A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase (msrA) Expression for Oxidative Stress Adaptation.

Reactive oxygen species (ROS) can cause destructive damage to biological macromolecules and protein dysfunction in bacteria. Methionine sulfoxide reductase (Msr) with redox-active Cys and/or seleno-cysteine (Sec) residues can restore physiological functions of the proteome, which is essential for oxidative stress tolerance of the extremophile Deinococcus radiodurans. However, the underlying mechanism regulating MsrA enzyme activity in D. radiodurans under oxidative stress has remained elusive. Here, we identified the function of MsrA in response to oxidative stress. msrA expression in D. radiodurans was significantly upregulated under oxidative stress. The msrA mutant showed a deficiency in antioxidative capacity and an increased level of dabsyl-Met-S-SO, indicating increased sensitivity to oxidative stress. Moreover, msrA mRNA was posttranscriptionally regulated by a small RNA, DsrO. Analysis of the molecular interaction between DsrO and msrA mRNA demonstrated that DsrO increased the half-life of msrA mRNA and then upregulated MsrA enzyme activity under oxidative stress compared to the wild type. msrA expression was also transcriptionally regulated by the DNA-repairing regulator DrRRA, providing a connection for further analysis of protein restoration during DNA repair. Overall, our results provide direct evidence that DsrO and DrRRA regulate msrA expression at two levels to stabilize msrA mRNA and increase MsrA protein levels, revealing the protective roles of DsrO signaling in D. radiodurans against oxidative stress. IMPORTANCE The repair of oxidized proteins is an indispensable function allowing the extremophile D. radiodurans to grow in adverse environments. Msr proteins and various oxidoreductases can reduce oxidized Cys and Met amino acid residues of damaged proteins to recover protein function. Consequently, it is important to investigate the molecular mechanism maintaining the high reducing activity of MsrA protein in D. radiodurans during stresses. Here, we showed the protective roles of an sRNA, DsrO, in D. radiodurans against oxidative stress. DsrO interacts with msrA mRNA to improve msrA mRNA stability, and this increases the amount of MsrA protein. In addition, we also showed that DrRRA transcriptionally regulated msrA gene expression. Due to the importance of DrRRA in regulating DNA repair, this study provides a clue for further analysis of MsrA activity during DNA repair. This study indicates that protecting proteins from oxidation is an effective strategy for extremophiles to adapt to stress conditions.

Luteolin increases susceptibility to macrolides by inhibiting MsrA efflux pump in Trueperella pyogenes.

Trueperella pyogenes (T. pyogenes) is an opportunistic pathogen associated with a variety of diseases in many domestic animals. Therapeutic treatment options for T. pyogenes infections are becoming limited due to antimicrobial resistance, in which efflux pumps play an important role. This study aims to evaluate the inhibitory activity of luteolin, a natural flavonoid, on the MsrA efflux pump and investigate its mechanism. The results of antimicrobial susceptibility testing indicated that the susceptibility of msrA-positive T. pyogenes isolates to six macrolides increased after luteolin treatment, while the susceptibility of msrA-negative isolates showed no change after luteolin treatment. It is suspected that luteolin may increase the susceptibility of T. pyogenes isolates by inhibiting MsrA activity. After 1/2 MIC luteolin treatment for 36 h, the transcription level of the msrA gene and the expression level of the MsrA protein decreased by 55.0-97.7% and 36.5-71.5%, respectively. The results of an affinity test showed that the equilibrium dissociation constant (KD) of luteolin and MsrA was 6.462 × 10-5 M, and hydrogen bonding was predominant in the interaction of luteolin and MsrA. Luteolin may inhibit the ATPase activity of the MsrA protein, resulting in its lack of an energy source. The current study illustrates the effect of luteolin on MsrA in T. pyogenes isolates and provides insight into the development of luteolin as an innovative agent in combating infections caused by antimicrobial-resistant bacteria.

Sarcopenia screening in elderly with Alzheimer's disease: performances of the SARC-F-3 and MSRA-5 questionnaires.

