Description: Publication date: 2015 Source: FEBS Open Bio, Volume 5 Author(s): Weihua Ye, Erika Spånning, Elzbieta Glaser, Lena Mäler Organellar proteins synthesized in the cytosol are usually selective for only one destination in a cell but some proteins are localized in more than one compartment, for example in both mitochondria and chloroplasts. The mechanism of dual targeting of proteins to mitochondria and chloroplasts is yet poorly understood. Previously, we observed that the dual targeting peptide of threonyl-tRNA synthetase in Arabidopsis thaliana ( At ThrRS-dTP) interacts with the mitochondrial receptor At Tom20 mainly through its N-terminal part. Here we report on the interaction of At ThrRS-dTP with the chloroplastic receptor At Toc34, presenting for the first time the mode of interactions of a dual targeting peptide with both Tom20 and Toc34. By NMR spectroscopy we investigated changes in 15 N HSQC spectra of At ThrRS-dTP as a function of At Toc34 concentration. Line broadening shows that the interaction with At Toc34 involves residues along the entire sequence, which is not the case for At Tom20. The N-terminal φχχφφ motif, which plays an important role in At Tom20 recognition, shows no specificity for At Toc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of At ThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of At ThrRS-dTP is essential for import into both organelles, while the C-terminal L30-P60 part is important for chloroplastic import efficiency. In conclusion, we have demonstrated that the recognition of the dual targeting peptide of At Thr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors. Graphical abstract

Description: Publication date: 2015 Source: FEBS Open Bio, Volume 5 Author(s): Yamixa Delgado, Moraima Morales-Cruz, Cindy M. Figueroa, José Hernández-Román, Glinda Hernández, Kai Griebenow Lipid–protein complexes comprised of oleic acid (OA) non-covalently coupled to human/bovine α-lactalbumin, named HAMLET/BAMLET, display cytotoxic properties against cancer cells. However, there is still a substantial debate about the role of the protein in these complexes. To shed light into this, we obtained three different BAMLET complexes using varying synthesis conditions. Our data suggest that to form active BAMLET particles, OA has to reach critical micelle concentration with an approximate diameter of 250 nm. Proteolysis experiments on BAMLET show that OA protects the protein and is probably located on the surface, consistent with a micelle-like structure. Native or unfolded α-lactalbumin without OA lacked any tumoricidal activity. In contrast, OA alone killed cancer cells with the same efficiency at equimolar concentrations as its formulation as BAMLET. Our data show unequivocally that the cytotoxicity of the BAMLET complex is exclusively due to OA and that OA alone, when formulated as a micelle, is as toxic as the BAMLET complex. The contradictory literature results on the cytotoxicity of BAMLET might be explained by our finding that it was imperative to sonicate the samples to obtain toxic OA. Graphical abstract

Description: Publication date: Available online 14 November 2015 Source: FEBS Open Bio Author(s): Ngonidzashe Faya, David L. Penkler, Özlem Tastan Bishop The treatment of protozoan parasitic diseases is challenging, and thus identification and analysis of new drug targets is important. Parasites survive within host organisms, and some need intermediate hosts to complete their life cycle. Changing host environment puts stress on parasites, and often adaptation is accompanied by the expression of large amounts of heat shock proteins (Hsps). Among Hsps, Hsp90 proteins play an important role in stress environments. Yet, there has been little computational research on Hsp90 proteins to analyze them comparatively as potential parasitic drug targets. Here, an attempt was made to gain detailed insights into the differences between host, vector and parasitic Hsp90 proteins by large-scale bioinformatics analysis. A total of 104 Hsp90 sequences were divided into three groups based on their cellular localizations; namely cytosolic, mitochondrial and endoplasmic reticulum (ER). Further, the parasitic proteins were divided according to the type of parasite (protozoa, helminth and ectoparasite). Primary sequence analysis, phylogenetic tree calculations, motif analysis and physicochemical properties of Hsp90 proteins suggested that despite the overall structural conservation of these proteins, parasitic Hsp90 proteins have unique features which differentiate them from human ones, thus encouraging the idea that protozoan Hsp90 proteins should be further analyzed as potential drug targets.

