Cell-type-specific transcriptomics uncovers spatial regulatory networks in bioenergy sorghum stems.

Bioenergy sorghum is a low-input, drought-resilient, deep-rooting annual crop that has high biomass yield potential enabling the sustainable production of biofuels, biopower, and bioproducts. Bioenergy sorghum's 4-5 m stems account for ~80% of the harvested biomass. Stems accumulate high levels of sucrose that could be used to synthesize bioethanol and useful biopolymers if information about cell-type gene expression and regulation in stems was available to enable engineering. To obtain this information, laser capture microdissection was used to isolate and collect transcriptome profiles from five major cell types that are present in stems of the sweet sorghum Wray. Transcriptome analysis identified genes with cell-type-specific and cell-preferred expression patterns that reflect the distinct metabolic, transport, and regulatory functions of each cell type. Analysis of cell-type-specific gene regulatory networks (GRNs) revealed that unique transcription factor families contribute to distinct regulatory landscapes, where regulation is organized through various modes and identifiable network motifs. Cell-specific transcriptome data was combined with known secondary cell wall (SCW) networks to identify the GRNs that differentially activate SCW formation in vascular sclerenchyma and epidermal cells. The spatial transcriptomic dataset provides a valuable source of information about the function of different sorghum cell types and GRNs that will enable the engineering of bioenergy sorghum stems, and an interactive web application developed during this project will allow easy access and exploration of the data (https://mc-lab.shinyapps.io/lcm-dataset/).

Redox regulation of m6A methyltransferase METTL3 in ß-cells controls the innate immune response in type 1 diabetes.

Type 1 diabetes (T1D) is characterized by the destruction of pancreatic ß-cells. Several observations have renewed the interest in ß-cell RNA sensors and editors. Here, we report that N6-methyladenosine (m6A) is an adaptive ß-cell safeguard mechanism that controls the amplitude and duration of the antiviral innate immune response at T1D onset. m6A writer methyltransferase 3 (METTL3) levels increase drastically in ß-cells at T1D onset but rapidly decline with disease progression. m6A sequencing revealed the m6A hypermethylation of several key innate immune mediators, including OAS1, OAS2, OAS3 and ADAR1 in human islets and EndoC-ßH1 cells at T1D onset. METTL3 silencing enhanced 2'-5'-oligoadenylate synthetase levels by increasing its mRNA stability. Consistently, in vivo gene therapy to prolong Mettl3 overexpression specifically in ß-cells delayed diabetes progression in the non-obese diabetic mouse model of T1D. Mechanistically, the accumulation of reactive oxygen species blocked upregulation of METTL3 in response to cytokines, while physiological levels of nitric oxide enhanced METTL3 levels and activity. Furthermore, we report that the cysteines in position C276 and C326 in the zinc finger domains of the METTL3 protein are sensitive to S-nitrosylation and are important to the METTL3-mediated regulation of oligoadenylate synthase mRNA stability in human ß-cells. Collectively, we report that m6A regulates the innate immune response at the ß-cell level during the onset of T1D in humans.

Signatures of cysteine oxidation on muscle structural and contractile proteins are associated with physical performance and muscle function in older adults: Study of Muscle, Mobility and Aging (SOMMA).

Oxidative stress is considered a contributor to declining muscle function and mobility during aging; however, the underlying molecular mechanisms remain poorly described. We hypothesized that greater levels of cysteine (Cys) oxidation on muscle proteins are associated with decreased measures of mobility. Herein, we applied a novel redox proteomics approach to measure reversible protein Cys oxidation in vastus lateralis muscle biopsies collected from 56 subjects in the Study of Muscle, Mobility and Aging (SOMMA), a community-based cohort study of individuals aged 70 years and older. We tested whether levels of Cys oxidation on key muscle proteins involved in muscle structure and contraction were associated with muscle function (leg power and strength), walking speed, and fitness (VO2 peak on cardiopulmonary exercise testing) using linear regression models adjusted for age, sex, and body weight. Higher oxidation levels of select nebulin Cys sites were associated with lower VO2 peak, while greater oxidation of myomesin-1, myomesin-2, and nebulin Cys sites was associated with slower walking speed. Higher oxidation of Cys sites in key proteins such as myomesin-2, alpha-actinin-2, and skeletal muscle alpha-actin were associated with lower leg power and strength. We also observed an unexpected correlation (R = 0.48) between a higher oxidation level of eight Cys sites in alpha-actinin-3 and stronger leg power. Despite this observation, the results generally support the hypothesis that Cys oxidation of muscle proteins impairs muscle power and strength, walking speed, and cardiopulmonary fitness with aging.

