A Cellular Mechanism to Detect and Alleviate Reductive Stress.

Metazoan organisms rely on conserved stress response pathways to alleviate adverse conditions and preserve cellular integrity. Stress responses are particularly important in stem cells that provide lifetime support for tissue formation and repair, but how these protective systems are integrated into developmental programs is poorly understood. Here we used myoblast differentiation to identify the E3 ligase CUL2FEM1B and its substrate FNIP1 as core components of the reductive stress response. Reductive stress, as caused by prolonged antioxidant signaling or mitochondrial inactivity, reverts the oxidation of invariant Cys residues in FNIP1 and allows CUL2FEM1B to recognize its target. The ensuing proteasomal degradation of FNIP1 restores mitochondrial activity to preserve redox homeostasis and stem cell integrity. The reductive stress response is therefore built around a ubiquitin-dependent rheostat that tunes mitochondrial activity to redox needs and implicates metabolic control in coordination of stress and developmental signaling.

Genetically encoded tools for measuring and manipulating metabolism.

Over the past few years, we have seen an explosion of novel genetically encoded tools for measuring and manipulating metabolism in live cells and animals. Here, we will review the genetically encoded tools that are available, describe how these tools can be used and outline areas where future development is needed in this fast-paced field. We will focus on tools for direct measurement and manipulation of metabolites. Metabolites are master regulators of metabolism and physiology through their action on metabolic enzymes, signaling enzymes, ion channels and transcription factors, among others. We hope that this Perspective will encourage more people to use these novel reagents or even join this exciting new field to develop novel tools for measuring and manipulating metabolism.

HPr antagonizes the anti-σ70 activity of Rsd in Escherichia coli.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA(Glc) (EIIA(Glc)) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA(Glc) in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ(70) in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ(70). Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ(70)-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ(70) and σ(S) for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ(70) activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.

Genetically encoded tool for manipulation of ΔΨm identifies the latter as the driver of integrative stress response induced by ATP Synthase dysfunction.

Mitochondrial membrane potential (ΔΨm) is one of the key parameters controlling cellular bioenergetics. Investigation of the role of ΔΨm in live cells is complicated by a lack of tools for its direct manipulation without off-target effects. Here, we adopted the uncoupling protein UCP1 from brown adipocytes as a genetically encoded tool for direct manipulation of ΔΨm. We validated the ability of exogenously expressed UCP1 to induce uncoupled respiration and lower ΔΨm in mammalian cells. UCP1 expression lowered ΔΨm to the same extent as chemical uncouplers but did not inhibit cell proliferation, suggesting that it manipulates ΔΨm without the off-target effects of chemical uncouplers. Using UCP1, we revealed that elevated ΔΨm is the driver of the Integrated Stress Response induced by ATP synthase inhibition in mammalian cells.

Structural insight into glucose repression of the mannitol operon.

Carbon catabolite repression is a regulatory mechanism to ensure sequential utilization of carbohydrates and is usually accomplished by repression of genes for the transport and metabolism of less preferred carbon compounds by a more preferred one. Although glucose and mannitol share the general components, enzyme I and HPr, of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for their transport, glucose represses the transport and metabolism of mannitol in a manner dependent on the mannitol operon repressor MtlR in Escherichia coli. In a recent study, we identified the dephosphorylated form of HPr as a regulator determining the glucose preference over mannitol by interacting with and augmenting the repressor activity of MtlR in E. coli. Here, we determined the X-ray structure of the MtlR-HPr complex at 3.5 Å resolution to understand how phosphorylation of HPr impedes its interaction with MtlR. The phosphorylation site (His15) of HPr is located close to Glu108 and Glu140 of MtlR and phosphorylation at His15 causes electrostatic repulsion between the two proteins. Based on this structural insight and comparative sequence analyses, we suggest that the determination of the glucose preference over mannitol solely by the MtlR-HPr interaction is conserved within  the Enterobacteriaceae family.

The general PTS component HPr determines the preference for glucose over mannitol.

Preferential sugar utilization is a widespread phenomenon in biological systems. Glucose is usually the most preferred carbon source in various organisms, especially in bacteria where it is taken up via the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The currently proposed model for glucose preference over non-PTS sugars in enteric bacteria including E. coli is strictly dependent on the phosphorylation state of the glucose-specific PTS component, enzyme IIAGlc (EIIAGlc). However, the mechanism of the preference among PTS sugars is largely unknown in Gram-negative bacteria. Here, we show that glucose preference over another PTS sugar, mannitol, is absolutely dependent on the general PTS component HPr, but not on EIIAGlc, in E. coli. Dephosphorylated HPr accumulates during the transport of glucose and interacts with the mannitol operon regulator, MtlR, to augment its repressor activity. This interaction blocks the inductive effect of mannitol on the mannitol operon expression and results in the inhibition of mannitol utilization.