Friedreich's ataxia, an autosomal cardio-and neurodegenerative disorder that affects 1 in 50,000 humans, is caused by decreased levels of the protein frataxin. Although nuclear encoded, frataxin is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting monomeric frataxin might function as the common iron donor.In order to provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron loaded monomer and the protein can bind 2 ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal binding sites is consistent with a 6 coordinate iron-(oxygen and nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. Based on our results, we have developed a model for how we believe yeast frataxin interacts with iron. Keywords Briefs:We present a characterization of monomeric yeast frataxin's iron binding ability. Various spectroscopic techniques were applied to help characterize the iron binding affinity of yeast frataxin, the oligomeric state of the protein, specific amino acids affected by the presence of iron and finally the metal-ligand coordination geometry. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 August 19. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript patients, is a direct result of a trinucleotide repeat expansion in the first intron of the gene for the protein frataxin; this expansion disrupts gene transcription leading to a frataxin deficiency in humans (4). The inability to produce frataxin is associated with mitochondrial accumulation of iron that is biologically unavailable. In humans, the breakdown in normal iron regulation pathways resulting from frataxin deficiency is exemplified physiologically by the degeneration of sensory neurons in the dorsal root ganglia, hypertrophic cardiomyopathy and diabetes mellitus (5). In yeast, reintroduction of frataxin into knockout cells results in restored bioavailability of the accumulated metal (6). These data indicate frataxin is required for retaining bi...

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.

Brain-derived neurotrophic factor signaling is defective in Angelman syndrome and can be rescued by disruption of Arc/PSD95 binding.

No abstract

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.

It is an accepted practice in ligand design to introduce conformational constraint with the expectation of improving affinity, justified by the theoretical possibility that an unfavorable change in binding entropy will be reduced. This rationale of minimizing the entropic penalty through imposing structural constraints upon a ligand, however, has been voiced more often than verified. Here we examine three modified cyclic peptides, along with multiple versions of their linear control analogs, and determine their thermodynamic parameters when binding the same host, the third PDZ domain (PDZ3) of the mammalian postsynaptic density-95 (PSD-95) protein. To begin a two-stage investigation, the initial evaluation involved solution binding studies with isothermal titration calorimetry (ITC), which provided the changes in Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) upon formation of the protein-ligand complex. In the second stage, a selected macrocycle along with two matched linear controls were subjected to more rigorous analysis by ITC, which included (1) change in heat of buffer ionization (DeltaH(ion)) titrations, to examine the role of proton transfer events; (2) change in heat capacity (DeltaC(p)) determinations, to indirectly probe the nature of the binding surface; and (3) osmotic stress experiments, to evaluate desolvation effects and quantitate water release. Together, these demonstrate that the entropic relationship between a macrocyclic ligand and a linear counterpart can be a complex one that is difficult to rationalize. Further, the addition of constraint can, counterintuitively, lead to a less favorable change in binding entropy. This underscores the need to use matched linear control ligands to assure that comparisons are made in a meaningful manner.

Purpose: Various studies have shown the importance of the GAIP interacting protein, COOHterminus (GIPC, also known as Synectin) as a central adaptor molecule in different signaling pathways and as an important mediator of receptor stability. GIPC/Synectin is associated with different growth-promoting receptors such as insulin-like growth factor receptor I (IGF-IR) and integrins. These interactions were mediated through its PDZ domain. GIPC/Synectin has been shown to be overexpressed in pancreatic and breast cancer. The goal of this study was to show the importance of GIPC/Synectin in pancreatic cancer growth and to evaluate a possible therapeutic strategy by using a GIPC-PDZ domain inhibitor. Furthermore, the effect of targeting GIPC on the IGF-I receptor as one of its associated receptors was tested. Experimental Design: The in vivo effects of GIPC/Synectin knockdown were studied after lentiviral transduction of luciferase-expressing pancreatic cancer cells with short hairpin RNA against GIPC/Synectin. Additionally, a GIPC-PDZ^targeting peptide was designed. This peptide was tested for its influence on pancreatic cancer growth in vitro and in vivo. Results: Knockdown of GIPC/Synectin led to a significant inhibition of pancreatic adenocarcinoma growth in an orthotopic mouse model. Additionally, a cell-permeable GIPC-PDZ inhibitor was able to block tumor growth significantly without showing toxicity in a mouse model. Targeting GIPC was accompanied by a significant reduction in IGF-IR expression in pancreatic cancer cells. Conclusions: Our findings show that targeting GIPC/Synectin and its PDZ domain inhibits pancreatic carcinoma growth and is a potential strategy for therapeutic intervention of pancreatic cancer.

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.