Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging

Stoichiometric Thiol Redox Proteomics for Quantifying Cellular Responses to Perturbations

Post-translational modifications regulate the structure and function of proteins that can result in changes to the activity of different pathways. These include modifications altering the redox state of thiol groups on protein cysteine residues, which are sensitive to oxidative environments. While mass spectrometry has advanced the identification of protein thiol modifications and expanded our knowledge of redox-sensitive pathways, the quantitative aspect of this technique is critical for the field of redox proteomics. In this review, we describe how mass spectrometry-based redox proteomics has enabled researchers to accurately quantify the stoichiometry of reversible oxidative modifications on specific cysteine residues of proteins. We will describe advancements in the methodology that allow for the absolute quantitation of thiol modifications, as well as recent reports that have implemented this approach. We will also highlight the significance and application of such measurements and why they are informative for the field of redox biology

Defining the S-Glutathionylation Proteome by Biochemical and Mass Spectrometric Approaches

Protein S-glutathionylation (SSG) is a reversible post-translational modification (PTM) featuring the conjugation of glutathione to a protein cysteine thiol. SSG can alter protein structure, activity, subcellular localization, and interaction with small molecules and other proteins. Thus, it plays a critical role in redox signaling and regulation in various physiological activities and pathological events. In this review, we summarize current biochemical and analytical approaches for characterizing SSG at both the proteome level and at individual protein levels. To illustrate the mechanism underlying SSG-mediated redox regulation, we highlight recent examples of functional and structural consequences of SSG modifications. Finally, we discuss the analytical challenges in characterizing SSG and the thiol PTM landscape, future directions for understanding of the role of SSG in redox signaling and regulation and its interplay with other PTMs, and the potential role of computational approaches to accelerate functional discovery

Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 min after the last stimulation and processed for redox proteomics assay of S-glutathionylation. Using selective reduction with a glutaredoxin enzyme cocktail and resin-assisted enrichment technique, we quantified the levels of site-specific protein S-glutathionylation at rest and following fatiguing contractions. Redox proteomics revealed over 2200 sites of S-glutathionylation modifications, of which 1290 were significantly increased after fatiguing contractions. Muscle contraction leads to the greatest increase in S-glutathionylation in the mitochondria (1.03%) and the smallest increase in the nucleus (0.47%). Regulatory cysteines were significantly S-glutathionylated on mitochondrial complex I and II, GAPDH, MDH1, ACO2, and mitochondrial complex V among others. Similarly, S-glutathionylation of RYR1, SERCA1, titin, and troponin I2 are known to regulate muscle contractility and were significantly S-glutathionylated after just 15 min of fatiguing contractions. The largest fold changes (> 1.6) in the S-glutathionylated proteome after fatigue occurred on signaling proteins such as 14-3-3 protein gamma and MAP2K4, as well as proteins like SERCA1, and NDUV2 of mitochondrial complex I, at previously unknown glutathionylation sites. These findings highlight the important role of redox control over muscle physiology, metabolism, and the exercise adaptive response. This study lays the groundwork for future investigation into the altered exercise adaptation associated with chronic conditions, such as sarcopenia. Keywords: Muscle contraction, Post-translational modification, S-glutathionylation, Redox signaling, Skeletal muscle, Thiol redox proteomic

All that is silver is not toxic: silver ion and particle kinetics reveals the role of silver ion aging and dosimetry on the toxicity of silver nanoparticles

Abstract Background When suspended in cell culture medium, nano-objects composed of soluble metals such as silver can dissolve resulting in ion formation, altered particle properties (e.g. mass, morphology, etc.), and modulated cellular dose. Cultured cells are exposed not just to nanoparticles but to a complex, dynamic mixture of altered nanoparticles, unbound ions, and ion-ligand complexes. Here, three different cell types (RAW 264.7 macrophages and bone marrow derived macrophages from wild-type C57BL/6 J mice and Scavenger Receptor A deficient (SR-A(−/−)) mice) were exposed to 20 and 110 nm silver nanoparticles, and RAW 264.7 cells were exposed to freshly mixed silver ions, aged silver ions (ions incubated in cell culture medium), and ions formed from nanoparticle dissolution. The In Vitro Sedimentation, Diffusion, Dissolution, and Dosimetry Model (ISD3) was used to predict dose metrics for each exposure scenario. Results Silver nanoparticles, freshly mixed ions, and ions from nanoparticle dissolution were toxic, while aged ions were not toxic. Macrophages from SR-A(−/−) mice did not take up 20 nm silver nanoparticles as well as wild-types but demonstrated no differences in silver levels after exposure to 110 nm nanoparticles. Dose response modeling with ISD3 predicted dose metrics suggest that amount of ions in cells and area under the curve (AUC) of ion amount in cells are the most predictive of cell viability after nanoparticle and combined nanoparticle/dissolution-formed-ions exposures, respectively. Conclusions Results of this study suggest that the unbound silver cation is the ultimate toxicant, and ions formed extracellularly drive toxicity after exposure to nanoparticles. Applying computational modeling (ISD3) to better understand dose metrics for soluble nanoparticles allows for better interpretation of in vitro hazard assessments

Discovery of potential pathways for biological conversion of poplar wood into lipids by co-fermentation of Rhodococci strains

Abstract Background Biological routes for utilizing both carbohydrates and lignin are important to reach the ultimate goal of bioconversion of full carbon in biomass into biofuels and biochemicals. Recent biotechnology advances have shown promises toward facilitating biological transformation of lignin into lipids. Natural and engineered Rhodococcus strains (e.g., R. opacus PD630, R. jostii RHA1, and R. jostii RHA1 VanA−) have been demonstrated to utilize lignin for lipid production, and co-culture of them can promote lipid production from lignin. Results In this study, a co-fermentation module of natural and engineered Rhodococcus strains with significant improved lignin degradation and/or lipid biosynthesis capacities was established, which enabled simultaneous conversion of glucose, lignin, and its derivatives into lipids. Although Rhodococci sp. showed preference to glucose over lignin, nearly half of the lignin was quickly depolymerized to monomers by these strains for cell growth and lipid synthesis after glucose was nearly consumed up. Profiles of metabolites produced by Rhodococcus strains growing on different carbon sources (e.g., glucose, alkali lignin, and dilute acid flowthrough-pretreated poplar wood slurry) confirmed lignin conversion during co-fermentation, and indicated novel metabolic capacities and unexplored metabolic pathways in these organisms. Proteome profiles suggested that lignin depolymerization by Rhodococci sp. involved multiple peroxidases with accessory oxidases. Besides the β-ketoadipate pathway, the phenylacetic acid (PAA) pathway was another potential route for the in vivo ring cleavage activity. In addition, deficiency of reducing power and cellular oxidative stress probably led to lower lipid production using lignin as the sole carbon source than that using glucose. Conclusions This work demonstrated a potential strategy for efficient bioconversion of both lignin and glucose into lipids by co-culture of multiple natural and engineered Rhodococcus strains. In addition, the involvement of PAA pathway in lignin degradation can help to further improve lignin utilization, and the combinatory proteomics and bioinformatics strategies used in this study can also be applied into other systems to reveal the metabolic and regulatory pathways for balanced cellular metabolism and to select genetic targets for efficient conversion of both lignin and carbohydrates into biofuels

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly