Methionine Sulfoxide Residues In Proteins Research Articles

Upregulation of cellular protective mechanisms against oxidative damage via pharmacological intervention

Oxidative damage is widely accepted as a major stress factor responsible for many age‐related diseases, as well as other degenerative conditions associated with normal aging. We initially became interested in activating or upregulating cellular protective mechanisms against oxidative damage after identifying methionine sulfoxide reductase A (MSRA), the first member of the MSR family of enzymes. The MSR system, MSRA and MSRB, reduces methionine sulfoxide residues in proteins, both the S and R epimers, back to methionine, repairing oxidative damage to proteins. MSRA can also reduce small molecules that contain a methyl sulfoxide S epimer, including the prodrug sulindac, that is converted to sulindac sulfide, the active NSAID (nonsteroidal anti‐inflammatory drug). We reported that sulindac can also protect both retinal pigmented epithelial (RPE) cells against chemical oxidative damage, and the heart against ischemia/reperfusion damage (I/R), by initiating a protective response, similar to ischemic preconditioning. This mechanism of sulindac protection was shown to be independent of its NSAID activity. In contrast, sulindac under similar conditions, sensitizes cancer cells to oxidative stress, resulting in enhanced killing of the cancer cells when they are exposed to additional stress. Because of sulindac's unique properties the National Institute of Aging recently selected sulindac to test for life span extension in mice in their Interventions Testing Program. In order to find a more active sulindac derivative that could protect cells against oxidative damage and lacked NSAID activity, we screened a series of sulindac derivatives containing a central indenyl core present in the sulindac scaffold. We have identified a compound, MCI‐100, that is not an NSAID, that is >20 fold more active than sulindac in protecting RPE cells from oxidative damage and also more active than sulindac in protecting the heart against I/R damage in a Langendorff ex‐vivo model. Additional experiments suggest that the protective mechanism of both sulndac and MCI‐100 may be related to their ability to inhibit PDE10, resulting in an increase in intracellular cGMP levels, that could initiate a pharmacological preconditioning response through PKG activation. However, unlike sulindac, MCI‐100 does not sensitize cancer cells to oxidative stress.Our eventual goal is to develop drug combinations that up‐regulate several different mechanisms that cells use to protect against oxidative damage, as a therapeutic approach to slow the progression of age related diseases, and treat other diseases that involve oxidative damage.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial gene expression in the human annulus: in vivo data from annulus cells and selectively harvested senescent annulus cells

Mitochondrial dysfunction is recognized during cell senescence and apoptosis, two important components of human disc aging/degeneration. We hypothesize that mitochondrial dysfunction is present in the degenerating and senescent annulus cells. The objective of the present study was to analyze gene expression profiles related to mitochondrial function invivo. This study had two objectives in the analysis of gene expression patterns related to mitochondria in the human annulus: First, to assess human annulus cells in a genome-wide microarray analysis approach to evaluate mitochondrial gene expression in annulus tissue from degenerated compared with healthier discs. Second, to use laser capture microdissection (LCM) to selectively isolate senescent versus nonsenescent annulus cells to evaluate their mitochondrial gene expression patterns. Following approval by our Human Subjects Institutional Review Board, annulus cells from 20 human lumbar discs were analyzed for gene groups related to mitochondrial function; a subset was also analyzed, which focused on senescent versus nonsenescent annulus cells in a study of annulus cells from 10 lumbar discs. Human annulus tissue was used in molecular studies following institutional review board approval. Gene expression levels identified with microarray analyses were statistically evaluated using GeneSifter Web-based software (VizX Labs, Seattle, WA, USA). Human annulus specimens were assessed for gene expression related to mitochondrial function. Approaches used whole annulus tissue and senescent or nonsenescent annulus cells selectively harvested using LCM. Microarray data were analyzed using gene ontology searches and GeneSifter Web-based software. Analysis of annulus cells compared mitochondrial gene expression patterns in annulus cells from more degenerated discs with patterns in annulus cells derived from healthier discs. Important findings included significant upregulation of p53 and several proapoptotic genes (including apoptosis-inducing factor, mitochondrion-associated 1, BCL2-like 11 [an apoptosis facilitator]; caspase 7 apoptosis-related cysteine peptidase; proteasome 26S subunit nonadenosine triphosphatase 10, programmed cell death 6, and reticulon 3). Methionine sulfoxide reductase (Msr), a repair enzyme that reduces methionine sulfoxide residues in proteins damaged by oxidation, was also significantly upregulated (2.02-fold increase). The gene "membrane-associated ring finger (C3HC4) 5" was significantly upregulated and relevant because it is believed to play a role in preventing cell senescence acting to regulate mitochondrial quality control. Nitric oxide synthase 3 (endothelial nitric oxide synthase [eNOS]) showed a 5.9-fold downregulation in more degenerated versus healthier annulus cells. In LCM-harvested senescent cells, Msr was significantly downregulated in senescent versus nonsenescent cells, a finding previously recognized in other types of senescent cells. Novel data showed that significant gene expression patterns are present in the human annulus related to mitochondrial dysfunction; changes were identified in important genes involving apoptosis, eNOS and Msr expressions, and solute carrier genes. Because current research efforts are focusing on bioactive compounds for mitochondria, we suggest that future biologic cell-based therapies for annulus degeneration should also consider mitochondrial-focused therapies.