In-Depth Proteome Profiling of Small Extracellular Vesicles Isolated from Cancer Cell Lines and Patient Serum.

Extracellular vesicle (EV) secretion has been observed in many types of both normal and tumor cells. EVs contain a variety of distinctive cargoes, allowing tumor-derived serum proteins in EVs to act as a minimally invasive method for clinical monitoring. We have undertaken a comprehensive study of the protein content of the EVs from several cancer cell lines using direct data-independent analysis. Several thousand proteins were detected, including many classic EV markers such as CD9, CD81, CD63, TSG101, and Syndecan-1, among others. We detected many distinctive cancer-specific proteins, including several known markers used in cancer detection and monitoring. We further studied the protein content of EVs from patient serum for both normal controls and pancreatic cancer and hepatocellular carcinoma. The EVs for these studies have been isolated by various methods for comparison, including ultracentrifugation and CD9 immunoaffinity column. Typically, 500-1000 proteins were identified, where most of them overlapped with the EV proteins identified from the cell lines studied. We were able to identify many of the cell-line EV protein markers in the serum EVs, in addition to the large numbers of proteins specific to pancreatic and HCC cancers.

Signatures of Cysteine Oxidation on Muscle Structural and Contractile Proteins Are Associated with Physical Performance and Muscle Function in Older Adults: Study of Muscle, Mobility and Aging (SOMMA).

Oxidative stress is considered a contributor to declining muscle function and mobility during aging; however, the underlying molecular mechanisms remain poorly described. We hypothesized that greater levels of cysteine (Cys) oxidation on muscle proteins are associated with decreased measures of mobility. Herein, we applied a novel redox proteomics approach to measure reversible protein Cys oxidation in vastus lateralis muscle biopsies collected from 56 subjects in the Study of Muscle, Mobility and Aging (SOMMA), a community-based cohort study of individuals aged 70 years and older. We tested whether levels of Cys oxidation on key muscle proteins involved in muscle structure and contraction were associated with muscle function (leg power and strength), walking speed, and fitness (VO2 peak on cardiopulmonary exercise testing) using linear regression models adjusted for age, sex, and body weight. Higher oxidation levels of select nebulin Cys sites were associated with lower VO2 peak, while greater oxidation of myomesin-1, myomesin-2, and nebulin Cys sites was associated with slower walking speed. Higher oxidation of Cys sites in key proteins such as myomesin-2, alpha-actinin-2, and skeletal muscle alpha-actin were associated with lower leg power and strength. We also observed an unexpected correlation (r = 0.48) between a higher oxidation level of 8 Cys sites in alpha-actinin-3 and stronger leg power. Despite this observation, the results generally support the hypothesis that Cys oxidation of muscle proteins impair muscle power and strength, walking speed, and cardiopulmonary fitness with aging.

Redox Regulation of m 6 A Methyltransferase METTL3 in Human ß-cells Controls the Innate Immune Response in Type 1 Diabetes.

Type 1 Diabetes (T1D) is characterized by autoimmune-mediated destruction of insulin-producing ß-cells. Several observations have renewed interest in the innate immune system as an initiator of the disease process against ß-cells. Here, we show that N 6 -Methyladenosine (m 6 A) is an adaptive ß-cell safeguard mechanism that accelerates mRNA decay of the 2'-5'-oligoadenylate synthetase (OAS) genes to control the antiviral innate immune response at T1D onset. m 6 A writer methyltransferase 3 (METTL3) levels increase drastically in human and mouse ß-cells at T1D onset but rapidly decline with disease progression. Treatment of human islets and EndoC-ßH1 cells with pro-inflammatory cytokines interleukin-1 ß and interferon α mimicked the METTL3 upregulation seen at T1D onset. Furthermore, m 6 A-sequencing revealed the m 6 A hypermethylation of several key innate immune mediators including OAS1, OAS2, and OAS3 in human islets and EndoC-ßH1 cells challenged with cytokines. METTL3 silencing in human pseudoislets or EndoC-ßH1 cells enhanced OAS levels by increasing its mRNA stability upon cytokine challenge. Consistently, in vivo gene therapy, to prolong Mettl3 overexpression specifically in ß-cells, delayed diabetes progression in the non-obese diabetic (NOD) mouse model of T1D by limiting the upregulation of Oas pointing to potential therapeutic relevance. Mechanistically, the accumulation of reactive oxygen species blocked METTL3 upregulation in response to cytokines, while physiological levels of nitric oxide promoted its expression in human islets. Furthermore, for the first time to our knowledge, we show that the cysteines in position C276 and C326 in the zinc finger domain of the METTL3 protein are sensitive to S-nitrosylation (SNO) and are significant for the METTL3 mediated regulation of OAS mRNA stability in human ß-cells in response to cytokines. Collectively, we report that m 6 A regulates human and mouse ß-cells to control the innate immune response during the onset of T1D and propose targeting METTL3 to prevent ß-cell death in T1D.

