Crystal structure of cardiac troponin C regulatory domain in complex with cadmium and deoxycholic acid reveals novel conformation

The amino-terminal regulatory domain of cardiac troponin C (cNTnC) plays an important role as the calcium sensor for the troponin complex. Calcium binding to cNTnC results in conformational changes that trigger a cascade of events that lead to cardiac muscle contraction. The cardiac N-terminal domain of TnC consists of two EF-hand calcium binding motifs, one of which is dysfunctional in binding calcium. Nevertheless, the defunct EF-hand still maintains a role in cNTnC function. For its structural analysis by X-ray crystallography, human cNTnC with the wild-type primary sequence was crystallized under a novel crystallization condition. The crystal structure was solved by the single-wavelength anomalous dispersion method and refined to 2.2 Å resolution. The structure displays several novel features. Firstly, both EF-hand motifs coordinate cadmium ions derived from the crystallization milieu. Secondly, the ion coordination in the defunct EF-hand motif accompanies unusual changes in the protein conformation. Thirdly, deoxycholic acid, also derived from the crystallization milieu, is bound in the central hydrophobic cavity. This is reminiscent of the interactions observed for cardiac calcium sensitizer drugs that bind to the same core region and maintain the “open” conformational state of calcium-bound cNTnC. The cadmium ion coordination in the defunct EF-hand indicates that this vestigial calcium binding site retains the structural and functional elements that allow it to coordinate a cadmium ion. However, it is a result of, or concomitant with, large and unusual structural changes in cNTnC.     

Singlet oxygen affects the activity of the thylakoid ATP synthase and has a strong impact on its γ subunit

Singlet oxygen is reported to have the most potent damaging effect upon the photosynthetic machinery. Usually this reactive oxygen molecule acts in concert with other ROS types under stressful conditions. To understand the specific role of singlet oxygen we took advantage of the conditional flu mutant of Arabidopsis thaliana. In flu, the negative feedback loop is abolished, which blocks chlorophyll biosynthesis in the dark. Therefore high amounts of free protochlorophyllide accumulate during darkness. If flu gets subsequently illuminated, free protochlorophyllide acts as a photosensitiser leading almost exclusively to high amounts of ¹O₂. Analysing the thylakoid protein pattern by using 2D PAGE and subsequent MALDI-TOF analysis, we could show, in addition to previous described effects on photosystem II, that singlet oxygen has a massive impact on the thylakoid ATP synthase, especially on its γ subunit. Additionally, it could be shown that the activity of the ATP synthase is reduced upon singlet oxygen exposure and that the rate of non-photochemical quenching is affected in flu mutants exposed to ¹O₂.     

Factors affecting trematode infection rates in freshwater mussels

Mussels are intermediate hosts of digenean trematodes, but determinants of these infections remain unknown. To address this problem, we collected duck mussels Anodonta anatina in eighteen lakes from northeastern Poland and examined how mussel age, sex, and the encrustation with zebra mussels Dreissena polymorpha and environmental conditions in lakes influenced infection rates. We also assessed parasitic preferences to host gonads and hepatopancreas and the impact of parasites on female fertility. Mussels were infected with Rhipidocotyle campanula and Phyllodistomum sp. Infection rates were higher in older and female mussels but were unrelated to the biomass of encrusting D. polymorpha and the trophy, thermal conditions, and Ca²⁺ availability. Parasites occupied gonads more often than hepatopancreas. Infected females were less likely to carry glochidia and incubated fewer glochidia. We suggest that the risk of infection by digenean trematodes increases with the amount of water processed by filter-feeding hosts and/or that parasites actively seek hosts which can provide them with abundant resources. This mechanism explains why parasites more often occupied older and female mussels and targeted their gonads. Future research on trematode-mussel interactions should integrate knowledge on different elements of the complex trematode life cycles, including effects of higher-order hosts such as fish.     

The crystal structure of the phosphatidylinositol 4‐kinase IIα

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4‐kinase type IIα (PI4K IIα), in complex with ATP solved by X‐ray crystallography at 2.8 Å resolution. The structure revealed a non‐typical kinase fold that could be divided into N‐ and C‐lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C‐lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.     

Biodistribution and Efficacy Studies of the Proteasome Inhibitor BSc2118 in a Mouse Melanoma Model

Inhibition of the proteasome offers many therapeutic possibilities in inflammation as well as in neoplastic diseases. However, clinical use of proteasome inhibitors is limited by the development of resistance or severe side effects. In our study we characterized the anti-tumor properties of the novel proteasome inhibitor BSc2118. The sensitivity of tumor lines to BSc2118 was analyzed in comparison to bortezomib using crystal violet staining in order to assess cell viability. The In Vivo distribution of BSc2118 in mouse tissues was tracked by a fluorescent-modified form of BSc2118 (BSc2118-FL) and visualized by confocal microscopy. Inhibition of the 20S proteasome was monitored both in cultured cell lines and in mice, respectively. Finally, safety and efficacy of BSc2118 was evaluated in a mouse melanoma model. BSc2118 inhibits proliferation of different tumor cell lines with a similar potency as compared with bortezomib. Systemic administration of BSc2118 in mice is well tolerated, even when given in a dose of 60 mg/kg body weight. After systemic injection of BSc2118 or bortezomib similar proteasome inhibition patterns are observed within the murine organs. Detection of BSc2118-FL revealed correlation of distribution pattern of BSc2118 with inhibition of proteasomal activity in cells or mouse tissues. Finally, administration of BSc2118 in a mouse melanoma model shows significant local anti-tumor effects. Concluding, BSc2118 represents a novel low-toxic agent that might be alternatively used for known proteasome inhibitors in anti-cancer treatment.     