Methionine sulfoxide reductase A is a stereospecific methionine oxidase

Methionine sulfoxide reductase A (MsrA) catalyzes the reduction of methionine sulfoxide to methionine and is specific for the S epimer of methionine sulfoxide. The enzyme participates in defense against oxidative stresses by reducing methionine sulfoxide residues in proteins back to methionine. Because oxidation of methionine residues is reversible, this covalent modification could also function as a mechanism for cellular regulation, provided there exists a stereospecific methionine oxidase. We show that MsrA itself is a stereospecific methionine oxidase, producing S-methionine sulfoxide as its product. MsrA catalyzes its own autooxidation as well as oxidation of free methionine and methionine residues in peptides and proteins. When functioning as a reductase, MsrA fully reverses the oxidations which it catalyzes.

Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2‐mediated oxidative stress

Methionine sulfoxide reductase A (MsrA), a member of the Msr gene family, can reduce methionine sulfoxide residues in proteins formed by oxidation of methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol-located and mitochondria-enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications.

The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species.

The PilB protein of Neisseria gonorrhoeae has been reported to be involved in the regulation of pilin gene transcription, but it also possesses significant homology to the peptide methionine sulfoxide reductase family of enzymes, specifically MsrA and MsrB from Escherichia coli. MsrA and MsrB in E. coli are able to reduce methionine sulfoxide residues in proteins to methionines. In addition, the gonococcal PilB protein encodes for both MsrA and MsrB activity associated with the repair of oxidative damage to proteins. In this work, we demonstrate that the PilB protein of Neisseria gonorrhoeae is not involved in pilus expression. Additionally, we show that wild-type N. gonorrhoeae produces two forms of this polypeptide, one of which contains a signal sequence and is secreted from the bacterial cytoplasm to the outer membrane; the other lacks a signal sequence and is cytoplasmic. Furthermore, we show that the secreted form of the PilB protein is involved in survival in the presence of oxidative damage.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins.

An enzyme that reduces methionine sulfoxide [Met(O)] residues in proteins [peptide Met(O) reductase (MsrA), EC 1.8.4.6; originally identified in Escherichia coli] was purified from bovine liver, and the cDNA encoding this enzyme was cloned and sequenced. The mammalian homologue of E. coli msrA (also called pmsR) cDNA encodes a protein of 255 amino acids with a calculated molecular mass of 25,846 Da. This protein has 61% identity with the E. coli MsrA throughout a region encompassing a 199-amino acid overlap. The protein has been overexpressed in E. coli and purified to homogeneity. The mammalian recombinant MsrA can use as substrate, proteins containing Met(O) as well as other organic compounds that contain an alkyl sulfoxide group such as N-acetylMet(O), Met(O), and dimethyl sulfoxide. Northern analysis of rat tissue extracts showed that rat msrA mRNA is present in a variety of organs with the highest level found in kidney. This is consistent with the observation that kidney extracts also contained the highest level of enzyme activity.

Enzymatic reduction of methionine sulfoxide residues in proteins and peptides

This chapter describes the purification and characteristics of an enzymatic system from Escherichia coli and human leukocytes that specifically reduces methionine sulfoxide, Met(O), residues in proteins and peptides to methionine. This enzymatic reduction can restore the biological activity of an inactive oxidized protein or peptide. In earlier studies, the assay for the presence of an enzyme, Met(O)-peptide reductase, capable of reducing Met(O) residues in proteins utilized oxidized E. coli ribosomal protein L12 as substrate. This assay is long and cumbersome and the substrate is not readily available. Because it has now been established that Met(O)-enkephalin could serve as a substrate, other Met(O)-containing compounds were tested in the study presented in the chapter. One of these, N-acetylMet(O), was found to be a good substrate and the product of the reaction, N-acetylMet, could be rapidly quantitated. The chapter shows the effect of protein concentration on the synthesis of N-acetylmethionine. The rate of reduction of N-acetylMet(O) by Met(O)-peptide reductase increases linearly with protein concentration.

Oxidation of methionine residues in proteins of activated human neutrophils.

A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins.

Biochemistry and physiological role of methionine sulfoxide residues in proteins

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability.