Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein

Fibrils from the Parkinson's-disease-related A53T mutant of α-synuclein were investigated by solid-state NMR spectroscopy, electron microscopy, and atomic force microscopy. Sequential solid-state NMR resonance assignments were obtained for a large fraction of the fibril core. Experiments conducted above and below the freezing point suggest that the fibrils contain regions with increased mobility and structural elements different from β-strand character, in addition to the rigid β-sheet-rich core region. As in earlier studies on wild-type α-synuclein, the C-terminus was found to be flexible and unfolded, whereas the main core region was highly rigid and rich in β-sheets. Compared to fibrils from wild-type α-synuclein, the well-ordered β-sheet region extends to at least L38 and L100. These results demonstrate that a disease-related mutant of α-synuclein differs in both aggregation kinetics and fibril structure.     

Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production

Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 ± 0.006 mol lysine (mol glucose)⁻¹, which was 12% higher than that of the empty vector control strain.     

The Thermodynamics of General and Local Anesthesia

General anesthetics are known to cause depression of the freezing point of transitions in biomembranes. This is a consequence of ideal mixing of the anesthetic drugs in the membrane fluid phase and exclusion from the solid phase. Such a generic law provides physical justification of the famous Meyer-Overton rule. We show here that general anesthetics, barbiturates, and local anesthetics all display the same effect on melting transitions. Their effect is reversed by hydrostatic pressure. Thus, the thermodynamic behavior of local anesthetics is very similar to that of general anesthetics. We present a detailed thermodynamic analysis of heat capacity profiles of membranes in the presence of anesthetics. Using this analysis, we are able to describe experimentally observed calorimetric profiles and predict the anesthetic features of arbitrary molecules. In addition, we discuss the thermodynamic origin of the cutoff effect of long-chain alcohols and the additivity of the effect of general and local anesthetics.     

Influence of manganese ions on cellular behavior of human osteoblasts in vitro

Divalent cations like Mn(2+) are known to strongly influence the integrin affinity to ligands and - in consequence - cell adhesion to extracellular matrix proteins. Therefore, divalent cation supplementation of biomaterials could be a promising approach to improve the ingrowth and the integration of implants. We were interested, whether manganese ions affect cellular functions like spreading, proliferation as well as gene expression in human osteoblasts. MG-63 osteoblastic cells were cultured in DMEM with 10% FCS. MnCl(2) was added at a concentration range of 0.01-0.5mM for 24h and 48 h. Spreading (cell area in microm(2)) of PKH26-stained cells (cell membrane dye) was analyzed using confocal microscopy. Cell proliferation was measured by flow cytometry. Quantification of the phosphorylation status of signaling proteins was estimated using the Bio-Plex 200 system. Gene expression of osteogenic markers at the mRNA and protein level was analyzed by quantitative real time RT-PCR and Western blot, respectively. The results demonstrated that at higher concentrations of Mn(2+) cells revealed a spindle shaped morphology. Further analyses indicated a reduced spreading, proliferation as well as phosphorylation of signaling proteins due to the influence of Mn(2+) in a concentration-dependent manner. Although expression of bone sialo protein (BSP) at the mRNA level increased both after 24h and 48 h in the presence of manganese, no increased expression of BSP was detected at the protein level. The expression of alkaline phosphatase (ALP) and collagen 1 (Col 1) mRNA decreased at >0.1mM MnCl(2). We speculate that the effect of manganese cations on cell functions is strongly concentration-dependent and the release of manganese when incorporated in a biomaterial surface has to be thoroughly adjusted.     

A cell-permeable fusion toxin as a tool to study the consequences of actin-ADP-ribosylation caused by the salmonella enterica virulence factor SpvB in intact cells

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.     

Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates – A Substudy

Background

Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates.

Objective

To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake.

Methods

A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m², age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-²H₅]phenylalanine and L-[ring-²H₂]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans.

Results

After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001). Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03), synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01) and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001) were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042±0.01 vs 0.045±0.01%/h;P = 0.620).

Conclusions

In the overnight fasted state, adaptation to a low-protein intake (0.4 g/kg/d) does not result in a more negative whole-body protein balance and does not lower basal muscle protein synthesis rates when compared to a high-protein intake.

Trial Registration

Clinicaltrials.gov NCT01551238.     

2-Oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase

Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.