BACKGROUND: The 3-item SARC-F (SARC-F-3) and the 5-item Mini Sarcopenia Risk Assessment (MSRA-5) questionnaires have been recently proposed to screen elderly people regarding the risk of sarcopenia. However, no studies have investigated their performances in Alzheimer's disease (AD). METHODS: We conducted a single-center observational study, including 130 consecutive AD patients (mean age: 70.71 ± 8.50 y, 54.6% women) who attended a center for neurodegenerative diseases. Sarcopenia was diagnosed using the European Working Group on Sarcopenia in Older People of 2010 (EWGSOP1) and of 2018 (EWGSOP2) criteria. Sensitivity, specificity, positive and negative likelihood ratio, and the area under the receiver operating characteristic curve (AUC) were used to assess the diagnostic performance of SARC-F-3 and MSRA-5. RESULTS: SARC-F-3 showed a sensitivity of 9.7%, a specificity of 82.8% and an AUC of 0.41 using EWGSOP1, whereas the sensitivity was of 16.7%, specificity of 84.7% and AUC of 0.58 using EWGSOP2. The MSRA-5 displayed a sensitivity of 3.2%, a specificity of 89.9% and an AUC of 0.41 using EWGSOP1, whereas sensitivity was of 0%, specificity of 91.1% and the AUC of 0.55 using EWGSOP2 criteria. The questionnaires showed a moderate agreement (Cohen's k = 0.53). CONCLUSIONS: In our sample of AD patients, a sizable number of sarcopenic individuals were misidentified by SARC-F-3 and MSRA-5, making those questionnaires unsuitable for sarcopenia screening. Considering that sarcopenia has a high prevalence in dementia and that its correct and timely identification is paramount for optimal management of patients, the development and validation of an ad-hoc sarcopenia screening tool for AD patients is highly desirable.

Recombinant Humanized IgG1 Antibody Protects against oxLDL-Induced Oxidative Stress and Apoptosis in Human Monocyte/Macrophage THP-1 Cells by Upregulation of MSRA via Sirt1-FOXO1 Axis.

Oxidized low-density lipoprotein (oxLDL)-induced oxidative stress and apoptosis are considered as critical contributors to cardiovascular diseases. Methionine sulfoxide reductase A (MSRA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. Here, we firstly provide evidence that recombinant humanized IgG1 antibody treatment upregulated the expression of MSRA in THP-1 cells to defend against oxLDL-induced oxidative stress and apoptosis. It was also observed that the upregulation of MSRA is regulated by the forkhead box O transcription factor (FOXO1), and the acetylation of FOXO1 increased when exposed to oxLDL but declined when treated with recombinant humanized IgG1 antibody. In addition, we identified that silent information regulator 1 (SIRT1) suppresses FOXO1 acetylation. Importantly, SIRT1 or FOXO1 deficiency impaired the anti-oxidative stress and anti-apoptotic effect of recombinant humanized IgG1 antibody. Together, our results suggest that recombinant humanized IgG1 antibody exerts its anti-oxidative stress and anti-apoptotic function by upregulation of MSRA via the Sirt1-FOXO1 axis.

Inhibition of Staphylococcus aureus TetK and MsrA efflux pumps by hydroxyamines derived from lapachol and norlachol.

The present study aimed to evaluate the in vitro efflux pump inhibitory capacity of hydroxyamines derived from lapachol and norlachol, where compounds 3, 4, and 5 were tested against the S. aureus strains: RN4220 carrying the pUL5054 plasmid; and IS-58, endowed with the PT181 plasmid. The substances were synthesized from 2-hydroxy-quinones, lapachol and nor-lapachol obtaining the corresponding 2-methoxylated derivatives via dimethyl sulfate alkylation in a basic medium, which then reacted chemoselectively with 2-ethanolamine and 3-propanolamine to form the corresponding amino alcohols. The antibacterial action of the substances was quantified by determining the Minimum Inhibitory Concentration (MIC), while a microdilution assay was carried out to ascertain efflux pump inhibition of Staphylococcus aureus strains carrying the MsrA macrolide and the TetK tetracycline efflux pumps with the substances at a sub-inhibitory concentration. The results were subjected to statistical analysis by an ANOVA test and Bonferroni post hoc test. The MIC from the substances exhibited a value ≥ 1024 µg/mL. However, a significant reduction (p < 0.0001) of the erythromycin, tetracycline and ethidium bromide MIC was demonstrated when these were in combination with the substances, with this effect being due to a supposed efflux pump inhibition. The tested substances demonstrated effectiveness at decreasing the MIC of erythromycin, tetracycline and ethidium bromide, potentially by inhibiting the MsrA macrolide and the TetK tetracycline efflux pumps present in the tested S. aureus strains.

