A specific fluorescent probe reveals compromised activity of methionine sulfoxide reductases in Parkinson's disease.
ABSTRACT: Oxidation of methionine residues to methionine sulfoxide (MetSO) may cause changes in protein structure and function, and may eventually lead to cell damage. Methionine sulfoxide reductases (Msrs) are the only known enzymes that catalyze the reduction of MetSO back to methionine by taking reducing equivalents from the thioredoxin system, and thus protect cells from oxidative damage. Nonetheless, a lack of convenient assays for the enzymes hampers the exploration of their functions. We report the discovery of Msr-blue, the first turn-on fluorescent probe for Msr with a >100-fold fluorescence increment from screening a rationally-designed small library. Intensive studies demonstrated the specific reduction of Msr-blue by the enzymes. Msr-blue is ready to determine Msr activity in biological samples and live cells. Importantly, we disclosed a decline of Msr activity in a Parkinson's model, thus providing a mechanistic linkage between the loss of function of Msrs and the development of neurodegeneration. The strategy for the discovery of Msr-blue would also provide guidance for developing novel probes with longer excitation/emission wavelengths and specific probes for Msr isoforms.
Project description:Methionine oxidation leads to the formation of S- and R-diastereomers of methionine sulfoxide (MetSO), which are reduced back to methionine by methionine sulfoxide reductases (MSRs) A and B, respectively. MSRBs are classified in two groups depending on the conservation of one or two redox-active Cys; 2-Cys MSRBs possess a catalytic Cys-reducing MetSO and a resolving Cys, allowing regeneration by thioredoxins. The second type, 1-Cys MSRBs, possess only the catalytic Cys. The biochemical mechanisms involved in activity regeneration of 1-Cys MSRBs remain largely elusive. In the present work we used recombinant plastidial Arabidopsis thaliana MSRB1 and MSRB2 as models for 1-Cys and 2-Cys MSRBs, respectively, to delineate the Trx- and glutaredoxin-dependent reduction mechanisms. Activity assays carried out using a series of cysteine mutants and various reductants combined with measurements of free thiols under distinct oxidation conditions and mass spectrometry experiments show that the 2-Cys MSRB2 is reduced by Trx through a dithiol-disulfide exchange involving both redox-active Cys of the two partners. Regarding 1-Cys MSRB1, oxidation of the enzyme after substrate reduction leads to the formation of a stable sulfenic acid on the catalytic Cys, which is subsequently glutathionylated. The deglutathionylation of MSRB1 is achieved by both mono- and dithiol glutaredoxins and involves only their N-terminal conserved catalytic Cys. This study proposes a detailed mechanism of the regeneration of 1-Cys MSRB activity by glutaredoxins, which likely constitute physiological reductants for this type of MSR.
Project description:Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine residues in proteins. This function implicated Msrs in antioxidant defense and the regulation of aging. There are two known Msr types in animals: MsrA specific for the reduction of methionine-S-sulfoxide, and MsrB that catalyzes the reduction of methionine-R-sulfoxide. In a previous study, overexpression of MsrA in the nervous system of Drosophila was found to extend lifespan by 70%. Overexpression of MsrA in yeast also extended lifespan, whereas MsrB overexpression did so only under calorie restriction conditions. The effect of MsrB overexpression on lifespan has not yet been characterized in animal model systems. Here, the GAL4-UAS binary system was used to drive overexpression of cytosolic Drosophila MsrB and mitochondrial mouse MsrB2 in whole body, fatbody, and the nervous system of flies. In contrast to MsrA, MsrB overexpression had no consistent effect on the lifespan of fruit flies on either corn meal or sugar yeast diets. Physical activity, fecundity, and stress resistance were also similar in MsrB-overexpressing and control flies. Thus, MsrA and MsrB, the two proteins with similar function in antioxidant protein repair, have different effects on aging in fruit flies.
Project description:SIGNIFICANCE: Selenium is utilized in the methionine sulfoxide reduction system that occurs in most organisms. Methionine sulfoxide reductases (Msrs), MsrA and MsrB, are the enzymes responsible for this system. Msrs repair oxidatively damaged proteins, protect against oxidative stress, and regulate protein function, and have also been implicated in the aging process. Selenoprotein forms of Msrs containing selenocysteine (Sec) at the catalytic site are found in bacteria, algae, and animals. RECENT ADVANCES: A selenoprotein MsrB1 knockout mouse has been developed. Significant progress in the biochemistry of Msrs has been made, which includes findings of a novel reducing system for Msrs and of an interesting reason for the use of Sec in the Msr system. The effects of mammalian MsrBs, including selenoprotein MsrB1 on fruit fly aging, have been investigated. Furthermore, it is evident that Msrs are involved in methionine metabolism and regulation of the trans-sulfuration pathway. CRITICAL ISSUES: This article presents recent progress in the Msr field while focusing on the physiological roles of mammalian Msrs, functions of selenoprotein forms of Msrs, and their biochemistry. FUTURE DIRECTIONS: A deeper understanding of the roles of Msrs in redox signaling, the aging process, and metabolism will be achieved. The identity of selenoproteome of Msrs will be sought along with characterization of the identified selenoprotein forms. Exploring new cellular targets and new functions of Msrs is also warranted.
