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.

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.

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis.

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

Cellular respiration is one of the main ways organisms make energy. It works by linking the oxidation of an electron donor (like sugar) to the reduction of an electron acceptor (like oxygen). Electrons pass between the two molecules along what is known as an 'electron transport chain'. This process generates a force that powers the production of adenosine triphosphate (ATP), a molecule that cells use to store energy. Respiration is a common way for cells to replenish their energy stores, but it is not the only way. A simpler process that does not require a separate electron acceptor or an electron transport chain is called fermentation. Many bacteria have the capacity to perform both respiration and fermentation and do so in a context-dependent manner. Research has shown that respiration can contribute to bacterial diseases, like tuberculosis and listeriosis (a disease caused by the foodborne pathogen Listeria monocytogenes). Indeed, some antibiotics even target bacterial respiration. Despite being often discussed in the context of generating ATP, respiration is also important for many other cellular processes, including maintaining the balance of reduced and oxidized nicotinamide adenine dinucleotide (NAD) cofactors. Because of these multiple functions, the exact role respiration plays in disease is unknown. To find out more, Rivera-Lugo, Deng et al. developed strains of the bacterial pathogen Listeria monocytogenes that lacked some of the genes used in respiration. The resulting bacteria were still able to produce energy, but they became much worse at infecting mammalian cells. The use of a genetic tool that restored the balance of reduced and oxidized NAD cofactors revived the ability of respiration-deficient L. monocytogenes to infect mammalian cells, indicating that this balance is what the bacterium requires to infect. Research into respiration tends to focus on its role in generating ATP. But these results show that for some bacteria, this might not be the most important part of the process. Understanding the other roles of respiration could change the way that researchers develop antibacterial drugs in the future. This in turn could help with the growing problem of antibiotic resistance.

Physical bioenergetics: Energy fluxes, budgets, and constraints in cells.

Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics.

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.

Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell.

Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR) but the underlying triggers remain unclear. We systematically combined acute mitochondrial inhibitors with genetic tools for compartment-specific NADH oxidation to trace mechanisms linking different forms of mitochondrial dysfunction to the ISR in proliferating mouse myoblasts and in differentiated myotubes. In myoblasts, we find that impaired NADH oxidation upon electron transport chain (ETC) inhibition depletes asparagine, activating the ISR via the eIF2α kinase GCN2. In myotubes, however, impaired NADH oxidation following ETC inhibition neither depletes asparagine nor activates the ISR, reflecting an altered metabolic state. ATP synthase inhibition in myotubes triggers the ISR via a distinct mechanism related to mitochondrial inner-membrane hyperpolarization. Our work dispels the notion of a universal path linking mitochondrial dysfunction to the ISR, instead revealing multiple paths that depend both on the nature of the mitochondrial defect and on the metabolic state of the cell.

A genetically encoded tool for manipulation of NADP+/NADPH in living cells.

The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.

Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.

A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.

Trimodal Control of Ion-Transport Activity on Cyclo-oligo-(1→6)-ß-D-glucosamine-Based Artificial Ion-Transport Systems.

Cyclo-oligo-(1→6)-ß-D-glucosamines functionalized with hydrophobic tails are reported as a new class of transmembrane ion-transport system. These macrocycles with hydrophilic cavities were introduced as an alternative to cyclodextrins, which are supramolecular systems with hydrophobic cavities. The transport activities of these glycoconjugates were manipulated by altering the oligomericity of the macrocycles, as well as the length and number of attached tails. Hydrophobic tails of 3 different sizes were synthesized and coupled with each glucosamine scaffold through the amide linkage to obtain 18 derivatives. The ion-transport activity increased from di- to tetrameric glucosamine macrocycles, but decreased further when flexible pentameric glucosamine was introduced. The ion-transport activity also increased with increasing length of attached linkers. For a fixed length of linkers, the transport activity decreased when the number of such tails was reduced. All glycoconjugates displayed a uniform anion-selectivity sequence: Cl(-) >Br(-) >I(-) . From theoretical studies, hydrogen bonding between the macrocycle backbone and the anion bridged through water molecules was observed.

Covalent modification of a cysteine residue in the XPB subunit of the general transcription factor TFIIH through single epoxide cleavage of the transcription inhibitor triptolide.

Triptolide is a key component of the traditional Chinese medicinal plant Thunder God Vine and has potent anticancer and immunosuppressive activities. It is an irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit of the general transcription factor TFIIH. Cys342 of XPB was identified as the residue that undergoes covalent modification by the 12,13-epoxide group of triptolide. Mutation of Cys342 of XPB to threonine conferred resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB with the Cys342Thr mutant in a HEK293T cell line rendered it completely resistant to triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these results deepen our understanding of the interaction between triptolide and XPB and have implications for the future development of new analogues of triptolide as leads for anticancer and immunosuppressive drugs.

Cyclo-oligo-(1 → 6)-ß-D-glucosamine based artificial channels for tunable transmembrane ion transport.

Unimolecular ion channels were designed by functionalization of a new type of cyclic oligosaccharides, cyclo-oligo-(1 → 6)-ß-d-glucosamines, with pentabutylene glycol chains. Their ion transporting activity was tuned by varying oligomericity. A halide selectivity sequence, Cl(-) > Br(-) > I(-) was observed.

Synthesis of multivalent carbohydrate-centered glycoclusters as nanomolar ligands of the bacterial lectin LecA from Pseudomonas aeruginosa.

