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.

One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killerFLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killerFLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killerFLIP are independent of its sequence similarity with c-FLIPL as killerFLIP-induced cell death was largely apoptosis and necroptosis independent. A killerFLIP-E variant containing a scrambled c-FLIPL motif indeed induced similar cell death, suggesting the importance of the c-FLIPL residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo.

The use of bacteriophage T7 is presented as a peptide display platform to identify short binding sequences for PDZ domain proteins. Two different domains are examined, the 10th PDZ domain (PDZ10) of the multi-PDZ domain protein 1 (MUPP1) and the third PDZ domain (PDZ3) of postsynaptic density-95 (PSD-95) protein. Using the T7Select 415-1b construct, which displays 415 peptides per phage particle, a random heptapeptide and focused octapeptide libraries were constructed and subjected to iterative selection-enrichment cycles against surface-immobilized PDZ3 and PDZ10 proteins. The derived consensus sequences, together with those of high-frequency clones, were used as the basis for individual chemically synthesized peptides. Each peptide was subjected to isothermal titration calorimetry binding determinations against the corresponding PDZ domain under standard solution conditions. For MUPP1 PDZ10, binding analysis demonstrated that one of the heptapeptides, Ac-IGRISRV, displayed a two-fold improved affinity over the octapeptide derived from the carboxy terminus of the hc-Kit protein, which we had recently demonstrated as among the highest affinity ligands reported to date for that domain. In the case of PSD-95 PDZ3, peptides were found that possessed low-micromolar dissociation constants, as well as those that rediscovered the C-terminal sequence (KQTSV) of the protein CRIPT, a known natural binding protein of PDZ3. These successful examples of ligand discovery against two distinctly different PDZ domains demonstrate that the T7 phage platform could prove broadly applicable to the numerous other PDZ domains for which binding peptides are absent or of insufficient affinity.

Post-synaptic density protein PSD-95 is emerging as a valid target for modulating nociception in animal studies. Based on the key role of PSD-95 in neuronal plasticity and the maintenance of pain behavior, we predicted that CN2097, a peptide-based macrocycle of 9 residues that binds to the PDZ domains of PSD-95, would interfere with physiologic phenomena in the spinal cord related to central sensitization. Furthermore, we tested whether spinal intrathecal injection of CN2097 attenuates thermal hyperalgesia in a rat model of sciatic neuropathy. Results demonstrate that spinal CN2097 reverses hyperexcitability of WDR neurons in the dorsal horn of neuropathic rats and decreases their evoked responses to peripheral stimuli (brush, low caliber von Frey and pressure), whereas CN5125 ('negative control') has no effect. CN2097 also blocks C-fiber LTP in the dorsal horn, which is linked to neuronal plasticity and central sensitization. At a molecular level, CN2097 attenuates the increase in phosphorylated p38 MAPK, a key intracellular signaling pathway in neuropathic pain. Moreover, spinal injection of CN2097 blocks thermal hyperalgesia in neuropathic rats. We conclude that CN2097 is a small molecule peptide with putative anti-nociceptive effects that modulates physiologic phenomena related to central sensitization under conditions of chronic pain.

Helix 69 of E. coli 23S rRNA has important roles in specific steps of translation, such as subunit association, translocation, and ribosome recycling. An M13 phage library was used to identify peptide ligands with affinity for helix 69. One selected sequence, NQVANHQ, was shown through a bead assay to interact with helix 69. Electrospray ionization mass spectroscopy revealed an apparent dissociation constant for the amidated peptide and helix 69 in the low micromolar range. This value is comparable to that of aminoglycoside antibiotics binding to the A site of 16S rRNA or helix 69. Helix 69 variants (human) and unrelated RNAs (helix 31 or A site of 16S rRNA) showed two- to four-fold lower affinity for NQVANHQ-NH2. These results suggest that the peptide has desirable features for development as a lead compound for novel antimicrobials.