Protein Repair Enzyme Research Articles

Adaptation mechanisms of Clostridioides difficile to auranofin and its impact on human gut microbiota

Auranofin (AF), a former rheumatoid polyarthritis treatment, gained renewed interest for its use as an antimicrobial. AF is an inhibitor of thioredoxin reductase (TrxB), a thiol and protein repair enzyme, with an antibacterial activity against several bacteria including C. difficile, an enteropathogen causing post-antibiotic diarrhea. Several studies demonstrated the effect of AF on C. difficile physiology, but the crucial questions of resistance mechanisms and impact on microbiota remain unaddressed. We explored potential resistance mechanisms by studying the impact of TrxB multiplicity and by generating and characterizing adaptive mutations. We showed that if mutants inactivated for trxB genes have a lower MIC of AF, the number of TrxBs naturally present in clinical strains does not impact the MIC. All stable mutations isolated after AF long-term exposure were in the anti-sigma factor of σB and strongly affect physiology. Finally, we showed that AF has less impact on human gut microbiota than vancomycin.

PROTEIN L‐ISOASPARTYL METHYLTRANSFERASE protects enolase dysfunction by repairing isoaspartyl‐induced damage and is positively implicated in agronomically important seed traits

SUMMARYThe protein‐repairing enzyme (PRE) PROTEIN L‐ISOASPARTYL METHYLTRANSFERASE (PIMT) influences seed vigor by repairing isoaspartyl‐mediated protein damage in seeds. However, PIMTs function in other seed traits, and the mechanisms by which PIMT affects such seed traits are still poorly understood. Herein, through molecular, biochemical, and genetic studies using overexpression and RNAi lines in Oryza sativa and Arabidopsis thaliana, we demonstrate that PIMT not only affects seed vigor but also affects seed size and weight by modulating enolase (ENO) activity. We have identified ENO2, a glycolytic enzyme, as a PIMT interacting protein through Y2H cDNA library screening, and this interaction was further validated by BiFC and co‐immunoprecipitation assay. We show that mutation or suppression of ENO2 expression results in reduced seed vigor, seed size, and weight. We also proved that ENO2 undergoes isoAsp modification that affects its activity in both in vivo and in vitro conditions. Further, using MS/MS analyses, amino acid residues that undergo isoAsp modification in ENO2 were identified. We also demonstrate that PIMT repairs such isoAsp modification in ENO2 protein, protecting its vital cellular functions during seed maturation and storage, and plays a vital role in regulating seed size, weight, and seed vigor. Taken together, our study identified ENO2 as a novel substrate of PIMT, and both ENO2 and PIMT in turn implicate in agronomically important seed traits.

Evaluation of supplemented protein-L-isoaspartate-O-methyltransferase (PIMT) gene of Carica papaya and Ricinus communis in stress survival of Escherichia coli BL21(DE3) cells

In growing plant population, effect of stress is a perturb issue affecting its physiological, biochemical, yield loss and developmental growth. Protein-L-isoaspartate-O-methyltransferase (PIMT) is a broadly distributed protein repair enzyme which actuate under stressful environment or aging. Stress can mediate damage converting protein bound aspartate (Asp) residues to isoaspartate (iso-Asp). This spontaneous and deleterious conversion occurs at an elevated state of stress and aging. Iso-Asp formation is associated with protein inactivation and compromised cellular survival. PIMT can convert iso-Asp back to Asp, thus repairing and contributing to cellular survival. The present work describes the isolation, cloning, sequencing and expression of PIMT genes of Carica papaya (Cp pimt) and Ricinus communis (Rc pimt) Using gene specific primers, both the pimts were amplified from their respective cDNAs and subsequently cloned in prokaryotic expression vector pProEXHTa. BL21(DE3) strain of E. coli cells were used as expression host. The expression kinetics of both the PIMTs were studied with various concentrations of IPTG and at different time points. Finally, the PIMT supplemented BL21(DE3) cells were evaluated against different stresses in comparison to their counterparts with the empty vector control.

