Bruce Morgan

Universität des Saarlandes | UKS · Institute of Biochemistry

Dimedone and its derivates are used as selective probes for the nucleophilic detection of sulfenic acids in biological samples. Qualitative analyses suggested that dimedone also reacts with cyclic sulfen-amides. Furthermore, under physiological conditions, dimedone must compete with the highly concentrated nucleophile glutathione. We therefore quan...

Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitocho...

The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo "steady state" redox potential imposed onto protein thiol disulfide pairs in the periplasm...

12 The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative 13 power and the DsbC/D system as its complement for isomerization of non-native disulfides. 14 While the standard redox potentials of those systems are known, the in vivo redox potential 15 imposed onto protein thiol disulfide pairs in the periplasm rem...

Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 pl...

Mitochondria are essential organelles that play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here we show that the yeast cytosoli...

Hydrogen peroxide (H2 O2 ) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2 O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2 O2 . Here, we employed...

Significance Leishmaniasis is one of the most dangerous, neglected tropical diseases. Oxidative phosphorylation is a known target of antipathogenic therapeutics. Herein, we addressed the contribution of type II NADH dehydrogenase (NDH2) and complex I activities to Leishmania physiology. Our results provide evidence that Leishmania NDH2 is essential...

H 2 O 2 performs central roles in signaling at physiological levels, while at elevated levels it causes molecular damage. Mitochondria are major producers of H 2 O 2 , which has been implied in regulating diverse processes inside and outside the organelle. However, it still remains unclear whether and how mitochondria in intact cells release H 2 O...

H2O2 is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H2O2 sensor that allows H2O2 monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H2O2 sensor roGFP2-Tsa2ΔCR to the cytosol...

The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a r...

Redox cycles have been reported in ultradian, circadian and cell cycle-synchronized systems. Redox cycles persist in the absence of transcription and cyclin-CDK activity, indicating that cells harbor multiple coupled oscillators. Nonetheless, the causal relationships and molecular mechanisms by which redox cycles are embedded within ultradian, circ...

Modern lifestyles, including lack of physical activity and poor nutritional habits, are driving the rapidly increasing prevalence of obesity and type 2 diabetes. Increased levels of free fatty acids (FFAs), particularly saturated FFAs, in obese individuals have been linked to pancreatic β-cell failure. This process, termed lipotoxicity, involves ac...

Amperometric biosensors have been widely utilized for the cost-effective and rapid analysis of various bioanalytes, for example glucose. However, a lack of standardization and validation procedures remains a major limitation in biosensor development. Therefore, despite rapid advances in material science driving the development of amperometric biose...

NADH and NAD+ are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD+ in plants usin...

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA...

Glutaredoxins are small proteins of the thioredoxin superfamily that are present throughout life. Most glutaredoxins fall into two major subfamilies. Class I glutaredoxins are glutathione-dependent thiol-disulfide oxidoreductases, whereas class II glutaredoxins coordinate Fe–S clusters. Class I glutaredoxins are typically dithiol enzymes with two a...

Background Free fatty acids (FFAs) are known for their dual effects on insulin secretion and pancreatic β-cell survival. Short-term exposure to FFAs, such as palmitate, increases insulin secretion. On the contrary, long-term exposure to saturated FFAs results in decreased insulin secretion, as well as triggering oxidative stress and endoplasmic ret...

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site...

The manufacturing of conventional enzymatic biosensors produced via a layer-by-layer (LbL) approach requires expensive instrumentation, and in most cases involves a complex, resource and time-consuming fabrication process. Moreover, LbL assemblies are prone to mechanical instability that leads to irreversible changes in sensor architecture and morp...

Dietary protein dilution (DPD) promotes metabolic remodelling and health but the precise nutritional components driving this response remain elusive. Here we demonstrate that dietary amino acids (AA) are sufficient and necessary to drive the response to DPD. In particular, the restriction of dietary essential AA (EAA) supply, but not non-EAA, drive...

Hydrogen peroxide (H2O2) plays important roles in cellular signaling, yet nonetheless is toxic at higher concentrations. Surprisingly, the mechanism(s) of cellular H2O2 toxicity remain poorly understood. Here, we reveal an important role for mitochondrial 1-Cys peroxiredoxin from budding yeast, Prx1, in regulating H2O2-induced cell death. We show t...

Glutathione (γ-l-glutamyl-l-cysteinylglycine) is a small tripeptide found at millimolar concentrations in nearly all eukaryotes as well as many prokaryotic cells. Glutathione synthesis is restricted to the cytosol in animals and fungi and to the cytosol and plastids in plants. Nonetheless, glutathione is found in virtually all subcellular compartme...

Significance: Genetically encoded H2O2 sensors, based upon fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2) have dramatically broadened the available 'toolbox' for monitoring cellular H2O2 changes. Recent advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2∆CR offer considerably impr...

Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are...

