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Gregory J Gatto

Publications and source records attributed to Gregory J Gatto.

5 recordsLinked to original sources
PubMed2004

Pex5p binding affinities for canonical and noncanonical PTS1 peptides.

The majority of proteins targeted to the peroxisomal lumen contain a C-terminal peroxisomal targeting signal-1 (PTS1) that is bound by the peroxin Pex5p. The PTS1 is generally regarded as a C-terminal tripeptide that adheres to the consensus (S/A/C)(K/R/H)(L/M). Previously, we studied the binding affinity of peptides of the form YQX(-3)X(-2)X(-1) to the peptide-binding domain of human Pex5p (referred to as Pex5p-C). Optimal affinity was found for YQSKL, which bound with an affinity of 200 +/- 40 nM. To extend this work, we investigated the properties of a peptide containing the last 9 residues of acyl-CoA oxidase (RHYLKPLQSKL) and discovered that it binds to Pex5p-C with a dissociation constant of 1.4 +/- 0.4 nM, 180 times tighter than YQSKL. Further analysis revealed that the enhanced affinity is primarily due to the presence of leucine in the (-5) position. In addition, a peptide corresponding to the luciferase C-terminus (YKGGKSKL) was found to bind Pex5p-C about 20 times tighter than YQSKL. The majority of this effect results from having lysine in position (-4). Catalase contains a noncanonical PTS1 (-AREKANL). The affinity of YQANL was found to be 3600 +/- 400 nM. This relatively weak binding is consistent with previous unsuccessful attempts to direct chloramphenicol acetyltransferase to the peroxisome by fusing -ANL to its C-terminus (-GGA-ANL). The peptides YKANL, YEKANL, YREKANL, and YAREKANL all bound Pex5p-C with higher affinities than did YQANL, but the affinities are still lower than peptides that correspond to functional targeting signals in other contexts. Because both catalase and Pex5p are tetramers (as opposed to the monomeric Pex5p-C and the peptides used in our studies), multidentate effects on binding affinity between Pex5p and other oligomeric proteins should be considered. Our study provides direct thermodynamic data revealing that peptide binding to Pex5p-C binding is favored by lysine in the (-4) position and leucine in the (-5) position. Our results suggest that peptides or proteins with optimized residues in the (-4) and/or (-5) positions can bind to Pex5p with affinities that are at least two orders of magnitude greater than that of YQSKL, and that this stabilization can compensates for otherwise weakly binding PTS1s.

Acyl-CoA Oxidase↗
PubMed2004

TC-1734: an orally active neuronal nicotinic acetylcholine receptor modulator with antidepressant, neuroprotective and long-lasting cognitive effects.

The development of selective ligands targeting neuronal nicotinic acetylcholine receptors to alleviate symptoms associated with neurodegenerative diseases presents the advantage of affecting multiple deficits that are the hallmarks of these pathologies. TC-1734 is an orally active novel neuronal nicotinic agonist with high selectivity for neuronal nicotinic receptors. Microdialysis studies indicate that TC-1734 enhances the release of acetylcholine from the cortex. TC-1734, by either acute or repeated administration, exhibits memory enhancing properties in rats and mice and is neuroprotective following excitotoxic insult in fetal rat brain in cultures and against alterations of synaptic transmission induced by deprivation of glucose and oxygen in hippocampal slices. At submaximal doses, TC-1734 produced additive cognitive effects when used in combination with tacrine or donepezil. Unlike (-)-nicotine, behavioral sensitization does not develop following repeated administration of TC-1734. Its pharmacokinetic (PK) profile (half-life of 2 h) contrasts with the long lasting improvement in working memory (18 h) demonstrating that cognitive improvement extends beyond the lifetime of the compound. The very low acute toxicity of TC-1734 and its receptor activity profile provides additional mechanistic basis for its suggested potential as a clinical candidate. TC-1734 was very well tolerated in acute and chronic oral toxicity studies in mice, rats and dogs. Phase I clinical trials demonstrated TC-1734's favorable pharmacokinetic and safety profile by acute oral administration at doses ranging from 2 to 320 mg. The bioavailability, pharmacological, pharmacokinetic, and safety profile of TC-1734 provides an example of a safe, potent and efficacious neuronal nicotinic modulator that holds promise for the management of the hallmark symptomatologies observed in dementia.

Administration, Oral↗
PubMed2003

Correlating structure and affinity for PEX5:PTS1 complexes.

Many proteins that are destined to reside within the lumen of the peroxisome contain the peroxisomal targeting signal-1 (PTS1), a C-terminal tripeptide approximating the consensus sequence -Ser-Lys-Leu-COO(-). The PTS1 is recognized by the tetratricopeptide repeat (TPR) domains of PEX5, a cytosolic receptor that cycles between the cytoplasm and the peroxisome. To gain insight into the energetics of PTS1 binding specificity and to correlate these with features from the recently determined structure of a PEX5:PTS1 complex, we used a fluorescence-based binding assay that enables the quantitation of the dissociation constants for PTS1-containing peptide complexes with the TPR region of human PEX5. Through application of this assay to a collection of pentapeptides containing different C-terminal tripeptide sequences, including both natural and unnatural amino acids, the thermodynamic effects of sequence variation were examined. PTS1 variants that correspond to known functional targeting signals bind to the PEX5 fragment with a change in the standard binding free energy within 1.8 kcal mol(-1) of that corresponding to the peptide ending with -Ser-Lys-Leu-COO(-). The results suggest that a binding energy threshold may determine the functionality of PTS1 sequences.

Amino Acid Substitution↗
PubMed2003

Nonrandom tripeptide sequence distributions at protein carboxyl termini.

The availability of complete genome sequences enables the statistical analysis of sequence features without significant database-imposed bias. The carboxyl termini of proteins often contain regions associated with protein targeting and enhanced translational termination. We analyzed the frequency of occurrence of C-terminal tripeptides in representative archaeal, bacterial, and eukaryotic genomes. The sequence distribution in prokaryotic genomes nearly matches that generated by the randomization of the observed tripeptide set. In contrast, eukaryotic genomes contain large numbers of overrepresented sequences. Some of these correspond to highly repeated sequences from either duplicated endogenous genes or transposon open reading frames. Gratifyingly, others represent previously known targeting signals or sequences associated with an increase in translational termination efficiency. However, a number of overrepresented tripeptides have not been previously noted and may represent novel functional sequences. For example, the sequence XSS may enhance translational termination efficiency in plants, whereas FWC may be a targeting or processing signal for certain amino acid permeases in yeast.

Amino Acid Motifs↗