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

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

The electric two-echelon vehicle routing problem

Two-echelon distribution systems are attractive from an economical standpoint and help to keep large vehicles out of densely populated city centers. Large trucks can be used to deliver goods to intermediate facilities in accessible locations, whereas smaller vehicles allow to reach the final customers. Due to their reduced size, pollution, and noise, multiple companies consider using an electric fleet of terrestrial or aerial vehicles for last-mile deliveries. Route planning in multi-tier logistics leads to notoriously difficult problems. This difficulty is accrued in the presence of an electric fleet since each vehicle operates on a smaller range and may require planned visits to recharging stations. To study these challenges, we introduce the electric two-echelon vehicle routing problem (E2EVRP) as a prototypical problem. We propose a large neighborhood search (LNS) metaheuristic as well as an exact mathematical programming algorithm, which uses decomposition techniques to enumerate promising first-level solutions in conjunction with bounding functions and route enumeration for the second-level routes. These algorithms produce optimal or near-optimal solutions for the problem and allow us to evaluate the impact of several defining features of optimized battery-powered distribution networks. We created representative E2EVRP benchmark instances to simulate realistic metropolitan areas. In particular, we observe that the detour miles due to recharging decrease proportionally to 1/ρx with x ≈ 5/4 as a function of the charging stations density ρ; e.g., in a scenario where the density of charging stations is doubled, recharging detours are reduced by 58%. Finally, we evaluate the trade-off between battery capacity and detour miles. This estimate is critical for strategic fleet-acquisition decisions, in a context where large batteries are generally more costly and less environment-friendly

Genetic variability and differentiation in roe deer (Capreolus capreolus L) of Central Europe

International audienc

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

Failure of RQC machinery causes protein aggregation and proteotoxic stress

Translation of messenger RNAs lacking a stop codon results in the addition of a carboxy-terminal poly-lysine tract to the nascent polypeptide, causing ribosome stalling. Non-stop proteins and other stalled nascent chains are recognized by the ribosome quality control (RQC) machinery and targeted for proteasomal degradation. Failure of this process leads to neurodegeneration by unknown mechanisms. Here we show that deletion of the E3 ubiquitin ligase Ltn1p in yeast, a key RQC component, causes stalled proteins to form detergent-resistant aggregates and inclusions. Aggregation is dependent on a C-terminal alanine/threonine tail that is added to stalled polypeptides by the RQC component, Rqc2p. Formation of inclusions additionally requires the poly-lysine tract present in non-stop proteins. The aggregates sequester multiple cytosolic chaperones and thereby interfere with general protein quality control pathways. These findings can explain the proteotoxicity of ribosome-stalled polypeptides and demonstrate the essential role of the RQC in maintaining proteostasis

The nucleolus functions as a phase-separated protein quality control compartment

The nuclear proteome is rich in stress-sensitive proteins, which suggests that effective protein quality control mechanisms are in place to ensure conformational maintenance. We investigated the role of the nucleolus in this process. In mammalian tissue culture cells under stress conditions, misfolded proteins entered the granular component (GC) phase of the nucleolus. Transient associations with nucleolar proteins such as NPM1 conferred low mobility to misfolded proteins within the liquid-like GC phase, avoiding irreversible aggregation. Refolding and extraction of proteins from the nucleolus during recovery from stress was Hsp70-dependent. The capacity of the nucleolus to store misfolded proteins was limited, and prolonged stress led to a transition of the nucleolar matrix from liquid-like to solid, with loss of reversibility and dysfunction in quality control. Thus, we suggest that the nucleolus has chaperone-like properties and can promote nuclear protein maintenance under stress.We acknowledge support by the MPIB Imaging facility and G. Cardone for providing the algorithm for image quantification