Pan msr gene deleted strain of Salmonella Typhimurium suffers oxidative stress, depicts macromolecular damage and attenuated virulence

Salmonella encounters but survives host inflammatory response. To defend host-generated oxidants, Salmonella encodes primary antioxidants and protein repair enzymes. Methionine (Met) residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO) which compromises protein functions and subsequently cellular survival. However, by reducing Met-SO to Met, methionine sulfoxide reductases (Msrs) enhance cellular survival under stress conditions. Salmonella encodes five Msrs which are specific for particular Met-SO (free/protein bound), and ‘R’/‘S’ types. Earlier studies assessed the effect of deletions of one or two msrs on the stress physiology of S. Typhimurium. We generated a pan msr gene deletion (Δ5msr) strain in S. Typhimurium. The Δ5msr mutant strain shows an initial lag in in vitro growth. However, the Δ5msr mutant strain depicts very high sensitivity (p < 0.0001) to hypochlorous acid (HOCl), chloramine T (ChT) and superoxide-generating oxidant paraquat. Further, the Δ5msr mutant strain shows high levels of malondialdehyde (MDA), protein carbonyls, and protein aggregation. On the other side, the Δ5msr mutant strain exhibits lower levels of free amines. Further, the Δ5msr mutant strain is highly susceptible to neutrophils and shows defective fitness in the spleen and liver of mice. The results of the current study suggest that the deletions of all msrs render S. Typhimurium highly prone to oxidative stress and attenuate its virulence.

Protein l-isoAspartyl Methyltransferase (PIMT) and antioxidants in plants.

All life forms, including plants, accumulate reactive oxygen species (ROS) as a byproduct of metabolism; however, environmental stresses, including abiotic stresses and pathogen attacks, cause enhanced accumulation of ROS in plants. The increased accumulation of ROS often causes oxidative damage to cells. Organisms are able to maintain levels of ROS below permissible limits by several mechanisms, including efficient antioxidant systems. In addition to antioxidant systems, recent studies suggest that protein l-isoaspartyl methyltransferase (PIMT), a highly conserved protein repair enzyme across evolutionary diverse organisms, plays a critical role in maintaining ROS homeostasis by repairing isoaspartyl-mediated damage to antioxidants in plants. Under stress conditions, antioxidant proteins undergo spontaneous isoaspartyl (isoAsp) modification which is often detrimental to protein structure and function. This reduces the catalytic action of antioxidants and disturbs the ROS homeostasis of cells. This chapter focuses on PIMT and its interaction with antioxidants in plants, where PIMT constitutes a secondary level of protection by shielding a primary level of antioxidants from dysfunction and permitting them to guard during unfavorable situations.

In vitro Anti-malignant Property of PCMT1 Silencing and Identification of the SNHG16/miR-195/PCMT1 Regulatory Axis in Breast Cancer Cells

BackgroundProtein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a highly conserved protein repair enzyme that participates in regulating the progression of human cancers. We therefore studied the function and the related mechanisms of PCMT1 in breast cancer cells. MethodsExpression profile and prognostic analysis of PCMT1 in breast cancer patients were analyzed using online databases. PCMT1 expression in breast cancer cells was detected by western blot analysis. Cell proliferation was determined by CCK-8 and colony formation assays. Apoptosis was evaluated using flow cytometry analysis and caspase-3/7 activity assay. Cell invasion was assessed by Transwell invasion assay. The small nucleolar RNA host gene 16 (SNHG16)/miR-195/PCMT1 regulatory axis was identified using bioinformatics analysis. ResultsPCMT1 expression was increased in breast cancer tissues and cells. High PCMT1 expression was correlated with poor prognosis in breast cancer patients. PCMT1 knockdown suppressed cell proliferation and colony formation ability in breast cancer cells. Moreover, PCMT1 knockdown induced apoptosis and restrained the invasive ability in breast cancer cells. PCMT1 overexpression increased the proliferative and invasive abilities of breast cancer cells. miR-195 was identified as the unique upstream miRNA of PCMT1. SNHG16 was identified as the unique upstream lncRNA of miR-195. SNHG16 knockdown downregulated PCMT1 by increasing miR-195 expression. Breast cancer cell proliferation was regulated by the SNHG16/miR-195/PCMT1 axis. ConclusionPCMT1 silencing inhibited cell proliferation and invasion and induced apoptosis in breast cancer cells and the SNHG16/miR-195/PCMT1 regulatory axis might serve as a potential therapeutic target for breast cancer.

Arabidopsis ABSCISIC ACID INSENSITIVE4 targets PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 in seed.

