Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein

The ATP-dependent Hsp70 chaperones (DnaK in E. coli) mediate protein folding in cooperation with J proteins and nucleotide exchange factors (E. coli DnaJ and GrpE, respectively). The Hsp70 system prevents protein aggregation and increases folding yields. Whether it also enhances the rate of folding remains unclear. Here we show that DnaK/DnaJ/GrpE accelerate the folding of the multi-domain protein firefly luciferase (FLuc) 20-fold over the rate of spontaneous folding measured in the absence of aggregation. Analysis by single-pair FRET and hydrogen/deuterium exchange identified inter-domain misfolding as the cause of slow folding. DnaK binding expands the misfolded region and thereby resolves the kinetically-trapped intermediates, with folding occurring upon GrpE-mediated release. In each round of release DnaK commits a fraction of FLuc to fast folding, circumventing misfolding. We suggest that by resolving misfolding and accelerating productive folding, the bacterial Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions. The Hsp70 system prevents protein aggregation and increases folding yields, but it is unknown whether it also enhances the rate of folding. Here the authors combine refolding assays, FRET and hydrogen/deuterium exchange-mass spectrometry measurements to study the folding of firefly luciferase and find that the bacterial Hsp70 actively promotes the folding of this multi-domain protein

Production and processing of organically grown fiber nettle (Urtica dioica L.) and its potential use in the natural textiles industry: A review

In Europe, the perennial stinging nettle was cultivated during the 19th century until the Second World War and has a long history as a fiber plant. Clone varieties dating back to the early 20th century are still maintained at European research institutions. The fiber content of clones ranges from 1.2 to 16% dry matter, and fiber yields range from 0.14 to 1.28 Mg/ha. Varietal purity of fiber nettle can only be achieved by planting cuttings. The harvesting of fiber starts in the second year of growth and the crop may produce well for several years. Several agronomic practices influence fiber quality, but causal relations are not yet well understood. Various parts of the fiber nettle plant can be used as food, fodder and as raw material for different purposes in cosmetics, medicine, industry and biodynamic agriculture. Organically produced fibers are in demand by the green textile industry and show potential that is economically promising

Improved recombinant expression and purification of functional plant Rubisco

Improving the performance of the key photosynthetic enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by protein engineering is a critical strategy for increasing crop yields. The extensive chaperone requirement of plant Rubisco for folding and assembly has long been an impediment to this goal. Production of plant Rubisco in Escherichia coli requires the coexpression of the chloroplast chaperonin and four assembly factors. Here, we demonstrate that simultaneous expression of Rubisco and chaperones from a T7 promotor produces high levels of functional enzyme. Expressing the small subunit of Rubisco with a C-terminal hexahistidine-tag further improved assembly, resulting in a similar to 12-fold higher yield than the previously published procedure. The expression system described here provides a platform for the efficient production and engineering of plant Rubisco

ER Stress-Induced eIF2-alpha Phosphorylation Underlies Sensitivity of Striatal Neurons to Pathogenic Huntingtin

A hallmark of Huntington's disease is the pronounced sensitivity of striatal neurons to polyglutamine-expanded huntingtin expression. Here we show that cultured striatal cells and murine brain striatum have remarkably low levels of phosphorylation of translation initiation factor eIF2 alpha, a stress-induced process that interferes with general protein synthesis and also induces differential translation of pro-apoptotic factors. EIF2 alpha phosphorylation was elevated in a striatal cell line stably expressing pathogenic huntingtin, as well as in brain sections of Huntington's disease model mice. Pathogenic huntingtin caused endoplasmic reticulum (ER) stress and increased eIF2 alpha phosphorylation by increasing the activity of PKR-like ER-localized eIF2 alpha kinase (PERK). Importantly, striatal neurons exhibited special sensitivity to ER stress-inducing agents, which was potentiated by pathogenic huntingtin. We could strongly reduce huntingtin toxicity by inhibiting PERK. Therefore, alteration of protein homeostasis and eIF2 alpha phosphorylation status by pathogenic huntingtin appears to be an important cause of striatal cell death. A dephosphorylated state of eIF2 alpha has been linked to cognition, which suggests that the effect of pathogenic huntingtin might also be a source of the early cognitive impairment seen in patients

The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli

Stress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantify similar to 1,500 soluble proteins and find similar to 500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an additional similar to 200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins containing the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addition to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions

An aperture masking mode for the MICADO instrument

MICADO is a near-IR camera for the Europea ELT, featuring an extended field (75" diameter) for imaging, and also spectrographic and high contrast imaging capabilities. It has been chosen by ESO as one of the two first-light instruments. Although it is ultimately aimed at being fed by the MCAO module called MAORY, MICADO will come with an internal SCAO system that will be complementary to it and will deliver a high performance on axis correction, suitable for coronagraphic and pupil masking applications. The basis of the pupil masking approach is to ensure the stability of the optical transfer function, even in the case of residual errors after AO correction (due to non common path errors and quasi-static aberrations). Preliminary designs of pupil masks are presented. Trade-offs and technical choices, especially regarding redundancy and pupil tracking, are explained.Comment: SPIE 2014 Proceeding -- Montrea

Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2

Plant RuBisCo, a complex of eight large and eight small subunits, catalyzes the fixation of CO2 in photosynthesis. The low catalytic efficiency of RuBisCo provides strong motivation to reengineer the enzyme with the goal of increasing crop yields. However, genetic manipulation has been hampered by the failure to express plant RuBisCo in a bacterial host. We achieved the functional expression of Arabidopsis thaliana RuBisCo in Escherichia coli by coexpressing multiple chloroplast chaperones. These include the chaperonins Cpn60/Cpn20, RuBisCo accumulation factors 1 and 2, RbcX, and bundle-sheath defective-2 (BSD2). Our structural and functional analysis revealed the role of BSD2 in stabilizing an end-state assembly intermediate of eight RuBisCo large subunits until the small subunits become available. The ability to produce plant RuBisCo recombinantly will facilitate efforts to improve the enzyme through mutagenesis

Condensin II Promotes the Formation of Chromosome Territories by Inducing Axial Compaction of Polyploid Interphase Chromosomes

The eukaryotic nucleus is both spatially and functionally partitioned. This organization contributes to the maintenance, expression, and transmission of genetic information. Though our ability to probe the physical structure of the genome within the nucleus has improved substantially in recent years, relatively little is known about the factors that regulate its organization or the mechanisms through which specific organizational states are achieved. Here, we show that Drosophila melanogaster Condensin II induces axial compaction of interphase chromosomes, globally disrupts interchromosomal interactions, and promotes the dispersal of peri-centric heterochromatin. These Condensin II activities compartmentalize the nucleus into discrete chromosome territories and indicate commonalities in the mechanisms that regulate the spatial structure of the genome during mitosis and interphase