Small molecule PGC-1α1 protein stabilizers induce adipocyte Ucp1 expression and uncoupled mitochondrial respiration.

OBJECTIVE: The peroxisome proliferator-activated receptor-γ coactivator-1α1 (PGC-1α1) regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1α1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1α1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1α1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1α1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions. METHODS: We designed a cell-based high-throughput screening system to identify PGC-1α1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1α1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes. RESULTS: Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1α1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes. CONCLUSIONS: We identify compounds that induce PGC-1α1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders.

What We Can Learn from Artificial Lateral Line Sensor Arrays.

The lateral line system of fish is important for many behaviors, including spatial orientation, prey detection, intraspecific communication, and entraining. With aid of the lateral line, fish perceive minute water motions. The smallest sensory unit of the lateral line is the neuromast, which occurs freestanding on the skin and in fluid-filled canals. We have built artificial lateral line canal systems that can be used to measure spatiotemporal flow patterns. Those patterns can, for instance, be used to distinguish between different environments and upstream objects.

Labelling of manganese-based magnetic resonance imaging (MRI) contrast agents with the positron emitter 51Mn, as exemplified by manganese-tetraphenyl-porphin-sulfonate (MnTPPS4).

The potential tumor seeking MRI contrast agent MnTPPS(4) was labelled with the positron emitting nuclide (51)Mn in no-carrier-added (n.c.a.) form. The complex formation kinetics were investigated and the apparent rate constants were determined under pseudo-first-order conditions. The derived bimolecular rate constants gave the Arrhenius parameters E(A)=84 kJ mol(-1) and A=2 x 10(12)s(-1)M(-1). Optimum labelling conditions were derived (radiochemical yields >99% possible, effective yields about 32%). Separation and purification of n.c.a. (51)MnTPPS(4) were performed for potential human use. All impurities were <1%.

Labelling of the solvent DMSO as side reaction of methylations with n.c.a. [11C]CH3I.

Competing labelling of solvent dimethyl sulfoxide (DMSO) can occur during the 11C-methylation of amine precursors. A kinetic analysis of the methylation reaction of DMSO with n.c.a. [11C]CH3I was performed at 120 degrees C resulting in rate constants. The rate constant for the formation of the intermediate, methylated DMSO ([11C]DMSO-M), is compared to the reaction of [11C]CH3I with two tertiary amines, namely Dexetimide and Desmethyloxotremorine-M. The specific activity of the labelled product is reduced due to partial 12C-methylation of the precursor amines by [11C]DMSO-M in cases of significant DMSO labelling as side reaction.

Recognition of peroxisomal targeting signal type 1 by the import receptor Pex5p.

We have studied how Pex5p recognizes peroxisomal targeting signal type 1 (PTS1)-containing proteins. A randomly mutagenized pex5 library was screened in a two-hybrid setup for mutations that disrupted the interaction with the PTS1 protein Mdh3p or for suppressor mutations that could restore the interaction with Mdh3p containing a mutation in its PTS1. All mutations localized in the tetratricopeptide repeat (TPR) domain of Pex5p. The Pex5p TPR domain was modeled based on the crystal structure of a related TPR protein. Mapping of the mutations on this structural model revealed that some of the loss-of-interaction mutations consisted of substitutions in alpha-helices of TPRs with bulky amino acids, probably resulting in local misfolding and thereby indirectly preventing binding of PTS1 proteins. The other loss-of-interaction mutations and most suppressor mutations localized in short, exposed, intra-repeat loops of TPR2, TPR3, and TPR6, which are predicted to mediate direct interaction with PTS1 amino acids. Additional site-directed mutants at conserved positions in intra-repeat loops underscored the importance of the loops of TPR2 and TPR3 for PTS1 interaction. Based on the mutational analysis and the structural model, we put forward a model as to how PTS1 proteins are selected by Pex5p.

The peroxisomal membrane protein Pex13p shows a novel mode of SH3 interaction.

Src homology 3 (SH3) domains are small non-catalytic protein modules capable of mediating protein-protein interactions by binding to proline-X-X-proline (P-X-X-P) motifs. Here we demonstrate that the SH3 domain of the integral peroxisomal membrane protein Pex13p is able to bind two proteins, one of which, Pex5p, represents a novel non-P-X-X-P ligand. Using alanine scanning, two-hybrid and in vitro interaction analysis, we show that an alpha-helical element in Pex5p is necessary and sufficient for SH3 interaction. Sup pressor analysis using Pex5p mutants located in this alpha-helical element allowed the identification of a unique site of interaction for Pex5p on the Pex13p-SH3 domain that is distinct from the classical P-X-X-P binding pocket. On the basis of a structural model of the Pex13p-SH3 domain we show that this interaction probably takes place between the RT- and distal loops. Thus, the Pex13p-SH3-Pex5p interaction establishes a novel mode of SH3 interaction.

Saccharomyces cerevisiae PTS1 receptor Pex5p interacts with the SH3 domain of the peroxisomal membrane protein Pex13p in an unconventional, non-PXXP-related manner.

A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p. In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain. Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p. Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way. To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3. Such mutations were all located in a small region in the N-terminal half of Pex5p. One of the altered residues (F208) was part of the sequence W(204)XXQF(208), that is conserved between Pex5 proteins of different species. Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain. The Pex5p mutants could only partially restore PTS1-protein import in pex5Delta cells in vivo. In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3. In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes. Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function.

Falls--hiding behind curtains.

Reported in this paper are the outcomes of a retrospective investigation into falls in toilets and injuries sustained by 29 elderly, frail, yet independently ambulatory aged people (mean age 86.21 years) in a residential aged care facility. It was hypothesised that the white or pale coloured curtains that had replaced walls in some toilet cubicles contributed to the falls and injuries. During the six months for which data were reviewed nine of the 29 independently mobile people fell in the toilets, seven in the cubicles with the curtains. This latter group sustained 100 percent of the injuries. It is concluded that curtained cubicles create an additional hazard to the elderly in this particular setting. Recommendations are made to improve the situation.

Case report: autonomic postganglionic denervation--sural nerve and saphenous vein biopsy.

The authors describe a 31-year-old woman of British Isle ancestry who developed a syndrome resembling familial dysautonomia in her early teenage years. Predominant manifestations included achalasia, severe orthostatic hypotension, and abnormal sweating. The study included resting and stimulated fractional catecholamines, which were almost nonexistent in both situations, and urinary catecholamines, demonstrating an increase in dopamine degradation products. Immunohistochemistry of saphenous vein was negative for dopamine beta-hydroxylase (DBH), serotonin (5-HT) and several vasoactive neuropeptides. The only neuropeptide detected at levels thought to be physiologically relevant was calcitonin gene-related peptide (CGRP), a vasodilator. This was in contrast to control veins, all of which had DBH and neuropeptide Y immunoreactive fibers but few CGRP fibers. Also in contrast to controls, electron microscopy of the saphenous vein indicated a close to total absence of terminals with norepinephrine containing vesicles. Sural nerve biopsy showed, on electron microscopy, a considerable reduction in the number of myelinated fibers, while unmyelinated fibers appeared to be in the normal range. The authors suggest, from the above findings, that the autonomic fibers were undergoing some form of distal axonal degeneration. Their findings differ from most biopsies performed in dysautonomic children, and they believe their patient has a different neurologic entity.