Project description:Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and together function as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes are fused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate how gene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressed the MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. For comparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a genetically engineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatory protein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymes together at 25 degrees C. A restoration of the normal protein fold is observed co-incident with the repair of MetSO in oxidized CaM (CaMox by MsrBA, as monitored by time-dependent increases in the anisotropy associated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). Underlying the efficient repair of MetSO in CaMox is the coordinate activity of the two catalytic domains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement in comparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of both domains of MsrBA permits the full repair of all MetSO in CaMox. The common expression of Msr fusion proteins in bacterial pathogens is consistent with an important role for this enzyme activity in the maintenance of protein function necessary for bacterial survival under highly oxidizing conditions associated with pathogenesis or bioremediation.
Project description:Oxidation of methionine to methionine sulfoxide is a type of posttranslational modification reversed by methionine sulfoxide reductases (Msrs), which present an exceptionally high number of gene copies in plants. The side-form general antioxidant function-specific role of each Msr isoform has not been fully studied. Thirty homologous genes of Msr type A (MsrA) and type B (MsrB) that originate from the genomes of Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa were analyzed in silico. From 109 to 201 transcription factors and responsive elements were predicted for each gene. Among the species, 220 and 190 common transcription factors and responsive elements were detected for the MsrA and MsrB isoforms, respectively. In a comparison of 14 MsrA and 16 MsrB genes, 424 transcription factors and responsive elements were reported in both types of genes, with almost ten times fewer unique elements. The transcription factors mainly comprised plant growth and development regulators, transcription factors important in stress responses with significant overrepresentation of the myeloblastosis viral oncogene homolog (MYB) and no apical meristem, Arabidopsis transcription activation factor and cup-shaped cotyledon (NAC) families and responsive elements sensitive to ethylene, jasmonate, sugar, and prolamine. Gene Ontology term-based functional classification revealed that cellular, metabolic, and developmental process terms and the response to stimulus term dominated in the biological process category. Available experimental transcriptomic and proteomic data, in combination with a set of predictions, gave coherent results validating this research. Thus, new manners Msr gene expression regulation, as well as new putative roles of Msrs, are proposed.
Project description:Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the K(m) lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.
Project description:Methionine residues and iron-sulphur (FeS) clusters are primary targets of reactive oxygen species in the proteins of micro-organisms. Here, we show that methionine redox modifications help to preserve essential FeS cluster activities in yeast. Mutants defective for the highly conserved methionine sulphoxide reductases (MSRs; which re-reduce oxidized methionines) are sensitive to many pro-oxidants, but here exhibited an unexpected copper resistance. This phenotype was mimicked by methionine sulphoxide supplementation. Microarray analyses highlighted several Cu and Fe homeostasis genes that were upregulated in the mxrDelta double mutant, which lacks both of the yeast MSRs. Of the upregulated genes, the Cu-binding Fe transporter Fet3p proved to be required for the Cu-resistance phenotype. FET3 is known to be regulated by the Aft1 transcription factor, which responds to low mitochondrial FeS-cluster status. Here, constitutive Aft1p expression in the wild-type reproduced the Cu-resistance phenotype, and FeS-cluster functions were found to be defective in the mxrDelta mutant. Genetic perturbation of FeS activity also mimicked FET3-dependent Cu resistance. 55Fe-labelling studies showed that FeS clusters are turned over more rapidly in the mxrDelta mutant than the wild-type, consistent with elevated oxidative targeting of the clusters in MSR-deficient cells. The potential underlying molecular mechanisms of this targeting are discussed. Moreover, the results indicate an important new role for cellular MSR enzymes in helping to protect the essential function of FeS clusters in aerobic settings.
Project description:The methionine sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the methionine sulfoxide in proteins back to methionine. A role of MsrA in animal aging was first demonstrated in Drosophila melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span. Subsequently, MsrA was also shown to be involved in the life span extension in Caenorhabditis elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB.
Project description:Seeds are in a natural oxidative context leading to protein oxidation. Although inevitable for proper progression from maturation to germination, protein oxidation at high levels is detrimental and associated with seed aging. Oxidation of methionine to methionine sulfoxide is a common form of damage observed during aging in all organisms. This damage is reversible through the action of methionine sulfoxide reductases (MSRs), which play key roles in lifespan control in yeast and animal cells. To investigate the relationship between MSR capacity and longevity in plant seeds, we first used two Medicago truncatula genotypes with contrasting seed quality. After characterizing the MSR family in this species, we analyzed gene expression and enzymatic activity in immature and mature seeds exhibiting distinct quality levels. We found a very strong correlation between the initial MSR capacities in different lots of mature seeds of the two genotypes and the time to a drop in viability to 50% after controlled deterioration. We then analyzed seed longevity in Arabidopsis thaliana lines, in which MSR gene expression has been genetically altered, and observed a positive correlation between MSR capacity and longevity in these seeds as well. Based on our data, we propose that the MSR repair system plays a decisive role in the establishment and preservation of longevity in plant seeds.
Project description:In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.