Sub-organellar mitochondrial hydrogen peroxide observed using a SNAP tag targeted coumarin-based fluorescent reporter.

Mitochondria are major sites of reactive oxygen species (ROS) production within cells. ROS are important signalling molecules, but excessive production can cause cellular damage and dysfunction. It is therefore crucial to accurately determine when, how and where ROS are produced within mitochondria.

Incorporating a Polyethyleneglycol Linker to Enhance the Hydrophilicity of Mitochondria-Targeted Triphenylphosphonium Constructs.

The targeting of bioactive molecules and probes to mitochondria can be achieved by coupling to the lipophilic triphenyl phosphonium (TPP) cation, which accumulates several hundred-fold within mitochondria in response to the mitochondrial membrane potential (Δψ ). Typically, a simple alkane links the TPP to its "cargo", increasing overall hydrophobicity. As it would be beneficial to enhance the water solubility of mitochondria-targeted compounds we explored the effects of replacing the alkyl linker with a polyethylene glycol (PEG).

Rapid and selective generation of HS within mitochondria protects against cardiac ischemia-reperfusion injury.

Mitochondria-targeted HS donors are thought to protect against acute ischemia-reperfusion (IR) injury by releasing HS that decreases oxidative damage. However, the rate of HS release by current donors is too slow to be effective upon administration following reperfusion. To overcome this limitation here we develop a mitochondria-targeted agent, MitoPerSulf that very rapidly releases HS within mitochondria.

Targeting Methylglyoxal in Diabetic Kidney Disease Using the Mitochondria-Targeted Compound MitoGamide.

Diabetic kidney disease (DKD) remains the number one cause of end-stage renal disease in the western world. In experimental diabetes, mitochondrial dysfunction in the kidney precedes the development of DKD. Reactive 1,2-dicarbonyl compounds, such as methylglyoxal, are generated from sugars both endogenously during diabetes and exogenously during food processing.

Photoactivated release of membrane impermeant sulfonates inside cells.

Photouncaging delivers compounds with high spatial and temporal control to induce or inhibit biological processes but the released compounds may diffuse out. We here demonstrate that sulfonate anions can be photocaged so that a membrane impermeable compound can enter cells, be uncaged by photoirradiation and trapped within the cell.

Tetra-arylborate lipophilic anions as targeting groups.

Tetraphenylborate (TPB) anions traverse membranes but are excluded from mitochondria by the membrane potential (Δψ). TPB-conjugates also distributed across membranes in response to Δψ, but surprisingly, they rapidly entered cells. They accumulated within lysosomes following endocystosis.

Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2.

Enhancing the Mitochondrial Uptake of Phosphonium Cations by Carboxylic Acid Incorporation.

There is considerable interest in developing drugs and probes targeted to mitochondria in order to understand and treat the many pathologies associated with mitochondrial dysfunction. The large membrane potential, negative inside, across the mitochondrial inner membrane enables delivery of molecules conjugated to lipophilic phosphonium cations to the organelle. Due to their combination of charge and hydrophobicity, quaternary triarylphosphonium cations rapidly cross biological membranes without the requirement for a carrier.

Confirmation of the Cardioprotective Effect of MitoGamide in the Diabetic Heart.

Purpose: HFpEF (heart failure with preserved ejection fraction) is a major consequence of diabetic cardiomyopathy with no effective treatments. Here, we have characterized Akita mice as a preclinical model of HFpEF and used it to confirm the therapeutic efficacy of the mitochondria-targeted dicarbonyl scavenger, MitoGamide.

Methods And Results: A longitudinal echocardiographic analysis confirmed that Akita mice develop diastolic dysfunction with reduced E peak velocity, E/A ratio and extended isovolumetric relaxation time (IVRT), while the systolic function remains comparable with wild-type mice.

Ester Prodrugs of Malonate with Enhanced Intracellular Delivery Protect Against Cardiac Ischemia-Reperfusion Injury In Vivo.

Purpose: Mitochondrial reactive oxygen species (ROS) production upon reperfusion of ischemic tissue initiates the ischemia/reperfusion (I/R) injury associated with heart attack. During ischemia, succinate accumulates and its oxidation upon reperfusion by succinate dehydrogenase (SDH) drives ROS production. Inhibition of succinate accumulation and/or oxidation by dimethyl malonate (DMM), a cell permeable prodrug of the SDH inhibitor malonate, can decrease I/R injury.

Succinate accumulation drives ischaemia-reperfusion injury during organ transplantation.