Variability in the antioxidant MSRA gene affects the psychopathology of patients with anorexia nervosa.

The objective is to determine whether variability in the MSRA gene, related to obesity and several psychiatric conditions, may be relevant for psychopathological symptoms common in Anorexia Nervosa (AN) and/or for the susceptibility to the disorder. A total of 629 women (233 AN patients and 396 controls) were genotyped for 14 tag-SNPs. Psychometric evaluation was performed with the EDI-2 and SCL-90R questionnaires. Genetic associations were carried out by logistic regression controlling for age and adjusting for multiple comparisons (FDR method). Two tag-SNPs, rs11249969 and rs81442 (with a pairwise r2 value of 0.41), were associated with the global EDI-2 score, which measures EDI-related psychopathology (adjusted FDR-q = 0.02 and 0.04, respectively). Moreover, rs81442 significantly modulated all the scales of the SCL-90R test that evaluates general psychopathology (FDR-q values ranged from 4.1E-04 to 0.011). A sliding-window analysis using adjacent 3-SNP haplotypes revealed a proximal region of the MSRA gene spanning 187.8 Kbp whose variability deeply affected psychopathological symptoms of the AN patients. Depression was the symptom that showed the strongest association with any of the constructed haplotypes (FDR-q = 3.60E-06). No variants were found to be linked to AN risk or anthropometric parameters in patients or controls. Variability in the MSRA gene locus modulates psychopathology often presented by AN patients.

Impact of Intrapartum Oral Azithromycin on the Acquired Macrolide Resistome of Infants' Nasopharynx: A Randomized Controlled Trial.

In a post hoc analysis of samples from an intrapartum azithromycin randomized clinical trial, we found that children whose mothers had been treated with the drug had higher prevalence of macrolide-resistance genes msr(A) and ermC at 28 days but not at 12 months. The 2 genes were positively associated in the nasopharynx. CLINICAL TRIALS REGISTRATION: NCT1800942.

MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220.

MsrA, an efflux pump belonging to ATP-binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2-8-fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8-fold. The real-time fluorometry-based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post-antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.

Phenylpiperazine 5,5-Dimethylhydantoin Derivatives as First Synthetic Inhibitors of Msr(A) Efflux Pump in Staphylococcus epidermidis.

Herein, 15 phenylpiperazine 3-benzyl-5,5-dimethylhydantoin derivatives (1-15) were screened for modulatory activity towards Msr(A) efflux pump present in S. epidermidis bacteria. Synthesis, crystallographic analysis, biological studies in vitro and structure-activity relationship (SAR) analysis were performed. The efflux pump inhibitory (EPI) potency was determined by employing ethidium bromide accumulation assay in both Msr(A) efflux pump overexpressed (K/14/1345) and deficient (ATCC 12228) S. epidermidis strains. The series of compounds was also evaluated for the capacity to reduce the resistance of K/14/1345 strain to erythromycin, a known substrate of Msr(A). The study identified five strong modulators for Msr(A) in S. epidermidis. The 2,4-dichlorobenzyl-hydantoin derivative 9 was found as the most potent EPI, inhibiting the efflux activity in K/14/1345 at a concentration as low as 15.63 µM. Crystallography-supported SAR analysis indicated structural properties that may be responsible for the activity found. This study identified the first synthetic compounds able to inhibit Msr(A) efflux pump transporter in S. epidermidis. Thus, the hydantoin-derived molecules found can be an attractive group in search for antibiotic adjuvants acting via Msr(A) transporter.

Macrolide-lincosamide-streptogramin B resistance phenotypes and their associated genotypes in Staphylococcus aureus isolates from a tertiary level public hospital of Uruguay.