Soil microarthropods respond differently to simulated drought in organic and conventional farming systems

In Central Europe, summer droughts are increasing in frequency which threatens production and biodiversity in agroecosystems. The potential of different farming systems to mitigate detrimental drought effects on soil animals is largely unknown. We investigated the effects of simulated drought on the abundance and community composition of soil microarthropods (Collembola and Oribatida and Meso‐, Pro‐, and Astigmata) in winter wheat fields under long‐term conventional and organic farming in the DOK trial, Switzerland. We simulated drought by excluding 65% of the ambient precipitation during the wheat‐growing season from March to June 2017. The abundance of Collembola and Oribatida declined more consistently in conventionally managed fields compared to organically managed fields under simulated drought. The abundance of Collembola as well as Meso‐, Pro‐ and Astigmata, but not the abundance of Oribatida, increased in deeper soil layers due to simulated drought, suggesting vertical migration as a drought avoidance strategy. The species composition of Oribatida communities, but not of Collembola communities, differed significantly between drought treatments and between farming systems. Soil carbon content was a major factor structuring Oribatida communities. Our results suggest that organic farming buffers negative effects of drought on soil microarthropods, presumably due to higher soil carbon content and associated higher soil moisture and improved soil structure. This potential of organic farming systems to mitigate consequences of future droughts on soil biodiversity is promising and needs further exploration across larger climatic and spatial scales and should be extended to other groups of soil biota.     

Loss of the orphan nuclear receptor SHP is more pronounced in fibrolamellar carcinoma than in typical hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a major problem in oncology. The molecular mechanisms which underlie its pathogenesis are poorly understood. Recently the Small Heterodimer Partner (SHP), an orphan nuclear receptor, was suggested to be involved as a tumor suppressor in hepatocellular carcinoma development. To date, there are no such studies regarding fibrolamellar carcinoma, a less common variant of HCC, which usually affects young people and displays distinct morphological features. The aim of our project was to evaluate the SHP levels in typical and fibrolamellar hepatocellular carcinoma with respect to the levels of one of the cell cycle regulators, cyclin D1. We assessed the immunoreactivity levels of SHP and cyclin D1 in 48 typical hepatocellular carcinomas, 9 tumors representing the fibrolamellar variant, 29 non malignant liver tissues and 7 macroregenerative nodules. We detected significantly lower SHP immunoreactivity in hepatocellular carcinoma when compared to non malignant liver tissue. Moreover, we found that SHP immunoreactivity is reduced in fibrolamellar carcinoma when compared to typical hepatocellular carcinoma. We also found that SHP is more commonly lost in HCC which arises in the liver with steatosis. The comparison between the cyclin D1 and SHP expression revealed the negative correlation between these proteins in the high grade HCC. Our results indicate that the impact of loss of SHP protein may be even more pronounced in fibrolamellar carcinoma than in a typical form of HCC. Further investigation of mechanisms through which the loss of SHP function may influence HCC formation may provide important information in order to design more effective HCC therapy.     

siRNA carriers based on carbosilane dendrimers affect zeta potential and size of phospholipid vesicles

One of the major limitations in gene therapy is an inability of naked siRNA to passively diffuse through negatively charged cell membranes. Therefore, the siRNA transport into a cell requires efficient carriers. In this work we analyzed the charge-dependent interaction of the complexes of cationic carbosilane dendrimers (CBD) and anti-HIV siRNA (dendriplexes) with the model membranes - large unilamellar vesicles (LUV). We used the second generation of branched with CBD carbon-silicon bonds (CBD-CS) which are water-stable and that of oxygen-silicon bonds (CBD-OS) which are slowly hydrolyzed in aqueous solutions. The LUVs were composed of zwitterionic dimyristoylphosphatidylcholine (DMPC), negatively charged dipalmitoylphosphatidylglycerol (DPPG) and their mixture (DMPC/DPPG, molar ratio 7:3). The interaction of dendriplexes with LUVs affected both zeta potential and size of the vesicles. The changes of these values were larger for the negatively charged LUV. CBD-CS resulted in the decrease of zeta potential values to more negative ones, whereas an opposite effect took place for CBD-OS suggesting a different kind of interaction between LUVs and the dendriplexes. The results indicate that both CBD-CS and CBD-OS can be used for transport of siRNA into the cells. However, CBD-CS are preferred due to a better stability in water and improved bioavailability of siRNA on their surface.