Control of a chemical chaperone by a universally conserved ATPase.

The universally conserved YchF/Ola1 ATPases regulate stress response pathways in prokaryotes and eukaryotes. Deletion of YchF/Ola1 leads to increased resistance against environmental stressors, such as reactive oxygen species, while their upregulation is associated with tumorigenesis in humans. The current study shows that in E. coli, the absence of YchF stimulates the synthesis of the alternative sigma factor RpoS by a transcription-independent mechanism. Elevated levels of RpoS then enhance the transcription of major stress-responsive genes. In addition, the deletion of ychF increases the levels of polyphosphate kinase, which in turn boosts the production of the evolutionary conserved and ancient chemical chaperone polyphosphate. This potentially provides a unifying concept for the increased stress resistance in bacteria and eukaryotes upon YchF/Ola1 deletion. Intriguingly, the simultaneous deletion of ychF and the polyphosphate-degrading enzyme exopolyphosphatase causes synthetic lethality in E. coli, demonstrating that polyphosphate production needs to be fine-tuned to prevent toxicity.

Quantitative proteomics identifies the universally conserved ATPase Ola1p as a positive regulator of heat shock response in Saccharomyces cerevisiae.

The universally conserved P-loop ATPase Ola1 is implicated in various cellular stress response pathways, as well as in cancer and tumor progression. However, Ola1p functions are divergent between species, and the involved mechanisms are only poorly understood. Here, we studied the role of Ola1p in the heat shock response of the yeast Saccharomyces cerevisiae using a combination of quantitative and pulse labeling-based proteomics approaches, in vitro studies, and cell-based assays. Our data show that when heat stress is applied to cells lacking Ola1p, the expression of stress-protective proteins is enhanced. During heat stress Ola1p associates with detergent-resistant protein aggregates and rapidly forms assemblies that localize to stress granules. The assembly of Ola1p was also observed in vitro using purified protein and conditions, which resembled those in living cells. We show that loss of Ola1p results in increased protein ubiquitination of detergent-insoluble aggregates recovered from heat-shocked cells. When cells lacking Ola1p were subsequently relieved from heat stress, reinitiation of translation was delayed, whereas, at the same time, de novo synthesis of central factors required for protein refolding and the clearance of aggregates was enhanced when compared with wild-type cells. The combined data suggest that upon acute heat stress, Ola1p is involved in the stabilization of misfolded proteins, which become sequestered in cytoplasmic stress granules. This function of Ola1p enables cells to resume translation in a timely manner as soon as heat stress is relieved.

Pyridinium Modified Anthracenes and Their Endoperoxides Provide a Tunable Scaffold with Activity against Gram-Positive and Gram-Negative Bacteria.

Due to the emergence of multidrug resistant bacteria, the development of new antibiotics is required. We introduce here asymmetrically modified positively charged bis(methylpyridinium) anthracenes as a novel tunable scaffold, in which the two positive charges can be placed at a defined distance and angle. Our structure-activity relationship reveals that coupling the methylpyridiniums with alkynyl linkers to the central anthracene unit yields antibacterial compounds against a wide range of bacteria, including Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis. Also, different mycobacteria, such as Mycobacterium smegmatis and Mycobacterium tuberculosis, are efficiently targeted by these compounds. The antibacterial activity depends on the number of alkynyl linkers and consequently also on the distance of the positive charges in the rigid anthracene scaffold. Additionally, the formation of an anthracene endoperoxide further increases the antibacterial activity, likely due to the release of toxic singlet oxygen that converts the endoperoxide back to the antibacterial anthracene scaffold with half-lives of several hours.

The Universally Conserved ATPase YchF Regulates Translation of Leaderless mRNA in Response to Stress Conditions.

The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, and intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promoter fusion experiments, we demonstrate that YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress, or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor 3 (IF3) and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs, and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation.

The Role of the Universally Conserved ATPase YchF/Ola1 in Translation Regulation during Cellular Stress.

The ability to respond to metabolic or environmental changes is an essential feature in all cells and involves both transcriptional and translational regulators that adjust the metabolic activity to fluctuating conditions. While transcriptional regulation has been studied in detail, the important role of the ribosome as an additional player in regulating gene expression is only beginning to emerge. Ribosome-interacting proteins are central to this translational regulation and include universally conserved ribosome interacting proteins, such as the ATPase YchF (Ola1 in eukaryotes). In both eukaryotes and bacteria, the cellular concentrations of YchF/Ola1 determine the ability to cope with different stress conditions and are linked to several pathologies in humans. The available data indicate that YchF/Ola1 regulates the stress response via controlling non-canonical translation initiation and via protein degradation. Although the molecular mechanisms appear to be different between bacteria and eukaryotes, increased non-canonical translation initiation is a common consequence of YchF/Ola1 regulated translational control in E. coli and H. sapiens. In this review, we summarize recent insights into the role of the universally conserved ATPase YchF/Ola1 in adapting translation to unfavourable conditions.

The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly.

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu-detoxification and Cu delivery for cytochrome c oxidase (cbb3 -Cox) assembly depend on two distinct Cu-exporting P1B -type ATPases. The low-affinity CopA is suggested to export excess Cu and the high-affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb3 -Cox biogenesis. In most organisms, CopA-like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI-like ATPases is unknown. Here we identified a CopZ-like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox-sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu-sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb3 -Cox activity is reduced, suggesting that CopZ not only supplies Cu to P1B -type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI-CopZ protein complex in native R. capsulatus membranes.