Arabidopsis ABSCISIC ACID INSENSITIVE4 (ABI4) positively regulates the protein repairing enzyme (PRE) PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 (PIMT1) in seed for its implication in seed vigor and longevity. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a protein repairing enzyme (PRE) and is implicated in seed vigor and longevity. PIMT has been shown to be induced by ABA, however, its detailed regulation by ABA signaling components is unknown. Herein, we report that ABSCISIC ACID INSENSITIVE4 (ABI4) directly binds to the PIMT1 promoter and regulates its expression in Arabidopsis seeds. AtPIMT1 promoter analysis demonstrated the presence of putative ABI4 binding sites. Our Y1H analysis revealed that AtABI4 transcription factor binds to the AtPIMT1 promoter. Dual luciferase assay also demonstrated the binding of the AtABI4 transcription factor to the AtPIMT1 promoter. Subsequently, we have generated AtPIMT1 promoter GUS lines and revealedthat ABA induced expression of GUS in Arabidopsis thaliana. Expression analyses exhibited reduced accumulation of PIMT1 protein and transcript with significant reduction in total PIMT activity in abi4-1 mutants as compared to that of the wild type. The AtPIMT1 promoter GUS expression in abi4-1 mutants was also found to be severely affected in both the control and ABA treatment. Hence, through molecular and genetic evidences we show that the AtABI4 plays a central role in regulating the expression of AtPIMT1 to impart seed vigor and longevity to orthodox seeds.

PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) in plants: regulations and functions.

Proteins are essential molecules that carry out key functions in a cell. However, as a result of aging or stressful environments, the protein undergoes a range of spontaneous covalent modifications, including the formation of abnormal l-isoaspartyl residues from aspartyl or asparaginyl residues, which can disrupt the protein's inherent structure and function. PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT: EC 2.1.1.77), an evolutionarily conserved ancient protein repairing enzyme (PRE), converts such abnormal l-isoaspartyl residues to normal l-aspartyl residues and re-establishes the protein's native structure and function. Although originally discovered in animals as a PRE, PIMT emerged as a key PRE in plants, particularly in seeds, in which PIMT plays a predominant role in preserving seed vigor and viability for prolonged periods of time. Interestingly, higher plants encode a second PIMT (PIMT2) protein which possesses a unique N-terminal extension, and exhibits several distinct features and far more complexity than non-plant PIMTs. Recent studies indicate that the role of PIMT is not restricted to preserving seed vigor and longevity but is also implicated in enhancing the growth and survivability of plants under stressful environments. Furthermore, expression studies indicate the tantalizing possibility that PIMT is involved in various physiological processes apart from its role in seed vigor, longevity and plant's survivability under abiotic stress. This review article particularly describes new insights and emerging interest in all facets of this enzyme in plants along with a concise comparative overview on isoAsp formation, and the role and regulation of PIMTs across evolutionary diverse species. Additionally, recent methods and their challenges in identifying isoaspartyl containing proteins (PIMT substrates) are highlighted.

Phenolic Extract of Seagrass, Halophila ovalis Activates Intrinsic Pathway of Apoptosis in Human Breast Cancer (MCF-7) Cells

The marine ecosystem is considered as a treasure of numerous novel biologically active molecules. We investigated the anticancer potential of the phenolic extract of Halophila ovalis in breast cancer (MCF-7) cells and characterized the possible underlying molecular mechanism. The phenolic extract (5 µl) of H. ovalis effectively inhibited the growth of MCF-7 cells. The results of DAPI staining indicated that this phenolic extract potently induces apoptosis in MCF-7 cells which was observed by increased chromatin condensation in the treated cells. An increased expression of the active fragments of an executioner caspase, caspase 3 in phenolic extract-treated MCF-7 cells further confirms this apoptosis induction. In consequence, the loss of mitochondrial membrane potential was noticed in treated cells. The protein expression analyzes show decreased expression of the anti-apoptotic protein, Bcl-2, and DNA repair enzyme, PARP in treated cells indicating the probable molecular targets of apoptosis. Further, the phenolic extract of H. ovalis blocked the antioxidant defense system in MCF-7 cells by down-regulating the protein expression of a major transcription factor, Nrf-2 and regulatory antioxidant enzymes, SOD-2 and HO-1. These results show the presence of chemopreventive compound(s) in the phenolic extract, which offers a platform for future studies to identify the active principles.

MSRB3 promotes the progression of clear cell renal cell carcinoma via regulating endoplasmic reticulum stress

