The thermodynamic parameters associated with the binding of several series of linear peptides to the third PDZ domain (PDZ3) of the postsynaptic density 95 protein (PSD-95) have been measured using isothermal titration calorimetry (ITC). Two strategies were pursued in developing these binding ligands: (1) systematic N-terminal truncation of sequences derived from the C-terminal regions of identified PDZ3-binding proteins (CRIPT, neuroligin-1, and citron) and (2) selective mutation of specific positions within a consensus hexapeptide (KKETEV) known to bind PDZ3. Each synthetically prepared peptide was used to titrate PDZ3, which yielded the changes in Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) for the binding event. Selected peptides were subjected to additional analysis, which entailed (1) measuring the change in heat capacity (DeltaCp) upon association, to assess the character of the binding interface, and (2) constructing thermodynamic double mutant cycles, to determine the presence of cooperative effects. From the first series, the CRIPT protein proved to be the better source for higher affinity sequences. From the second series, enhanced binding was associated with peptides that closely adhered to the established motif for class I PDZ domain C-termini, X-(T/S)-X-(V/I/L), and more specifically to a narrower motif of X-T-X-V. Further, in both series a length of six residues was necessary and sufficient to capture maximal affinity. In addition, there were significant influences upon binding by modifying the abutting "X" positions. The cumulative results provide greater detail into the specific nature of ligand binding to PDZ3 and will assist in the development of selective molecular probes for the study of this and structurally homologous PDZ domains.

Intracellular membrane trafficking of glutamate receptors at excitatory synapses is critical for synaptic function. However, little is known about the specialized trafficking events occurring at the postsynaptic membrane. We have found that two components of the exocyst complex, Sec8 and Exo70, separately control synaptic targeting and insertion of AMPA-type glutamate receptors. Sec8 controls the directional movement of receptors towards synapses through PDZ-dependent interactions. In contrast, Exo70 mediates receptor insertion at the postsynaptic membrane, but it does not participate in receptor targeting. Thus, interference with Exo70 function accumulates AMPA receptors inside the spine, forming a complex physically associated, but not yet fused with the postsynaptic membrane. Electron microscopic analysis of these complexes indicates that Exo70 mediates AMPA receptor insertion directly within the postsynaptic density, rather than at extrasynaptic membranes. Therefore, we propose a molecular and anatomical model that dissects AMPA receptor sorting and synaptic delivery within the spine, and uncovers new functions of the exocyst at the postsynaptic membrane.

Background: NMDA receptor hyperactivity results in mitochondrial dysfunction in neurons promoting neurodegenerative disorders. Results: Short polyarginine peptides target mitochondria to promote neuronal survival. Conclusion: Short polyarginine peptides reduce mitochondrial respiration, membrane hyperpolarization, and generation of reactive oxygen species. Significance: Treatment with polyarginine has the potential to minimize neuronal damage resulting from stroke or traumatic brain injury and may be therapeutic to ameliorate multiple sclerosis and Parkinson disease.

GIPC (GAIP-interacting protein, C terminus) represents a new target class for the discovery of chemotherapeutics. While many of the current generation of anticancer agents function by directly binding to intracellular kinases or cell surface receptors, the disruption of cytosolic protein-protein interactions mediated by non-enzymatic domains is an underdeveloped avenue for inhibiting cancer growth. One such example is the PDZ domain of GIPC. Previously we developed a molecular probe, the cell permeable octapeptide CR1023 (N-myristoyl-PSQSSSEA), which diminished proliferation of pancreatic cancer cells. We have expanded upon that discovery using a chemical modification approach, and here report a series of cell permeable, side chain-modified lipopeptides that target the GIPC PDZ domain in vitro and in vivo. These peptides exhibit significant activity against pancreatic and breast cancers, both in vitro and in animal models. CR1166 (N-myristoyl-PSQSK(εN-4-bromobenzoyl)SK(εN-4-bromobenzoyl)A), bearing two halogenated aromatic units on alternate side chains, was found to be the most active compound, with pronounced down-regulation of EGFR/1GF-1R expression. We hypothesize that these organic acid-modified residues extend the productive reach of the peptide beyond the canonical binding pocket, which defines the limit of accessibility for the native proteinogenic sequences that the PDZ domain has evolved to recognize. Cell permeability is achieved with N-terminal lipidation using myristate, rather than a larger CPP (cell-penetrating peptide) sequence. This, in conjunction with optimization of targeting through side chain modification, has yielded an approach that will allow the discovery and development of next-generation cellular probes for GIPC PDZ as well as other PDZ domains.