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.

Elamipretide effects on the skeletal muscle phosphoproteome in aged female mice.

The age-related decline in skeletal muscle mass and function is known as sarcopenia. Sarcopenia progresses based on complex processes involving protein dynamics, cell signaling, oxidative stress, and repair. We have previously found that 8-week treatment with elamipretide improves skeletal muscle function, reverses redox stress, and restores protein S-glutathionylation changes in aged female mice. This study tested whether 8-week treatment with elamipretide also affects global phosphorylation in skeletal muscle consistent with functional improvements and S-glutathionylation. Using female 6-7-month-old mice and 28-29-month-old mice, we found that phosphorylation changes did not relate to S-glutathionylation modifications, but that treatment with elamipretide did partially reverse age-related changes in protein phosphorylation in mouse skeletal muscle.

A deep redox proteome profiling workflow and its application to skeletal muscle of a Duchenne Muscular Dystrophy model.

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In-depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5,608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where a ∼2% shift in the median oxidation occupancy was observed. Pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining greater insight into oxidative stress regulation and pathways/processes that are perturbed in an oxidizing environment.

Capturing an Early Gene Induction Event during Wood Decay by the Brown Rot Fungus Rhodonia placenta.

Brown rot fungi dominate wood decomposition in coniferous forests, and their carbohydrate-selective mechanisms are of commercial interest. Brown rot was recently described as a two-step, sequential mechanism orchestrated by fungi using differentially expressed genes (DEGs) and consisting of oxidation via reactive oxygen species (ROS) followed by enzymatic saccharification. There have been indications, however, that the initial oxidation step itself might require induction. To capture this early gene regulation event, here, we integrated fine-scale cryosectioning with whole-transcriptome sequencing to dissect gene expression at the single-hyphal-cell scale (tens of micrometers). This improved the spatial resolution 50-fold, relative to previous work, and we were able to capture the activity of the first 100 µm of hyphal front growth by Rhodonia placenta in aspen wood. This early decay period was dominated by delayed gene expression patterns as the fungus ramped up its mechanism. These delayed DEGs included many genes implicated in ROS pathways (lignocellulose oxidation [LOX]) that were previously and incorrectly assumed to be constitutively expressed. These delayed DEGs, which include those with and without predicted functions, also create a focused subset of target genes for functional genomics. However, this delayed pattern was not universal, with a few genes being upregulated immediately at the hyphal front. Most notably, this included a gene commonly implicated in hydroquinone and iron redox cycling: benzoquinone reductase. IMPORTANCE Earth's aboveground terrestrial biomass is primarily wood, and fungi dominate wood decomposition. Here, we studied these fungal pathways in a common "brown rot"-type fungus, Rhodonia placenta, that selectively extracts sugars from carbohydrates embedded within wood lignin. Using a space-for-time design to map fungal gene expression at the extreme hyphal front in wood, we made two discoveries. First, we found that many genes long assumed to be "on" (constitutively expressed) from the very beginning of decay were instead "off" before being upregulated, when mapped (via transcriptome sequencing [RNA-seq]) at a high resolution. Second, we found that the gene encoding benzoquinone reductase was "on" in incipient decay and quickly downregulated, implying a key role in "kick-starting" brown rot.

Distinctive carbon repression effects in the carbohydrate-selective wood decay fungus Rhodonia placenta.