During heart transplantation, storage in cold preservation solution is thought to protect the organ by slowing metabolism; by providing osmotic support; and by minimising ischaemia-reperfusion (IR) injury upon transplantation into the recipient. Despite its widespread use our understanding of the metabolic changes prevented by cold storage and how warm ischaemia leads to damage is surprisingly poor. Here, we compare the metabolic changes during warm ischaemia (WI) and cold ischaemia (CI) in hearts from mouse, pig, and human.

Correction to: The Mitochondria-Targeted Methylglyoxal Sequestering Compound, MitoGamide, Is Cardioprotective in the Diabetic Heart.

The article "The Mitochondria-Targeted Methylglyoxal Sequestering Compound, MitoGamide, Is Cardioprotective in the Diabetic Heart".

A sensitive mass spectrometric assay for mitochondrial CoQ pool redox state in vivo.

Coenzyme Q (CoQ) is an essential cofactor, primarily found in the mitochondrial inner membrane where it functions as an electron carrier in the respiratory chain, and as a lipophilic antioxidant. The redox state of the CoQ pool is the ratio of its oxidised (ubiquinone) and reduced (ubiquinol) forms, and is a key indicator of mitochondrial bioenergetic and antioxidant status. However, the role of CoQ redox state in vivo is poorly understood, because determining its value is technically challenging due to redox changes during isolation, extraction and analysis.

The Mitochondria-Targeted Methylglyoxal Sequestering Compound, MitoGamide, Is Cardioprotective in the Diabetic Heart.

Purpose: Methylglyoxal, a by-product of glycolysis and a precursor in the formation of advanced glycation end-products, is significantly elevated in the diabetic myocardium. Therefore, we sought to investigate the mitochondria-targeted methylglyoxal scavenger, MitoGamide, in an experimental model of spontaneous diabetic cardiomyopathy.

Methods: Male 6-week-old Akita or wild type mice received daily oral gavage of MitoGamide or vehicle for 10 weeks.

Selective mitochondrial superoxide generation in vivo is cardioprotective through hormesis.

Reactive oxygen species (ROS) have an equivocal role in myocardial ischaemia reperfusion injury. Within the cardiomyocyte, mitochondria are both a major source and target of ROS. We evaluate the effects of a selective, dose-dependent increase in mitochondrial ROS levels on cardiac physiology using the mitochondria-targeted redox cycler MitoParaquat (MitoPQ).

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation.

Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice.

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues.

Methionine sulfoxide reductase B3 requires resolving cysteine residues for full activity and can act as a stereospecific methionine oxidase.

The oxidation of methionine residues in proteins occurs during oxidative stress and can lead to an alteration in protein function. The enzyme methionine sulfoxide reductase (Msr) reverses this modification. Here, we characterise the mammalian enzyme Msr B3.

Synthesis of an azido-tagged low affinity ratiometric calcium sensor.

Changes in high localised concentrations of Ca ions are fundamental to cell signalling. The synthesis of a dual excitation, ratiometric calcium ion sensor with a of 90 μM, is described. It is tagged with an azido group for bioconjugation, and absorbs in the blue/green and emits in the red region of the visible spectrum with a large Stokes shift.

Protein-mediated dethreading of a biotin-functionalised pseudorotaxane.

In this article, we describe the synthesis of new biotin-functionalised naphthalene derivatives 3 and 4 and their complexation behaviour with avidin and neutravidin using a range of analytical techniques. We have shown using 2-(4'-hydroxyazobenzene)benzoic acid displacement and ITC experiments, that compounds 3 and 4 have the propensity to form reasonably high-affinity bioconjugates with avidin and neutravidin. We have also demonstrated using (1)H NMR, UV-vis and fluorescence spectroscopy that the naphthalene moiety of 3 and 4 facilitates the formation of pseudorotaxane-like structures with 1 in water.

Excited state charge redistribution and dynamics in the donor-π-acceptor flavin derivative ABFL.

Chromophores containing a donor-π-acceptor (D-π-A) motif have been shown to exhibit many interesting photophysical properties. The lowest electronic transition of a flavin derivative containing this motif, azobenzylflavin (ABFL), has previously been shown to be highly sensitive to solvent environment and hydrogen bonding ligands. To better understand this sensitivity, we have investigated the excited state charge redistribution and dynamics of ABFL in a low-dielectric, non-hydrogen bonding solvent by steady-state Stark and femtosecond optical transient absorption spectroscopies.