A family of fifteen glycoclusters based on a cyclic oligo-(1→6)-ß-D-glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme-linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub-micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.

Stereochemistry of intramolecular cyclization of tetra-ß-(1→6)-D-glucosamines and related tetrasaccharides: the role of the conformational stereocontrol and the neighboring group participation.

The effect of reaction conditions, the nature of a leaving group, and a substituent at C-2 in the glycosylating monosaccharide on the stereochemical outcome of cyclization of linear tetra-ß-(1→6)-d-glucosamines and some 'mixed' tetrasaccharides comprising glucose and glucosamine residues has been examined. Toluene and nitrile solvents improved the ß-stereoselectivity of cyclization, however, the overall efficiency of the formation of cyclic products in these solvents was lower than that in dichloromethane. The use of bromide or pentenyl glycoside as leaving groups instead of the thioglycoside did not increase the ß-stereoselectivity. Replacement of the N-phthaloyl group in the glycosylating unit by N-Troc one did not affect the stereoselectivity of cyclization, while the tetrasaccharides, which contained 2-O-benzoyl glucose instead of glucosamine as the glycosyl donor moiety, were found to provide ß-linked cyclic products exclusively. Using this finding, two cyclic tetrasaccharides with alternate or adjacent arrangement of two glucose and two glucosamine units have been efficiently synthesized. These cycles were intended for the preparation of divalent glycoclusters with different ligand orientation. The difference in the stereoselectivity of cyclization of glucose and glucosamine precursors was accounted for by more effective anchimeric participation of the O-benzoyl group as compared to N-Phth and N-Troc counterparts that was confirmed by calculations of the stabilization energy and rotational barriers around C2-O/N bond in the corresponding glycosyl cations. Only the 2-O-benzoylated glycosyl cation proved to be able to form a stabilized dioxalenium intermediate, which provides stereospecific ß-glycosylation.

Identification and validation of protein targets of bioactive small molecules.

Identification and validation of protein targets of bioactive small molecules is an important problem in chemical biology and drug discovery. Currently, no single method is satisfactory for this task. Here, we provide an overview of common methods for target identification and validation that historically were most successful. We have classified for the first time the existing methods into two distinct and complementary types, the 'top-down' and 'bottom-up' approaches. In a typical top-down approach, the cellular phenotype is used as a starting point and the molecular target is approached through systematic narrowing down of possibilities by taking advantage of the detailed existing knowledge of cellular pathways and processes. In contrast, the bottom-up approach entails the direct detection and identification of the molecular targets using affinity-based or genetic methods. A special emphasis is placed on target validation, including correlation analysis and genetic methods, as this area is often ignored despite its importance.

NMR and conformational studies of linear and cyclic oligo-(1→6)-ß-D-glucosamines.

The conformational behavior of a series of linear and cyclic oligo-(1→6)-ß-D-glucosamines and their N-acetylated derivatives, which are related to fragments of natural poly-N-acetylglucosamine, was studied by theoretical molecular modeling and experimental determination of transglycosidic vicinal coupling constants (3)J(C,H) and (3)J(H,H). Molecular dynamics simulations were performed under several types of conditions varying in the consideration of ionization of amino groups, solvent effect, and temperature. Neural network clustering and asphericity calculations were performed on the basis of molecular dynamics data. It was shown that disaccharide fragments in the studied linear oligosaccharides were not rigid, and tended to have several conformers, thus determining the overall twisted shape with helical elements. In addition, it was found that the behavior of C5-C6 bond depended significantly upon the simulation conditions. The cyclic di-, tri-, and tetrasaccharides mostly had symmetrical ring-shaped conformations. The larger cycles tended to adopt more complicated shapes, and the conformational behavior of their disaccharide fragments was close to that in the linear oligosaccharides.

XPB, a subunit of TFIIH, is a target of the natural product triptolide.

Triptolide (1) is a structurally unique diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. Its molecular mechanism of action, however, has remained largely elusive to date. We report that triptolide covalently binds to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II-mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the majority of the known biological activities of triptolide. These findings also suggest that triptolide can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.

Regulator of Ras depalmitoylation and retrograde trafficking: a new hat for FKBP.

In this issue of Molecular Cell, Ahearn et al. (2011) identified FKBP12 as a novel regulator of Ras signaling through its modulation of depalmitoylation of H-Ras and its recycling from plasma membrane to the Golgi.

Analyzing protein kinase dynamics in living cells with FRET reporters.

Genetically encoded reporters based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of kinase activities in living cells, as the activities of this class of enzymes are often dynamically regulated and spatially compartmentalized within specific signaling context. Here we describe a general modular design and engineering strategies for the development of activity reporters for kinases of interest, using A-kinase activity reporter (AKAR) as an illustrative example. Discussed here are basic structure of such reporters, design considerations, reporter gene construction, cellular and in vitro characterization. Strategies for improving specificity, dynamic range or sensitivity, reversibility and integrity of the reporter as well as basic methods for live-cell time-lapse imaging using these reporters are summarized. Discussion of using this approach in the study of MAPK cascades is also provided. These FRET-based kinase activity reporters, along with analogous probes based on alternative designs, provide real-time tracking of kinase dynamics with subcellular resolution, which should complement other methods and offer great opportunities to delineate the molecular mechanisms underlying the complex regulation of kinases.