BackgroundRenal cancer represents about 3 % of all human cancers. Clear cell renal cell carcinoma (ccRCC) is the main type of renal cancer. Methionine sulfoxide reductase B3 (MSRB3) is a protein repair enzyme that specifically catalyzes the reduction of methionine-R-sulfoxide residues and has an antioxidant function. However, MSRB3’s role in ccRCC is still obscure. MethodsImmunohistochemical staining and Real-time PCR were used to compare the expression level of MSRB3 in ccRCC tissues and adjacent tissues. Western blot was used to detect the expression of MSRB3 in cell lines. Chi-square test were applied to evaluate the potential of MSRB3 to function as a cancer biomarker. RNA interference was used to inhibit MSRB3 expression in ccRCC cells, followed by detecting cell proliferation, apoptosis, migration and invasion. The markers of endoplasmic reticulum stress were then detected by western blot. ResultsIn this study, we validated that MSRB3 was significantly up-regulated in ccRCC samples and cell lines. It was also demonstrated that the up-regulation of MSRB3 was associated with several clinicopathologic features. Knockdown of MSRB3 remarkably arrested the proliferation, migration and invasion, while promoted apoptosis, and induced the changes of markers of endoplasmic reticulum stress. ConclusionIn conclusion, we demonstrated that MSRB3 was an oncogene of ccRCC associated with patients’ pathological characteristics and modulated endoplasmic reticulum stress of cancer cells.

The Oxidized Protein Repair Enzymes Methionine Sulfoxide Reductases and Their Roles in Protecting against Oxidative Stress, in Ageing and in Regulating Protein Function

Cysteine and methionine residues are the amino acids most sensitive to oxidation by reactive oxygen species. However, in contrast to other amino acids, certain cysteine and methionine oxidation products can be reduced within proteins by dedicated enzymatic repair systems. Oxidation of cysteine first results in either the formation of a disulfide bridge or a sulfenic acid. Sulfenic acid can be converted to disulfide or sulfenamide or further oxidized to sulfinic acid. Disulfide can be easily reversed by different enzymatic systems such as the thioredoxin/thioredoxin reductase and the glutaredoxin/glutathione/glutathione reductase systems. Methionine side chains can also be oxidized by reactive oxygen species. Methionine oxidation, by the addition of an extra oxygen atom, leads to the generation of methionine sulfoxide. Enzymatically catalyzed reduction of methionine sulfoxide is achieved by either methionine sulfoxide reductase A or methionine sulfoxide reductase B, also referred as to the methionine sulfoxide reductases system. This oxidized protein repair system is further described in this review article in terms of its discovery and biologically relevant characteristics, and its important physiological roles in protecting against oxidative stress, in ageing and in regulating protein function.

Mechanisms of Persistence of the Ammonia-Oxidizing Bacteria Nitrosomonas to the Biocide Free Nitrous Acid.

Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of wastewater treatment processes (WWTPs), nitrite-oxidizing bacteria (NOB) are more susceptible to FNA compared to ammonia-oxidizing bacteria (AOB). This selective inhibition of NOB over AOB in WWTPs bypasses nitrate production and improves the efficiency and costs of the nitrogen removal process in both the activated sludge and anaerobic ammonium oxidation (Anammox) system. However, the molecular mechanisms governing this atypical tolerance of AOB to FNA have yet to be understood. Herein we investigate the varying effects of the antimicrobial FNA on activated sludge containing AOB and NOB using an integrated metagenomics and label-free quantitative sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) metaproteomic approach. The Nitrosomonas genus of AOB, on exposure to FNA, maintains internal homeostasis by upregulating a number of known oxidative stress enzymes, such as pteridine reductase and dihydrolipoyl dehydrogenase. Denitrifying enzymes were upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Interestingly, proteins involved in stress response mechanisms, such as DNA and protein repair enzymes, phage prevention proteins, and iron transport proteins, were upregulated on exposure to FNA. In addition enzymes involved in energy generation were also upregulated on exposure to FNA. The total proteins specifically derived from the NOB genus Nitrobacter was low and, as such, did not allow for the elucidation of the response mechanism to FNA exposure. These findings give us an understanding of the adaptive mechanisms of tolerance within the AOB Nitrosomonas to the biocidal agent FNA.

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.

Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation

Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically catalyzes the reduction of methionine-R-sulfoxide residues and has an antioxidant function. We have previously shown that depletion of MsrB3 suppresses the proliferation of normal mammalian cells by arresting cell cycle. In this study, we report the crucial role of MsrB3 in cancer cell death. Deficiency of MsrB3 induced cancer cell death, while MsrB3 overexpression stimulated cancer cell proliferation. MsrB3 depletion resulted in apoptotic cancer cell death through the activation of the intrinsic mitochondrial pathway. MsrB3 deficiency increased the levels of cellular reactive oxygen species (ROS) and led to redox imbalance, and also increased the Bax to Bcl-2 ratio and cytochrome c release, leading to caspase activation. Treatment of MsrB3-depleted cells with N-acetylcysteine, an ROS scavenger, prevented cell death, suggesting that MsrB3 deficiency-induced cell death is associated with increased ROS production. In addition, MsrB3 depletion activated poly(ADP ribose) polymerase-1 (PARP-1) and led to the translocation of apoptosis-inducing factor (AIF) to the nucleus. Taken together, our results suggest that MsrB3 plays an important role in cancer cell survival through the modulation of the intrinsic apoptosis pathway.

