The atypical G beta-like/RACK1 Gib2 protein promotes cAMP signalling that plays a central role in regulating the virulence of Cryptococcus neoformans. Gib2 contains a seven-bladed beta transducin structure and is emerging as a scaffold protein interconnecting signalling pathways through interactions with various protein partners. Here, we present the crystal structure of Gib2 at a 2.2-angstrom resolution. The structure allows us to analyse the association between Gib2 and the ribosome, as well as to identify the Gib2 amino acid residues involved in ribosome binding. Our studies not only suggest that Gib2 has a role in protein translation but also present Gib2 as a physical link at the crossroads of various regulatory pathways important for the growth and virulence of C. neoformans.
Background. Reversion:inducing-cysteine-rich protein with Kazal motifs (RECK) is critical for the invasiveness and metastasis of tumor cells; however, its role in regulating the endoplasmic reticulum (ER) stress response remains unclear In this study we investigated the protein that interacts with RECK and the effects of RECK overexpression on the ER stress response and on cisplatin-induced cell death in neuroblastoma cells. Methods. Full-length RECK (FL-RECK) or a C-terminus-deleted mutant of RECK (del-C-RECK) was transfected into neuroblastoma cells. An immunoprecipitation (IF) assay and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis were used to identify the RECK-interacting proteins. The interaction between RECK and these proteins was confirmed using co-IP and an immunofluorescence assay. Phosphorylation of double-stranded, RNA-activated protein kinase-like, ER-localized eukaryotic initiation factor-2 alpha (eIF-2 alpha) kinase (PERK) and eIF-2 alpha, and expression of ER stress-related apoptotic factors were studied by Western blot analysis. Results. Glucose-legulated protein 78 (GRP78) was identified as the RECK-interacting protein in neuroblastoma cells, and the C-terminus region of the RECK protein was shown to interact with GRP78. Overexpression of FL-RECK, but not of del-C-RECK, increased the phosphorylation of PERK and eIF-2 alpha in neuroblastoma cells. With cisplatin treatment, the expression of phosphorylated PERK and eIF-2 alpha, CCAAT/enhancer-binding protein-homologous protein, Bax, and caspase-4 and -7 was higher and the cell viability was lower (P < .01) in FL-RECK-overexpressing cells than in del-C-RECK-overexpressing or vector control cells. Conclusion. RECK regulated the cellular ER stress response through interaction with GRP78 and enhanced cisplatin-induced cell death in neuroblastoma cells.
Since organelle preparations often contain more than one organelle type (e.g., acidic organelles and mitochondria), techniques that measure the properties of a given organelle type while avoiding biases caused by ancillary subcellular compartments are highly desirable. We report here the use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) dual-channel detection to identify acidic organelles containing doxorubicin (DOX) in crude subcellular fractions from CCRF-CEM and CEM/C2 cell fines. As confirmed by confocal microscopy, acidic organelles are identified by their accumulation of fluorescently labeled nanospheres. Using CE-LIF analysis, individually detected organelles are classified into three kinds: acidic organelles containing only nanospheres, acidic organelles containing nanospheres and DOX, and other organelles containing DOX (e.g., mitochondria) with no detectable nanospheres. Electrophoretic mobility, DOX fluorescence intensity, and nanosphere fluorescence intensity distributions of individual acidic organelles and other organelles containing DOX are determined in the same CE-LIF run. The acidic organelle mobilities range from (-0.7 to -2.0) x 10(-4) cm(2) V-1 s(-1) while those of the other organelles spread from (-0.6 to -3.5) x 10(-4) cm(2) V-1 s(-1). In addition, by calibrating the detector response, DOX content in individual acidic organelles and other organelles can be estimated. The average amounts of DOX per acidic organelle in CEM/C2 and CCRF-CEM cells are 11.1 +/- 0.5 and 10.6 +/- 0.4 zmol, respectively. This first report of an analysis of the accumulation of DOX in individual acidic organelles presents a procedure for analyzing the accumulation of fluorescent compounds in acidic organelles that could be useful for investigating acidic organelle maturation and the role of these organelles in drug resistance.
The invention relates to substrates based on vinylcyclohexane homopolymers, copolymers and blockpolymers, wherein comonomers are selected from the group consisting of olefins, acrylic acid derivatives, maleic acid derivatives, vinyl ether and vinyl ester or mixtures thereof and from at least two comonomers. The invention is characterized in that the moment of inertia of the substrate ranges from 280 to 50 g?.¿cm?2¿ and specific density from 1 to 0.8 g/cm?3¿.
Chen, Yun
Bao, Jichen
Kim, Il-Kwon
Siewers, Verena
Nielsen, Jens
3-Hydroxypropionic acid (3-HP) is an attractive platform chemical, which can be used to produce a variety of commodity chemicals, such as acrylic acid and acrylamide. For enabling a sustainable alternative to petrochemicals as the feedstock for these commercially important chemicals, fermentative production of 3-HP is widely investigated and is centered on bacterial systems in most cases. However, bacteria present certain drawbacks for large-scale organic acid production. In this study, we have evaluated the production of 3-HP in the budding yeast Saccharomyces cerevisiae through a route from malonyl-CoA, because this allows performing the fermentation at low pH thus making the overall process cheaper. We have further engineered the host strain by increasing availability of the precursor malonyl-CoA and by coupling the production with increased NADPH supply we were able to substantially improve 3-HP production by five-fold, up to a final titer of 463 mg l(-1). Our work thus led to a demonstration of 3-HP production in yeast via the malonyl-CoA pathway, and this opens for the use of yeast as a cell factory for production of bio-based 3-HP and derived acrylates in the future. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved
Bio-based production of chemical building blocks from renewable resources is an attractive alternative to petroleum-based platform chemicals. Metabolic pathway and strain engineering is the key element in constructing robust microbial chemical factories within the constraints of cost effective production. Here we discuss how the development of computational algorithms, novel modules and methods, omics-based techniques combined with modeling refinement are enabling reduction in development time and thus advance the field of industrial biotechnology. We further discuss how recent technological developments contribute to the development of novel cell factories for the production of the building block chemicals: adipic acid, succinic acid and 3-hydroxypropionic acid.