Significance: Mitochondrial glutathione fulfils crucial roles in a number of processes including iron-sulfur cluster biosynthesis and peroxide detoxification. Recent Advances: Genetically-encoded fluorescent probes for the glutathione redox potential (EGSH) have permitted extensive new insights into the regulation of mitochondrial glutathione redo...

A disulfide bond is a covalent bond between two sulfur atoms. Disulfide bonds are prevalent in biology and found in a range of biological molecules, for example between the sulfur atoms of protein cysteine residues. Protein disulfide bonds can fulfil a wide range of functions including promoting protein stability and regulating protein activity or...

Peroxisomes are cellular organelles with vital functions in lipid, amino acid, and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes, however, the regulation of peroxisome abundance is yet poorly understood. Here we use a high-content microscopy screen to identify novel regulators of peroxisome size and...

Rationale: Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity and the possibility to analyze subcellular domains. Recent advances to quantitativ...

Redox signalling is increasingly recognized to play a crucial role in cellular and organismal physiology. Whilst we have made considerable advances in recent years with respect to understanding the roles and regulation of many different cellular redox species, research remains restricted by a lack of appropriate tools and techniques. Here we discus...

Fluorescent protein sensors have re-vitalized the field of redox biology by revolutionizing the study of redox processes in living cells and organisms. Recent Advances: Within one decade a set of fundamental new insights has been gained, driven by the rapid technical development of in vivo redox sensing. Redox-sensitive yellow and green fluorescent...

Genetically encoded probes based on the H2O2-sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H2O2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H2O2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxin-bas...

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to reversible oxidative inactivation by hydrogen peroxide (H2O2). Here we show that H2O2 reactivity of the active site thiolate (C152) is catalyzed by a previously unrecognized mechanism based on a dedicated proton relay promoting leaving group departure. Disruption of the peroxidatic re...

Abstract Cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.12) is present in all organisms and catalyzes the oxidation of triose phosphate during glycolysis. GAPDH is one of the most prominent cellular targets of oxidative modifications when reactive oxygen and nitrogen species are formed during metabolism and under stress condi...

Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial; several studies suggest that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen et al.1 provided apparent mech...

Glutathione is the most abundant small molecule thiol in nearly all eukaryotes. Whole-cell levels of oxidized (GSSG) and reduced (GSH) glutathione are variable and responsive to genetic and chemical manipulations, which has led to their relative levels being widely used as a marker of the 'cellular redox state' and to indicate the level of 'oxidati...

Glutathione, the most abundant small-molecule thiol in eukaryotic cells, is synthesized de-novo solely in the cytosol and must subsequently be transported to other cellular compartments. The mechanisms of glutathione transport into and out of organelles remain largely unclear. We show that budding yeast Opt2, a close homolog of the plasma membrane...

Redox signalling plays an important role in many aspects of physiology, including that of the cardiovascular system. Perturbed redox regulation has been associated with numerous pathological conditions, nevertheless, the causal relationships between redox changes and pathology often remain unclear. Redox signalling involves the production of specif...

Redox processes play a crucial role in many aspects of physiology and changes in cellular redox species are increasingly being linked to a wide range of pathological conditions. Redox species can change dynamically at the subcellular compartment-, cell- and tissue-level and different redox species likely convey different biological information. The...

Glutathione is central to cellular redox chemistry. The majority of glutathione redox research has been based on the chemical analysis of whole-cell extracts, which unavoidably destroy subcellular compartment-specific information. Compartment-specific real-time measurements based on genetically encoded fluorescent probes now suggest that the cytoso...

Aims: To identify yeast mutants that show a strong redox dependence of the ability to respire, we systematically screened a yeast deletion library for mutants that require the presence of reductants for growth on nonfermentable carbon sources. Results: Respirative growth of 44 yeast mutants was significantly improved by the addition of dithiothr...

Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (E(GSH)) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with E(GSH)-specific fluorescent reporters, we inv...

Redox biochemistry plays an important role in a wide range of cellular events. However, investigation of cellular redox processes is complicated by the large number of cellular redox couples, which are often not in equilibrium with one another and can vary significantly between subcellular compartments and cell types. Further, it is becoming increa...

Ion gradients across intracellular membranes contribute to the physicochemical environment inside compartments. CLC anion transport proteins that localise to intracellular organelles are anion-proton exchangers involved in anion sequestration or vesicular acidification. By homology, the only CLC protein of Saccharomyces cerevisiae, Gef1, belongs to...

Zinc is an essential cofactor required for the function of ∼8% of the yeast and 10% of the human proteome. All of the “small Tim” proteins of the mitochondrial intermembrane space contain a strictly conserved “twin CX3C” zinc finger motif, which can bind zinc ions in the Cys-reduced form. We have shown previously that although disulfide bond format...

All small Tim proteins of the mitochondrial intermembrane space contain two conserved CX(3)C motifs, which form two intramolecular disulfide bonds essential for function, but only the cysteine-reduced, but not oxidized, proteins can be imported into mitochondria. We have shown that Tim10 can be oxidized by glutathione under cytosolic concentrations...