Brown rot fungi dominate the carbon degradation of northern terrestrial conifers. These fungi adapted unique genetic inventories to degrade lignocellulose and to rapidly release a large quantity of carbohydrates for fungal catabolism. We know that brown rot involves "two-step" gene regulation to delay most hydrolytic enzyme expression until after harsh oxidative pretreatments. This implies the crucial role of concise gene regulation to brown rot efficacy, but the underlying regulatory mechanisms remain uncharacterized. Here, using the combined transcriptomic and enzyme analyses we investigated the roles of carbon catabolites in controlling gene expression in model brown rot fungus Rhodonia placenta. We identified co-regulated gene regulons as shared transcriptional responses to no-carbon controls, glucose, cellobiose, or aspen wood (Populus sp.). We found that cellobiose, a common inducing catabolite for fungi, induced expression of main chain-cleaving cellulases in GH5 and GH12 families (cellobiose vs. no-carbon > 4-fold, Padj < 0.05), whereas complex aspen was a universal inducer for Carbohydrate Active Enzymes (CAZymes) expression. Importantly, we observed the attenuated glucose-mediated repression effects on cellulases expression, but not on hemicellulases and lignin oxidoreductases, suggesting fungi might have adapted diverged regulatory routes to boost cellulase production for the fast carbohydrate release. Using carbon regulons, we further predicted the cis- and trans-regulatory elements and assembled a network model of the distinctive regulatory machinery of brown rot. These results offer mechanistic insights into the energy efficiency traits of a common group of decomposer fungi with enormous influence on the carbon cycle.

Elamipretide (SS-31) treatment attenuates age-associated post-translational modifications of heart proteins.

It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes. Aging led to an increase in oxidation of protein thiols demonstrated by increased S-glutathionylation of cysteine residues on proteins from Old (24 months old at the start of the study) mouse hearts compared to Young (5-6 months old). This shift in the oxidation state of the proteome was almost completely reversed by 8 weeks of treatment with elamipretide. Many of the significant changes that occurred were in proteins involved in mitochondrial or cardiac function. We also found changes in the mouse heart phosphoproteome that were associated with age, some of which were partially restored with elamipretide treatment. Parallel reaction monitoring of a subset of phosphorylation sites revealed that the unmodified peptide reporting for Myot S231 increased with age, but not its phosphorylated form and that both phosphorylated and unphosphorylated forms of the peptide covering cMyBP-C S307 increased, but that elamipretide treatment did not affect these changes. These results suggest that changes to thiol redox state and phosphorylation status are two ways in which age may affect mouse heart function, which can be restored by treatment with elamipretide.

Mass spectrometry-based direct detection of multiple types of protein thiol modifications in pancreatic beta cells under endoplasmic reticulum stress.

Thiol-based post-translational modifications (PTMs) play a key role in redox-dependent regulation and signaling. Functional cysteine (Cys) sites serve as redox switches, regulated through multiple types of PTMs. Herein, we aim to characterize the complexity of thiol PTMs at the proteome level through the establishment of a direct detection workflow. The LC-MS/MS based workflow allows for simultaneous quantification of protein abundances and multiple types of thiol PTMs. To demonstrate its utility, the workflow was applied to mouse pancreatic ß-cells (ß-TC-6) treated with thapsigargin to induce endoplasmic reticulum (ER) stress. This resulted in the quantification of >9000 proteins and multiple types of thiol PTMs, including intra-peptide disulfide (S-S), S-glutathionylation (SSG), S-sulfinylation (SO2H), S-sulfonylation (SO3H), S-persulfidation (SSH), and S-trisulfidation (SSSH). Proteins with significant changes in abundance were observed to be involved in canonical pathways such as autophagy, unfolded protein response, protein ubiquitination pathway, and EIF2 signaling. Moreover, ~500 Cys sites were observed with one or multiple types of PTMs with SSH and S-S as the predominant types of modifications. In many cases, significant changes in the levels of different PTMs were observed on various enzymes and their active sites, while their protein abundance exhibited little change. These results provide evidence of independent translational and post-translational regulation of enzyme activity. The observed complexity of thiol modifications on the same Cys residues illustrates the challenge in the characterization and interpretation of protein thiol modifications and their functional regulation.

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation.

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

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.

Enhancement of polyhydroxyalkanoate production by co-feeding lignin derivatives with glycerol in Pseudomonas putida KT2440.

BACKGROUND: Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy-co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440-for the first time, which simultaneously improved both cell biomass and PHA production. RESULTS: Co-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4-16.1% and PHA content by 29.0-63.2%, respectively, compared with feeding glycerol alone. GC-MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long-chain monomers (C10 and C12) by 0.4-4.4% and increased the distribution of short-chain monomers (C6 and C8) by 0.8-3.5%. The 1H-13C HMBC, 1H-13C HSQC, and 1H-1H COSY NMR analysis confirmed that the PHA monomers (C6-C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, 1H, 13C and 31P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD+/NADH and NADPH/NADP+ ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner-Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid ß-oxidation, and PHA biosynthesis. CONCLUSIONS: This work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin break-down products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favours further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.

Characterization of cellular oxidative stress response by stoichiometric redox proteomics.

The thiol redox proteome refers to all proteins whose cysteine thiols are subjected to various redox-dependent posttranslational modifications (PTMs) including S-glutathionylation (SSG), S-nitrosylation (SNO), S-sulfenylation (SOH), and S-sulfhydration (SSH). These modifications can impact various aspects of protein function such as activity, binding, conformation, localization, and interactions with other molecules. To identify novel redox proteins in signaling and regulation, it is highly desirable to have robust redox proteomics methods that can provide global, site-specific, and stoichiometric quantification of redox PTMs. Mass spectrometry (MS)-based redox proteomics has emerged as the primary platform for broad characterization of thiol PTMs in cells and tissues. Herein, we review recent advances in MS-based redox proteomics approaches for quantitative profiling of redox PTMs at physiological or oxidative stress conditions and highlight some recent applications. Considering the relative maturity of available methods, emphasis will be on two types of modifications: 1) total oxidation (i.e., all reversible thiol modifications), the level of which represents the overall redox state, and 2) S-glutathionylation, a major form of reversible thiol oxidation. We also discuss the significance of stoichiometric measurements of thiol PTMs as well as future perspectives toward a better understanding of cellular redox regulatory networks in cells and tissues.

Protein thiol oxidation in the rat lung following e-cigarette exposure.

E-cigarette (e-cig) aerosols are complex mixtures of various chemicals including humectants (propylene glycol (PG) and vegetable glycerin (VG)), nicotine, and various flavoring additives. Emerging research is beginning to challenge the "relatively safe" perception of e-cigarettes. Recent studies suggest e-cig aerosols provoke oxidative stress; however, details of the underlying molecular mechanisms remain unclear. Here we used a redox proteomics assay of thiol total oxidation to identify signatures of site-specific protein thiol modifications in Sprague-Dawley rat lungs following in vivo e-cig aerosol exposures. Histologic evaluation of rat lungs exposed acutely to e-cig aerosols revealed mild perturbations in lung structure. Bronchoalveolar lavage (BAL) fluid analysis demonstrated no significant change in cell count or differential. Conversely, total lung glutathione decreased significantly in rats exposed to e-cig aerosol compared to air controls. Redox proteomics quantified the levels of total oxidation for 6682 cysteine sites representing 2865 proteins. Protein thiol oxidation and alterations by e-cig exposure induced perturbations of protein quality control, inflammatory responses and redox homeostasis. Perturbations of protein quality control were confirmed with semi-quantification of total lung polyubiquitination and 20S proteasome activity. Our study highlights the importance of redox control in the pulmonary response to e-cig exposure and the utility of thiol-based redox proteomics as a tool for elucidating the molecular mechanisms underlying this response.

Stochiometric quantification of the thiol redox proteome of macrophages reveals subcellular compartmentalization and susceptibility to oxidative perturbations.

Posttranslational modifications of protein cysteine thiols play a significant role in redox regulation and the pathogenesis of human diseases. Herein, we report the characterization of the cellular redox landscape in terms of quantitative, site-specific occupancies of both S-glutathionylation (SSG) and total reversible thiol oxidation (total oxidation) in RAW 264.7 macrophage cells under basal conditions. The occupancies of thiol modifications for ~4000 cysteine sites were quantified, revealing a mean site occupancy of 4.0% for SSG and 11.9% for total oxidation, respectively. Correlations between site occupancies and structural features such as pKa, relative residue surface accessibility, and hydrophobicity were observed. Proteome-wide site occupancy analysis revealed that the average occupancies of SSG and total oxidation in specific cellular compartments correlate well with the expected redox potentials of respective organelles in macrophages, consistent with the notion of redox compartmentalization. The lowest average occupancies were observed in more reducing organelles such as the mitochondria (non-membrane) and nucleus, while the highest average occupancies were found in more oxidizing organelles such as endoplasmic reticulum (ER) and lysosome. Furthermore, a pattern of subcellular susceptibility to redox changes was observed under oxidative stress induced by exposure to engineered metal oxide nanoparticles. Peroxisome, ER, and mitochondria (membrane) are the organelles which exhibit the most significant redox changes; while mitochondria (non-membrane) and Golgi were observed as the organelles being most resistant to oxidative stress. Finally, it was observed that Cys residues at enzymatic active sites generally had a higher level of occupancy compared to non-active Cys residues within the same proteins, suggesting site occupancy as a potential indicator of protein functional sites. The raw data are available via ProteomeXchange with identifier PXD019913.

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.