Description: Publication date: Available online 10 November 2015 Source: FEBS Open Bio Author(s): Karel Olavarria, Marina Pupke Marone, Henrique da Costa Oliveira, Juan Camilo Roncallo, Fernanda Nogales da Costa Vasconcelos, Luiziana Ferreira da Silva, José Gregório Cabrera Gomez Despite the lack of biochemical information, all available in silico metabolic models of Pseudomonas putida KT2440 consider NADP as the only cofactor accepted by the glucose-6-phosphate dehydrogenases. Because the Entner–Doudoroff pathway is the main glycolytic route in this bacterium, determining how much NADH and NADPH are produced in the reaction catalyzed by these enzymes is very important for the correct interpretation of metabolic flux distributions. To determine the actual cofactor preference of the glucose-6-phosphate dehydrogenase encoded by the zwf-1 gene (PputG6PDH-1), the major isoform during growth on glucose, we purified this protein and studied its kinetic properties. Based on simple kinetic principles, we estimated the in vivo relative production of NADH and NADPH during the oxidation of glucose-6-phosphate (G6P). Contrary to the general assumption, our calculations showed that the reaction catalyzed by PputG6PDH-1 yields around 1/3 mol of NADPH and 2/3 mol of NADH per mol of oxidized G6P. Additionally, we obtained data suggesting that the reaction catalyzed by the 6-phosphogluconate dehydrogenase is active during growth on glucose, and it also produces NADH. These results indicate that the stoichiometric matrix of in silico models of P. putida KT2440 must be corrected and highlight the importance of considering the physiological concentrations of the involved metabolites to estimate the actual proportion of NADH and NADPH produced by a dehydrogenase.

Description: Publication date: Available online 10 November 2015 Source: FEBS Open Bio Author(s): Kexin Sun, Tiancai Zeng, Dong Huang, Zizhong Liu, Shang Huang, Jiong Liu, Zhenfan Qu MicroRNA-431 (miR-431) has been recognized as an oncogenic miRNA, being implicated in the initiation and development of human cancers. Recently, deregulation of miR-431 has been reported in several tumors. However, the clinical significance of miR-431 and its underlying role in human hepatocellular carcinoma (HCC) are poorly explored. Herein, we found that miR-431 expression was reduced in HCC tissues compared to noncancerous tissues. Otherwise, down-regulation of miR-431 was observed in aggressive tumor tissues. The levels of miR-431 expression in HCC cell lines were significantly lower than that in a nontransformed hepatic cell line. Clinical association analyses disclosed that a low level of miR-431 was prominently associated with poor prognostic features of HCC including venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage. Our in vitro studies showed that up-regulation of miR-431 expression reduced cell invasion and migration in HCCLM3 cells. In contrast, down-regulation of miR-431 expression promoted SMMC-7721 cell invasion and migration. We found that up-regulation of miR-431 expression decreased zinc finger E-box binding homeobox 1 (ZEB1) expression and inhibited the epithelial–mesenchymal transition (EMT) with increased E-cadherin expression and decreased vimentin expression in HCCLM3 cells. Otherwise, down-regulation of miR-431 expression increased ZEB1 expression and promoted EMT in SMMC-7721 cells. Significantly, ZEB1 was identified as a target of miR-431 in HCC. ZEB1 knockdown abrogated the effect of miR-431 silencing on EMT and cell mobility in SMMC-7721 cells. In conclusion, miR-431 inhibits migration and invasion of HCC cells by suppressing ZEB1-mediated EMT.

Description: Publication date: Available online 7 November 2015 Source: FEBS Open Bio Author(s): Junwei Shao, Jun Cao, Yong Liu, Hongliang Mei, Yang Zhang, Weitian Xu Recent studies report that microRNA-519a (miR-519a) is a novel oncomir, which facilitates the onset and progression of human cancers. However, the clinical significance of miR-519a and its functional role and underlying mechanisms in hepatocellular carcinoma (HCC) are poorly investigated. In the present study, elevated expression of miR-519a was observed in HCC tissues compared with adjacent non-tumor tissues. The increased level of miR-519a expression was significantly correlated with adverse clinical features of HCC including hepatitis B virus (HBV) infection, large tumor size, cirrhosis and advanced tumor-node-metastasis tumor stage. Furthermore, high expression of miR-519a was prominently associated with a poorer 5-year overall survival and recurrence-free survival of HCC patients. Gain- and loss-of function experiments showed that miR-519a overexpression enhanced proliferation and reduced apoptosis of Huh7 cells. By contrast, miR-519a knockdown inhibited SMMC-7721 cell proliferation and induced apoptosis. Importantly, up-regulation of miR-519a reduced the expression of FOXF2 mRNA and protein in Huh7 cells, while down-regulation of miR-519a resulted in increased expression of FOXF2 in SMMC-7721 cells. An inverse correlation between mRNA levels of miR-519a and FOXF2 was observed in HCC tissues. Thus, Forkhead box F2 (FOXF2) was identified as a downstream target of miR-519a in HCC. Mechanistically, the effects of miR-519a knockdown on SMMC-7721 cells were abrogated by FOXF2 repression. In conclusion, miR-519a is a novel prognostic predictor for HCC patients and it may potentiate proliferation and inhibits apoptosis of HCC cells by targeting FOXF2.

Description: Publication date: Available online 27 October 2015 Source: FEBS Open Bio Author(s): Luuk J.G.W. van Wilderen, Gary Silkstone, Maria Mason, Jasper J. van Thor, Michael T. Wilson Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non-covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo . Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped-flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence. In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADH . Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH − ). Reaction of this with dioxygen leads to transient radical (FADH ) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer. Graphical abstract

Description: Publication date: Available online 23 October 2015 Source: FEBS Open Bio Author(s): Michael S. Parker, Floyd R. Sallee, Edwards A. Park, Steven L. Parker Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks.

Description: Publication date: Available online 23 October 2015 Source: FEBS Open Bio Author(s): Yi-Lian Wang, Li-Ming Sun, Li Zhang, Hai-Tao Xu, Zheng Dong, Luo-Qing Wang, Ming-Lang Wang Published data regarding the association between Apolipoprotein E ( ApoE ) genetic variation and myocardial infarction (MI) risk were not always consistent. Therefore, the current meta-analysis was conducted to derive a more precise estimation of the association between ApoE polymorphism and MI risk. PubMed and Web of Science were searched to identify relevant studies. Summary odds ratio (ORs) and 95% confidence intervals (CIs) were calculated using random-effect or fixed-effect models based on the heterogeneity of included studies. All the tests were performed using Stata 11.0. A total of 22 eligible studies were identified in this meta-analysis. The results show that ApoE ε2 and ε4 alleles were associated with MI risk. The study suggests that there is close association between ApoE polymorphism and MI risk. It shows that ApoE ε2 allele is a protective factor of MI, while ε4 allele is a risk factor of MI, especially in Caucasian and Asian population. Nevertheless, well-designed, unbiased and larger sample size studies are required to confirm the results.

Description: Publication date: Available online 23 October 2015 Source: FEBS Open Bio Author(s): Yuki Numakura, Takashi Miura Resonance Raman spectra of azobenzene derivatives were examined in the presence of lipid membranes to find a probe that can distinguish different membrane phases. The NO 2 symmetric stretching band of 4-(4-nitrophenylazo)aniline, also known as Disperse Orange 3 (DO3), is downshifted by about 4 cm −1 on the phase transition of phosphatidylcholine membranes from the liquid crystalline to the gel phase. A comparable downshift also occurs when DO3 is bound to cholesterol-containing membranes in the liquid-ordered phase. Our results demonstrate that Raman spectrum of DO3 is a unique tool for measuring the molecular order of lipids in membranes.