Membrane binding and pore forming insertion of PEX5 into horizontal lipid bilayer.

The assembly of the peroxisomal translocon involves the transition of a soluble form of the peroxisomal targeting receptor PEX5 into a membrane-bound form, which becomes an integral membrane component of the import pore for peroxisomal matrix proteins. How this transition occurs is still a mystery. We addressed this question using a artificial horizontal bilayer in combination with fluorescence time-correlated single photon counting (TCSPC) and electrophysiological channel recording. Purified human isoform PEX5L and truncated PEX5L(1-335) lacking the cargo binding domain were selectively labeled with thiol-reactive Atto-dyes. Diffusion coefficients of labeled protein in solution show that PEX5L is monomeric with a rather compact spherical conformation, while the truncated protein appeared in a more extended conformation. Labeled PEX5L and the truncated PEX5L(1-335) bind stably to horizontal bilayer thereby accumulating around 100-fold. The diffusion coefficients of the membrane-bound PEX5L forms are 3-4 times lower than in solution, indicating the formation of larger complexes. Electrophysiological single channel recording shows that membrane-bound labeled and non-labeled PEX5L, but not the truncated PEX5L(1-335), can form ion conducting membrane channels. The data suggest that PEX5L is the pore-forming component of the oligomeric peroxisomal translocon and that spontaneous PEX5L membrane surface binding might be an important step in its assembly.

Interneuronal growth and expression of interneuronal markers in visual cortex of mice with transgenic activation of Ras.

The synRas transgenic mice express constitutively activated Valin12-Harvey Ras in postnatal neocortical pyramidal neurons. This leads to somatodendritic hypertrophy, higher densities of spines and synapses, and an enhancement of synaptic long-term potentiation associated with an increased glutamate receptor-mediated activity. It was less clear how the interneurons respond to these alterations, and this prompted the quantitative assessment of interneuron neurochemistry. Interneurons rarely expressed the transgene, however, several interneuron types displayed a transient somatic hypertrophy. Furthermore, NPY mRNA expression was persistently increased as were the laminar percentages of labeled neurons. The expression of parvalbumin and voltage-gated potassium channels Kv3.1b/3.2 was unchanged. A significant decline of GAD-67, but not GAD-65, mRNA expressing neurons was observed in layer VI in animals older than P60. This suggested that subtle deficits in inhibition and enhanced excitation evoke the interneuronal changes in the synRastransgenic mouse cortex.

A molecular mechanism for direct sirtuin activation by resveratrol.

Sirtuins are protein deacetylases regulating metabolism, stress responses, and aging processes, and they were suggested to mediate the lifespan extending effect of a low calorie diet. Sirtuin activation by the polyphenol resveratrol can mimic such lifespan extending effects and alleviate metabolic diseases. The mechanism of Sirtuin stimulation is unknown, hindering the development of improved activators. Here we show that resveratrol inhibits human Sirt3 and stimulates Sirt5, in addition to Sirt1, against fluorophore-labeled peptide substrates but also against peptides and proteins lacking the non-physiological fluorophore modification. We further present crystal structures of Sirt3 and Sirt5 in complex with fluorogenic substrate peptide and modulator. The compound acts as a top cover, closing the Sirtuin's polypeptide binding pocket and influencing details of peptide binding by directly interacting with this substrate. Our results provide a mechanism for the direct activation of Sirtuins by small molecules and suggest that activators have to be tailored to a specific Sirtuin/substrate pair.