoyl-CoA hydratase (ECH) are cellular targets that can be inhibited by MCPF-CoA and/or MCPG. To gain more insights with respect to the target specificity and the mode of action, we have carried out a detailed investigation of the effects of MCPF-CoA on a variety of enzymes involved in fatty acid metabolism. Our studies confirmed that MCPF-CoA is a potent inactivator for ECHs but shows little effect on other β-oxidation enzymes tested in this study. Our results also revealed that MCPF-CoA manifests distinct modes of inhibition among ECHs isolated from different sources, being a competitive inhibitor for rat liver ECH and an irreversible inactivator for the bovine liver as well as pig kidney ECH. Given the high sequence homology of the mammalian ECH genes studied so far, the structures of these proteins are expected to be similar. Thus, the effects of MCPF-CoA toward different ECHs must be governed in part by the interaction of MCPF-CoA with the active site of each ECH whose architecture may be subtly different. More importantly, the incubation results with bovine liver ECH established that MCPF-CoA inactivates this enzyme via a mechanism involving the covalent trapping of an active site nucleophile by the methylenecyclopropane ring. Since MCPF-CoA is a rare irreversible inhibitor for ECHs, it could serve as a new lead for designing more effective agents for modulating ECH activity so as to control and/or regulate fatty acid metabolism.Methylenecyclopropylglycine (1, MCPG) was first isolated in 1962 from the kernels of litchi fruits by Gray and Fowden and has been shown to cause hypoglycemia in mice 1 and fasting rats. 2,3 MCPG is a homologue of hypoglycin A (2), the better known and more potent hypoglycemic amino acid, and the