This study was undertaken to investigate the resistance phenotypes to macrolide-lincosamide-streptogramin B (MLSB) antibiotics and their associated genotypes in isolates of Staphylococcus aureus. We analyzed one hundred, consecutive, non-duplicate isolates (methicillin-susceptible MSSA, n=53 and methicillin-resistant MRSA, n=47) obtained from various clinical samples between July 2012 to December 2013. The resistance profile to MLSB antibiotics was determined by phenotypic methods and the resistance genes were detected by PCR assays. All of the isolates were subjected to pulsed-field gel electrophoresis (SmaI-PFGE). The overall prevalence of resistance to MLSB antibiotics was 38% and the resistance phenotype distribution was as follows: cMLSB, 22%; iMLSB, 10%; MSB, 5% and L, 1%. We detected ermA, ermC, ermB and mrsA/B genes in these resistant isolates. The single ermA gene was commonly observed mainly in those with a cMLSB R phenotype, whereas the combination ermA and ermC was more commonly observed in isolates with inducible expression. The patterns of SmaI-PFGE suggest a great genetic diversity in both MRSA and MSSA resistant to MLSB antibiotics. The results demonstrate the local presence of S. aureus resistant to MLSB antibiotics and its most frequently described responsible genes. Some of these isolates, especially those with the iMLSB phenotype, may be associated with therapeutic failure. Therefore, efforts should be directed to the correct detection of all MLSB resistant isolates using appropriate laboratory tests. PFGE results reveal a wide spread of resistance genes rather than the circulation of S. aureus clones resistant to MLSB antibiotics.

Enhancing disease resistance in poplar through modification of its natural defense pathway.

KEY MESSAGE: Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva. Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.

Higher Circulating Trimethylamine N-oxide Sensitizes Sevoflurane-Induced Cognitive Dysfunction in Aged Rats Probably by Downregulating Hippocampal Methionine Sulfoxide Reductase A.

Gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) has recently been shown to promote oxidative stress and inflammation in the peripheral tissues, contributing to the pathogenesis of many diseases. Here we examined whether pre-existing higher circulating TMAO would influence cognitive function in aged rats after anesthetic sevoflurane exposure. Aged rats received vehicle or TMAO treatment for 3 weeks. After 2 weeks of treatment, these animals were exposed to either control or 2.6% sevoflurane for 4 h. One week after exposure, freezing as measured by fear conditioning test, microglia activity, proinflammatory cytokine expression and NADPH oxidase-dependent reactive oxygen species (ROS) production in the hippocampus (a key brain structure involved in learning and memory) were comparable between vehicle-treated rats exposed to control and vehicle-treated rats exposed to sevoflurane. TMAO treatment, which increased plasma TMAO before and 1 week after control or sevoflurane exposure, significantly reduced freezing to contextual fear conditioning, which was associated with increases in microglia activity, proinflammatory cytokine expression and NADPH oxidase-dependent ROS production in the hippocampus in rats exposed to sevoflurane but not in rats exposed to control. Moreover, hippocampal expression of antioxidant enzyme methionine sulfoxide reductase A (MsrA) was reduced by TMAO treatment in both groups, and TMAO-induced reduction in MsrA expression was negatively correlated with increased proinflammatory cytokine expression in rats exposed to SEV. These findings suggest that pre-existing higher circulating TMAO downregulates antioxidant enzyme MsrA in the hippocampus, which may sensitize the hippocampus to oxidative stress, resulting in microglia-mediated neuroinflammation and cognitive impairment in aged rats after sevoflurane exposure.

Mobile macrolide resistance genes in staphylococci.

Macrolide resistance in staphylococci is based on the expression of a number of genes which specify four major resistance mechanisms: (i) target site modification by methylation of the ribosomal target site in the 23S rRNA, (ii) ribosome protection via ABC-F proteins, (iii) active efflux via Major Facilitator Superfamily (MFS) transporters, and (iv) enzymatic inactivation by phosphotransferases or esterases. So far, 14 different classes of erm genes, which code for 23S rRNA methylases, have been reported to occur in staphylococci from humans, animals and environmental sources. Inducible or constitutive expression of the erm genes depends on the presence and intactness of a regulatory region known as translational attenuator. The erm genes commonly confer resistance not only to macrolides, but also to lincosamides and streptogramin B compounds. In contrast, the msr(A) gene codes for an ABC-F protein which confers macrolide and streptogramin B resistance whereas the mef(A) gene codes for a Major Facilitator Superfamily protein that can export only macrolides. Enzymatic inactivation of macrolides may be due to the macrolide phosphotransferase gene mph(C) or the macrolide esterase genes ere(A) or ere(B). Many of these macrolide resistance genes are part of either plasmids, transposons, genomic islands or prophages and as such, can easily be transferred across strain, species and genus boundaries. The co-location of other antimicrobial or metal resistance genes on the same mobile genetic element facilitates co-selection and persistence of macrolide resistance genes under the selective pressure of metals or other antimicrobial agents.

