Lars Dreier

In applications of NMR to biological macromolecules in solution, the concentration of the NMR sample is an important parameter describing the sample and providing information for the selection and planning of experiments. Although concentrations can be measured directly by NMR spectroscopy, other methods are usually preferred to measure the concentration of macromolecules in NMR samples. The reasons are the difficulties in the correlation of the sample of interest with the signal intensity representing a known concentration. This correlation is usually obtained by adding to the sample a reference compound with known concentration and comparing the integral over resolved resonance lines of the molecules with known and unknown concentrations. For solutions of biological macromolecules it is very difficult to find a compound that does not interact with the macromolecules and has a resonance outside their spectral range. We introduce PULCON which is a method that correlates the absolute intensities of two spectra measured in different solution conditions. PULCON is easy to implement and apply on all NMR spectrometers; it does not need any special hardware or software. PULCON is very robust and at the same time delivers accurate concentrations of samples in the NMR tube. We demonstrate that PULCON has the potential to replace UV spectroscopy for concentration measurements of NMR samples.

BACKGROUND: Parkin is recruited to defective mitochondria to promote degradation by an autophagy mechanism (mitophagy). RESULTS: VDACs specifically interact with Parkin on defective mitochondria and are required for efficient targeting of Parkin to mitochondria and subsequent mitophagy. CONCLUSION: VDACs recruit Parkin to defective mitochondria. SIGNIFICANCE: A novel mechanistic aspect of Parkin-dependent mitophagy is proposed that may be relevant to Parkinson disease. Mutations in the ubiquitin ligase Parkin and the serine/threonine kinase PINK1 can cause Parkinson disease. Both proteins function in the elimination of defective mitochondria by autophagy. In this process, activation of PINK1 mediates translocation of Parkin from the cytosol to mitochondria by an unknown mechanism. To better understand how Parkin is targeted to defective mitochondria, we purified affinity-tagged Parkin from mitochondria and identified Parkin-associated proteins by mass spectrometry. The three most abundant interacting proteins were the voltage-dependent anion channels 1, 2, and 3 (VDACs 1, 2, and 3), pore-forming proteins in the outer mitochondrial membrane. We demonstrate that Parkin specifically interacts with VDACs when the function of mitochondria is disrupted by treating cells with the proton uncoupler carbonyl cyanide p-chlorophenylhydrazone. In the absence of all three VDACs, the recruitment of Parkin to defective mitochondria and subsequent mitophagy are impaired. Each VDAC is sufficient to support Parkin recruitment and mitophagy, suggesting that VDACs can function redundantly. We hypothesize that VDACs serve as mitochondrial docking sites to recruit Parkin from the cytosol to defective mitochondria.

The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking.