Brian McDonagh

Differential cysteine labeling and global label-free proteomics reveals an altered metabolic state in skeletal muscle aging.

The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054.

Ubiquitination and carbonylation as markers of oxidative-stress in Ruditapes decussatus.

Environmental pollutants, such as metals, are widespread in aquatic environments and can lead to the formation of reactive oxygen species (ROS). ROS are highly toxic in marine species since they can cause serious reversible and irreversible changes in proteins including ubiquitination and modifications such as carbonylation. This study aimed to confirm the potential of ubiquitination and carbonylation as markers of oxidative stress in the clam Ruditapes decussatus (Veneroida, Veneridae) exposed to cadmium (40 microg/L). After 21 days of exposure clams were dissected into gills and digestive gland. Cytosolic proteins of both tissues were separated by two-dimensional electrophoresis (2-D SDS-PAGE) and analysed by immunobloting. Higher ubiquitination and carbonylation levels were in digestive gland of contaminated organisms. These results confirm the potential of ubiquitination and carbonylation as a sensitive and specific marker of oxidative stress in marine bivalves. In this approach, changes in protein structure provide options for affinity selection of sub-proteomes for 2D SDS-PAGE, simplifying the detection of protein biomarkers using proteomic approach.

Effects of oxidative stress on protein thiols and disulphides in Mytilus edulis revealed by proteomics: Actin and protein disulphide isomerase are redox targets

Many proteins contain cysteines which are sensitive to oxidation. This is sometimes reversible through interaction with glutathione, glutaredoxin or thioredoxin systems making these proteins potential sensors of oxidative stress. In this study we analysed whether there was an increase in mixed disulphide bond (-S-S-) formation in the blue mussel Mytilus edulis in response to menadione. This was achieved by initially blocking reduced thiols with N-ethylmaleimide, -S-S- were then reduced with dithiothreitol (DTT) and labelled with 5-iodoacetamidofluorescein (5-IAF). Free -SHs were also labelled directly with 5-IAF. Separations were performed on 1D or 2D SDS PAGE and images analysed. There was an increase in -S-S- in response to menadione and detection of changes in oxidised proteins was easier than that of changes in the amount of reduced proteins. Protein disulphide isomerase (PDI) was labelled both as -SH and -S-S-, underlining its involvement in the redox status of the animal. A glutathione transferase (GST P1-1) forms an inter-chain disulphide bridge in response to menadione.

Application of redox proteomics to skeletal muscle aging and exercise

Skeletal muscle represents a physiologically relevant model for the application of redox proteomic techniques to dissect its response to exercise and aging. Contracting skeletal muscles generate ROS (reactive oxygen species) and RNS (reactive nitrogen species) necessary for the regulation of many proteins involved in excitation-contraction coupling. The magnitude and species of ROS/RNS generated by contracting muscles will have downstream effects on specific protein targets and cellular redox signalling. Redox modifications on specific proteins are essential for the adaptive response to exercise and skeletal muscle can develop a dysregulated redox response during aging. In the present article, we discuss how redox proteomics can be applied to identify and quantify the reversible modifications on susceptible cysteine residues within those redox-sensitive proteins, and the integration of oxidative and non-oxidative protein modifications in relation to the functional proteome.

Proteomic Profiling of Perturbed Protein Sulfenation in Renal Medulla of the Spontaneously Hypertensive Rat

Protein sulfenic acids have been proposed as potential biochemical switches for redox signaling. This post-translational modification (PTM) is readily reversible, in contrast to some other types of oxidative PTM. Enhanced oxidative stress has been reported as a feature of hypertension, and renal function has been implicated in the development and progression of the disease in animal models such as the spontaneously hypertensive rat (SHR). However, reactive oxygen species (ROS) are also signaling molecules and may play a role in vascular function. To investigate protein sulfenation under hypertensive conditions, we examined protein extracts of SHR kidney medulla in comparison to medulla from normotensive Wistar rats. Total free thiol content of the SHR medulla was significantly lower than that of Wistar medulla, indicating enhanced oxidation of sulfhydryls. Protein sulfenation was also significantly greater in the medulla of hypertensive animals. Thioredoxin reductase activity was also reduced in SHR medulla and this may account, in part, for enhanced protein sulfenation. Purification of sulfenated proteins from SHR medulla revealed several proteins involved in processes such as metabolism, antioxidant defense, and regulation of nitric oxide synthase. Enhanced sulfenation may represent perturbed redox signaling in SHR medulla, or simply enhanced ROS generation.

Selection of thiol- and disulfide-containing proteins of Escherichia coli on activated thiol-Sepharose

Activated thiol-Sepharose (ATS) facilitates selection of thiol-containing proteins. In control- and menadione-treated Escherichia coli, batch selection performed under denaturing conditions revealed distinct two-dimensional electrophoresis (2DE) patterns. Using shotgun proteomics, 183 thiol-containing proteins were identified in control ATS-selected extracts and 126 were identified in menadione-treated E. coli, with 85 proteins being common to both. More than 90% of identified proteins contained one or more cysteines. Blocking with N-ethyl maleimide followed by reduction facilitated ATS-based selection of disulfide-containing proteins. In total, 62 proteins were unique to control cells and 164 were identified in menadione-treated E. coli cells, with 29 proteins being common to both. Proteins from menadione-treated cells were excised from 2DE gels, digested with trypsin, and identified by peptide mass fingerprinting. This revealed 19 unique proteins, 14 of which were identified by shotgun proteomics. Outer membrane proteins A, C, W, and X and 30S ribosomal protein S1 were found in 2DE but not by shotgun proteomics. Foldases, ribosomal proteins, aminoacyl transfer RNA (tRNA) synthetases, and metabolic and antioxidant enzymes were prominent among identified proteins, and many had previously been found to respond to, and be targets for, oxidative stress in E. coli. ATS provides a convenient and rapid way to select thiol-containing proteins.

Ubiquitination and carbonylation of proteins in the clam Ruditapes decussatus, exposed to nonylphenol using redox proteomics

Ubiquitination and carbonylation of proteins were investigated in the gill and digestive gland of Ruditapes decussatus exposed to NP (nonylphenol) (100 μgL(-1)) using redox proteomics. After 21 d of exposure, clams were dissected and cytosolic proteins of both tissues separated by 2DE SDS-PAGE. Protein expression profiles were tissue-dependent and differently affected by NP exposure. Ubiquitination and carbonylation were also tissue-specific. NP exposure induced significantly more ubiquitinated proteins in gills than in digestive glands, compared to controls. Digestive gland showed a significant higher number of carbonylated proteins than gills after NP exposure. Protein ubiquitination and carbonylation are therefore independent processes. Results showed that NP exposure generated ROS in gill and digestive gland of R. decussatus that significantly altered the proteome. Results also highlighted the advantage of using redox proteomics in the assessment of protein ubiquitination and carbonylation, which may be markers of oxidative stress in R. decussatus.

Structure and function of yeast glutaredoxin 2 depend on postranslational processing and are related to subcellular distribution.

We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H(2)O(2) as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide.

Nitrogen starvation induces extensive changes in the redox proteome of Prochlorococcus sp. strain SS120

Very low nitrogen concentration is a critical limitation in the oligotrophic oceans inhabited by the cyanobacterium Prochlorococccus, one of the main primary producers on Earth. It is well known that nitrogen starvation affects redox homeostasis in cells. We have studied the effect of nitrogen starvation on the thiol redox proteome in the Prochlorococcus sp. SS120 strain, by using shotgun proteomic techniques to map the cysteine modified in each case and to quantify the ratio of reversibly oxidized/reduced species. We identified a number of proteins showing modified cysteines only under either control or N-starvation, including isocitrate dehydrogenase and ribulose phosphate 3-epimerase. We detected other key enzymes, such as glutamine synthetase, transporters and transaminases, showing that nitrogen-related pathways were deeply affected by nitrogen starvation. Reversibly oxidized cysteines were also detected in proteins of other important metabolic pathways, such as photosynthesis, phosphorus metabolism, ATP synthesis and nucleic acids metabolism. Our results demonstrate a wide effect of nitrogen limitation on the redox status of the Prochlorococcus proteome, suggesting that besides previously reported transcriptional changes, this cyanobacterium responds with post-translational redox changes to the lack of nitrogen in its environment.

Biosynthetic and Iron Metabolism Is Regulated by Thiol Proteome Changes Dependent on Glutaredoxin-2 and Mitochondrial Peroxiredoxin-1 in Saccharomyces cerevisiae

Redoxins are involved in maintenance of thiol redox homeostasis, but their exact sites of action are only partly known. We have applied a combined redox proteomics and transcriptomics experimental strategy to discover specific functions of two interacting redoxins: dually localized glutaredoxin 2 (Grx2p) and mitochondrial peroxiredoxin 1 (Prx1p). We have identified 139 proteins showing differential postranslational thiol redox modifications when the cells do not express Grx2p, Prx1p, or both and have mapped the precise cysteines involved in each case. Some of these modifications constitute functional switches that affect metabolic and signaling pathways as the primary effect, leading to gene transcription remodeling as the secondary adaptive effect as demonstrated by a parallel high throughput gene expression analysis. The results suggest that in the absence of Grx2p, the metabolic flow toward nucleotide and aromatic amino acid biosynthesis is slowed down by redox modification of the key enzymes Rpe1p (d-ribulose-5-phosphate 3-epimerase), Tkl1p (transketolase) and Aro4p (3-deoxy-d-arabino-heptulosonate-7-phosphate synthase). The glycolytic mainstream is then diverted toward carbohydrate storage by induction of trehalose and glycogen biosynthesis genes. Porphyrin biosynthesis may also be compromised by inactivation of the redox-sensitive cytosolic enzymes Hem12p (uroporphyrinogen decarboxylase) and Sam1p (S-adenosyl methionine synthetase) and a battery of respiratory genes sensitive to low heme levels are induced. Genes of the Aft1p-dependent iron regulon were induced specifically in the absence of Prx1p despite optimal mitochondrial Fe-S biogenesis, suggesting dysfunction of the mitochondria to the cytosol signaling pathway. Strikingly, requirement of Grx2p for these events places dithiolic Grx2 in the framework of iron metabolism.