Methionine sulfoxide reductase A protects hepatocytes against acetaminophen-induced toxicity via regulation of thioredoxin reductase 1 expression.

Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA-/-) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA+/+) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA-/- than in MsrA+/+ hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA-/- than in MsrA+/+ hepatocytes, while TXNRD1 depletion in both MsrA-/- and MsrA+/+ cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA-/- hepatocytes, while no significant change was observed in MsrA+/+ cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRD1 expression levels in APAP-treated MsrA-/- hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRD1 expression.

Methionine sulfoxide reductase A deficiency exacerbates acute liver injury induced by acetaminophen.

Acetaminophen (APAP) overdose induces acute liver injury via enhanced oxidative stress and glutathione (GSH) depletion. Methionine sulfoxide reductase A (MsrA) acts as a reactive oxygen species scavenger by catalyzing the cyclic reduction of methionine-S-sulfoxide. Herein, we investigated the protective role of MsrA against APAP-induced liver damage using MsrA gene-deleted mice (MsrA-/-). We found that MsrA-/- mice were more susceptible to APAP-induced acute liver injury than wild-type mice (MsrA+/+). The central lobule area of the MsrA-/- liver was more impaired with necrotic lesions. Serum alanine transaminase, aspartate transaminase, and lactate dehydrogenase levels were significantly higher in MsrA-/- than in MsrA+/+ mice after APAP challenge. Deletion of MsrA enhanced APAP-induced hepatic GSH depletion and oxidative stress, leading to increased susceptibility to APAP-induced liver injury in MsrA-deficient mice. APAP challenge increased Nrf2 activation more profoundly in MsrA-/- than in MsrA+/+ livers. Expression and nuclear accumulation of Nrf2 and its target gene expression were significantly elevated in MsrA-/- than in MsrA+/+ livers after APAP challenge. Taken together, our results demonstrate that MsrA protects the liver from APAP-induced toxicity. The data provided herein constitute the first in vivo evidence of the involvement of MsrA in hepatic function under APAP challenge.

Methionine sulfoxide reductase A protects against lipopolysaccharide-induced septic shock via negative regulation of the proinflammatory responses.

Methionine sulfoxide reductase A (MsrA) is a major antioxidant enzyme that specifically catalyzes the reduction of methionine S-sulfoxide. In this study, we used MsrA gene-knockout (MsrA-/-) mice and bone marrow-derived macrophages (BMDMs) to investigate the role of MsrA in the regulation of inflammatory responses induced by lipopolysaccharide (LPS). MsrA-/- mice were more susceptible to LPS-induced lethal shock than wild-type (MsrA+/+) mice. Serum levels of the proinflammatory cytokines IL-6 and TNF-α induced by LPS were higher in MsrA-/- than in MsrA+/+ mice. MsrA deficiency in the BMDMs also increased the LPS-induced cytotoxicity as well as TNF-α level. Basal and LPS-induced reactive oxygen species (ROS) levels were higher in MsrA-/- than in MsrA+/+ BMDMs. Phosphorylation levels of p38, JNK, and ERK were higher in MsrA-/- than in MsrA+/+ BMDMs in response to LPS, suggesting that MsrA deficiency increases MAPK activation. Furthermore, MsrA deficiency increased the expression and nuclear translocation of NF-κB and the expression of inducible nitric oxide synthase, a target gene of NF-κB, in response to LPS. Taken together, our results suggest that MsrA protects against LPS-induced septic shock, and negatively regulates proinflammatory responses via inhibition of the ROS-MAPK-NF-κB signaling pathways.

Methionine oxidation activates a transcription factor in response to oxidative stress.

