Thermodynamic and spectroscopic investigation on the role of Met residues in Cu(II) binding to the non-octarepeat site of the human prion protein

Among the common features shared by neurodegenerative diseases there is the central role played by specific proteins or peptides which accumulate in neurons as insoluble plaques or tangles, containing abnormal amounts of redox-active metal ions, like copper and iron. In the case of transmissible spongiform encephalopathies (TSE), the involved protein is known as "prion protein" (PrP(C)) since "prions" (proteinaceous and infectious) are the agents which make TSE transmissible. It is widely accepted that PrP(C), in its wild-type form, can bind up to six Cu(II) ions, four of them in the so-called "octarepeat domain" and the others in the "fifth (non-octarepeat) binding-site". The latter domain contains two His residues, acting as anchoring sites for Cu(II) ions, and other potential binding residues, such as Lys and Met. While it is widely accepted that Lys residues do not take part in complex-formation, the role of methionines is still debated. In order to shed light on this issue, some peptides have been synthesized, either directly mimicking the sequence of the second half of the fifth binding site of human-PrP(C) (apo-form) or analogues where Met residues have been substituted by n-leucine. In addition, a series of short peptides, containing both His and Met residues in different relative positions, have been investigated, for the sake of comparison. Spectroscopic results, including NMR spectra of systems containing Ni(II) as a probe for the paramagnetic Cu(II) ion, agree on the exclusion of any direct interaction between the sulphur atom of Met residues and the Cu(II) ion already bound to His-imidazole side-chains. However, thermodynamic data show that Met-109 somewhat contributes to stability of complex species and this can be attributed to different electronic and steric effects.     

UV reflecting vole scent marks attract a passerine, the great grey shrike Lanius excubitor

Diurnal raptors have been shown to use ultraviolet vision and UV-reflecting vole scent marks to find suitable hunting areas. We studied if a passerine species, the great grey shrike Lanius excubitor that uses voles as its primary food, may also detect prey-patches in the same way. We conducted a laboratory experiment with ten shrikes. Each individual shrike had four options to choose from: (1) scent marks with UV light, (2) scent marks without UV light, (3) clean arena with UV light, and (4) clean arena without UV light. The birds preferred the scent-marked arena with UV light as measured by the number of scans and the time spent above it. Therefore, we suggest that great grey shrike probably uses UV cues to gain information on vole locations and abundance.     

Bedaquiline,an FDA-approved drug,inhibits mitochondrial ATP production and metastasis in vivo,by targeting the gamma subunit (ATP5F1C) of the ATP synthase

Here, we provide evidence that high ATP production by the mitochondrial ATP-synthase is a new therapeutic target for anticancer therapy, especially for preventing tumor progression. More specifically, we isolated a subpopulation of ATP-high cancer cells which are phenotypically aggressive and demonstrate increases in proliferation, stemness, anchorage-independence, cell migration, invasion and multi-drug resistance, as well as high antioxidant capacity. Clinically, these findings have important implications for understanding treatment failure and cancer cell dormancy. Using bioinformatic analysis of patient samples, we defined a mitochondrial-related gene signature for metastasis, which features the gamma-subunit of the mitochondrial ATP-synthase (ATP5F1C). The relationship between ATP5F1C protein expression and metastasis was indeed confirmed by immunohistochemistry. Next, we used MDA-MB-231 cells as a model system to functionally validate these findings. Importantly, ATP-high MDA-MB-231 cells showed a nearly fivefold increase in metastatic capacity in vivo. Consistent with these observations, ATP-high cells overexpressed (i) components of mitochondrial complexes I–V, including ATP5F1C, and (ii) markers associated with circulating tumor cells (CTCs) and metastasis, such as EpCAM and VCAM1. Knockdown of ATP5F1C expression significantly reduced ATP-production, anchorage-independent growth, and cell migration, as predicted. Similarly, therapeutic administration of the FDA-approved drug, Bedaquiline, downregulated ATP5F1C expression in vitro and prevented spontaneous metastasis in vivo. In contrast, Bedaquiline had no effect on the growth of non-tumorigenic mammary epithelial cells (MCF10A) or primary tumors in vivo. Taken together, our results suggest that mitochondrial ATP depletion is a new therapeutic strategy for metastasis prophylaxis, to avoid treatment failure. In summary, we conclude that mitochondrial ATP5F1C is a promising new biomarker and molecular target for future drug development, for the prevention of metastatic disease progression.Subject terms: Metastasis, Tumour heterogeneity     

Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man₅GlcNAc₂-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30–100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.     