Oxidant-mediated antibacterial response systems are broadly used to control bacterial proliferation. Hypochlorite (HOCl) is an important component of the innate immune system produced in neutrophils and specific epithelia. Its antimicrobial activity is due to damaging cellular macromolecules. Little is known about how bacteria escape HOCl-inflicted damage. Recently, the transcription factor YjiE was identified that specifically protects Escherichia coli from HOCl killing. According to its function, YjiE is now renamed HypT (hypochlorite-responsive transcription factor). Here we unravel that HypT is activated by methionine oxidation to methionine sulfoxide. Interestingly, so far only inactivation of cellular proteins by methionine oxidation has been reported. Mutational analysis revealed three methionines that are essential to confer HOCl resistance. Their simultaneous substitution by glutamine, mimicking the methionine sulfoxide state, increased the viability of E. coli cells upon HOCl stress. Triple glutamine substitution generates a constitutively active HypT that regulates target genes independently of HOCl stress and permanently down-regulates intracellular iron levels. Inactivation of HypT depends on the methionine sulfoxide reductases A/B. Thus, microbial protection mechanisms have evolved along the evolution of antimicrobial control systems, allowing bacteria to survive within the host environment.

Oxidant stress promotes disease by activating CaMKII.

CaMKII is activated by oxidation of methionine residues residing in the regulatory domain. Oxidized CaMKII (ox-CaMKII) is now thought to participate in cardiovascular and pulmonary diseases and cancer. This invited review summarizes current evidence for the role of ox-CaMKII in disease, considers critical knowledge gaps and suggests new areas for inquiry.

Mechanism of cleavage of alpha-synuclein by the 20S proteasome and modulation of its degradation by the RedOx state of the N-terminal methionines.

Alpha-synuclein is a small protein implicated in the pathophysiology of Parkinson's disease (PD). We have investigated the mechanism of cleavage of alpha-synuclein by the 20S proteasome. Alpha-synuclein interacts with the C8 (α7) subunit of the proteasome. The N-terminal part of alpha-synuclein (amino acids 1-60) is essential for its proteasomal degradation and analysis of peptides released from proteasomal digestion allows concluding that initial cleavages occur within the N-terminal region of the molecule. Aggregated alpha-synucleins are also degraded by the proteasome with a reduced rate, likely due to Met oxidation. In fact, mild oxidation of alpha-synuclein with H2O2 resulted in the inhibition of its degradation by the proteasome, mainly due to oxidation of Met 1 and 5 of alpha-synuclein. The inhibition was reversed by treatment of the oxidized protein with methionine sulfoxide reductases (MsrA plus MsrB). Similarly, treatment with H2O2 of N2A cells transfected with alpha-synuclein resulted in the inhibition of its degradation that was also reverted by co-transfection of MsrA plus MsrB. These results clearly indicate that oxidative stress, a common feature of PD and other synucleinopathies, promotes a RedOx change in the proteostasis of alpha-synuclein due to Met oxidation and reduced proteasomal degradation; compromised reversion of those oxidative changes would result in the accumulation of oxidative damaged alpha-synuclein likely contributing to the pathogenesis of PD.

Mechanism of 1-Cys type methionine sulfoxide reductase A regeneration by glutaredoxin.

Glutaredoxin (Grx), a major redox regulator, can act as a reductant of methionine sulfoxide reductase A (MsrA). However, the biochemical mechanisms involved in MsrA activity regeneration by Grx remain largely unknown. In this study, we investigated the regeneration mechanism of 1-Cys type Clostridium oremlandii MsrA (cMsrA) lacking a resolving Cys residue in a Grx-dependent assay. Kinetic analysis showed that cMsrA could be reduced by both monothiol and dithiol Grxs as efficiently as by in vitro reductant dithiothreitol. Our data revealed that the catalytic Cys sulfenic acid intermediate is not glutathionylated in the presence of the substrate, and that Grx instead directly formed a complex with cMsrA. Mass spectrometry analysis identified a disulfide bond between the N-terminal catalytic Cys of the active site of Grx and the catalytic Cys of cMsrA. This mixed disulfide bond could be resolved by glutathione. Based on these findings, we propose a model for regeneration of 1-Cys type cMsrA by Grx that involves no glutathionylation on the catalytic Cys of cMsrA. This mechanism contrasts with that of the previously known 1-Cys type MsrB.