The rice coleoptile: an example of anaerobic nitrate assimilation

Nitrate present in rice caryopses can be reduced to ammonium and the ammonium subsequently assimilated by the coleoptile during anaerobic germination. All the enzymes of nitrate reduction and ammonia assimilation are present in the coleoptile. The supply of ¹⁵NO₃ confirms that the nitrate nitrogen is anaerobically incorporated into amino acids. Under anoxia, nitrate and nitrite reductase activities are increased in the coleoptile by exogenous nitrate. The importance of nitrate utilization during the anaerobic germination of rice caryopses is discussed.     

Abscisic acid induced stress-like polyamine pattern in wheat seedlings, and its reversal by potassium ions

In 3-day-old wheat ( Triticum aestivum L. cv. Marinat) seedlings, 100 μ M ABA blocked the growth and altered the level of K⁺ in both the shoot and root. The presence of ABA increased the putrescine titer during a 24-h treatment. Increasing the endogenous level of K⁺ by the addition of 10 m M KCl to the ABA-treated seedlings, inhibited the effect of ABA on growth and putrescine level. In both tissues, ABA increased putrescine content at low concentrations (1 μ M ), reaching the maximal effect at 100 μ M . Putrescine increase induced by ABA was inhibited by both α-difluoromethylarginine (DFMA) and α-difluoromethylornithine (DFMO) in shoots while only the inhibitor of arginine decarboxylase was effective in the root. The presence of ABA modulated, in opposite ways, ornithine and arginine decarboxylase activities. These results are discussed in relation to ion balance under stress.     

Comparison of rates of hydrolysis of N-oleoyl and N-stearoyl glucocerebroside in patients with Gaucher's disease

The deficiency of oleic acid as one of the fatty acids in glucocerebrosides that accumulate (31--77 mg/g dry weight) in the spleen in patients with Gaucher's disease was confirmed in 9 cases. In an effort to account for the 10-fold difference between the oleoyl glycocerebroside content of glucocerebrosides in spleen from controls and patients with Gaucher's disease, we compared the ability of extracts of spleen and fibroblasts from individuals with various forms of Gaucher's disease and controls to hydrolyze [14C]stearoyl and [3H]oleoyl glucocerebroside. The residual glucosylceramidase activity in patients with Gaucher's disease hydrolyzes the glucose moiety of oleoyl glucocerebroside at approximately the same rate as that of stearoyl glucocerebroside. Similarly, the more active glucosylceramidase of control tissue acts upon both oleoyl and stearoyl glucocerebrosides with equal efficiency. These observations indicate that a mutation affecting the substrate specificity of glucosylceramidase cannot account for the lack of oleic acid-containing glucocerebrosides in patients with Gaucher's disease. Thus, the hypothesis that the difference in fatty acid composition found in glucocerebroside is obtained as a result of a mutation affecting the specificity of the residual glucosylceramidase must be rejected.     

Effects of genetically modified potatoes with increased zeaxanthin content on the abundance and diversity of rhizobacteria with in vitro antagonistic activity do not exceed natural variability among cultivars

To assess potential effects of genetically modified (GM) potatoes on the abundance and diversity of rhizobacteria with in vitro antagonistic activity in relation to natural variability among cultivars, two GM potato lines accumulating the carotenoid zeaxanthin in their tubers, the parental cultivar and four additional commercial cultivars were planted at two field sites in Germany. Rhizosphere samples were taken at three developmental stages of the plants. A total of 3,985 bacteria isolated from the rhizosphere were screened for their in vitro antagonistic activity towards Rhizoctonia solani, Verticillium dahliae and Phytophthora infestans using a dual-culture assay. Genotypic characterisation, 16S rRNA gene sequencing and antifungal metabolite analysis was performed to characterize the 595 antagonists obtained. The 16S rRNA gene-based identification of in vitro antagonists revealed strong site-dependent differences in their taxonomic composition. This study showed that the site was the overriding factor determining the proportion and diversity of antagonists from the rhizosphere of potato while the effect of the genetic modification on the proportion of antagonists obtained did not exceed natural variability among the five commercial cultivars tested.