184 - Redox-Markers in the European MARK-AGE Study

Redox markers have been associated with age and disease status in various human studies. It is known that life expectancy is increasing, whereas health span, i.e. the disease-free period in a person’s life, remains constant. This means that although humans reach higher age, the disease-ridden period may prevail. Biomarkers reflecting ageing and health span are of increasing interest to monitor and possibly even intervene in these processes. However, so far redox markers have never been assessed in a large number of samples in the general healthy population in relation to age. We here present results on oxidative stress markers of the European MARK-AGE study, an observational cross-sectional study in seven countries with 3,000 participants aiming to identify a set of biomarkers of ageing. The study comprised three different groups: 1) subjects aged 35-74 recruited in an age-stratified manner from the healthy general population (HG; n = 2,200), 2) offspring from centenarians (CO; n = 500) as well as 3) spouses of centenarian-offspring (SCO; n = 300). Our results indicate that the three groups differ significantly in biomarkers of protein oxidation, lipid peroxidation, glycation and also the activity of protein degradation systems and repair enzymes. Interestingly, none of the analyzed parameters differed significantly between CO and SCO. In contrast, most parameters were statistically significantly different between HG and CO as well as between HG and SCO. Since CO and SCO did not differ in any of the assessed biomarkers, lifestyle is one likely contributor for the altered redox status, offering a promising approach for intervention studies. However, before intervention studies are carried out, the reason for the altered redox markers in these groups should be further investigated as they might also be influenced by metabolism and genetics.

215 - Methionine Sulfoxide Reductase B3 Deficiency Stimulates Heme Oxygenase-1 Expression Via ROS-Dependent and Nrf2 Activation Pathways

Methionine sulfoxide reductase B3 (MsrB3), which is primarily found in the endoplasmic reticulum (ER), is an important protein repair enzyme that stereospecifically reduces methionine- R -sulfoxide residues. We previously found that MsrB3 deficiency arrests the cell cycle at the G 1 /S stage through up-regulation of p21 and p27. In this study, we report a critical role of MsrB3 in gene expression of heme oxygenase-1 (HO-1), which has an anti-proliferative effect associated with p21 up-regulation. Depletion of MsrB3 elevated HO-1 expression in mammalian cells, whereas MsrB3 overexpression had no effect. MsrB3 deficiency increased cellular reactive oxygen species (ROS), particularly in the mitochondria. ER stress, which is associated with up-regulation of HO-1, was also induced by depletion of MsrB3. Treatment with N -acetylcysteine as an ROS scavenger reduced augmented HO-1 levels in MsrB3-depleted cells. MsrB3 deficiency activated Nrf2 transcription factor by enhancing its expression and nuclear import. The activation of Nrf2 induced by MsrB3 depletion was confirmed by increased expression levels of its other target genes, such as γ-glutamylcysteine ligase. Taken together, these data suggest that MsrB3 attenuates HO-1 induction by inhibiting ROS production, ER stress, and Nrf2 activation.

Methionine sulfoxide reductase B3 deficiency stimulates heme oxygenase-1 expression via ROS-dependent and Nrf2 activation pathways

Methionine sulfoxide reductase B3 (MsrB3), which is primarily found in the endoplasmic reticulum (ER), is an important protein repair enzyme that stereospecifically reduces methionine-R-sulfoxide residues. We previously found that MsrB3 deficiency arrests the cell cycle at the G1/S stage through up-regulation of p21 and p27. In this study, we report a critical role of MsrB3 in gene expression of heme oxygenase-1 (HO-1), which has an anti-proliferative effect associated with p21 up-regulation. Depletion of MsrB3 elevated HO-1 expression in mammalian cells, whereas MsrB3 overexpression had no effect. MsrB3 deficiency increased cellular reactive oxygen species (ROS), particularly in the mitochondria. ER stress, which is associated with up-regulation of HO-1, was also induced by depletion of MsrB3. Treatment with N-acetylcysteine as an ROS scavenger reduced augmented HO-1 levels in MsrB3-depleted cells. MsrB3 deficiency activated Nrf2 transcription factor by enhancing its expression and nuclear import. The activation of Nrf2 induced by MsrB3 depletion was confirmed by increased expression levels of its other target genes, such as γ-glutamylcysteine ligase. Taken together, these data suggest that MsrB3 attenuates HO-1 induction by inhibiting ROS production, ER stress, and Nrf2 activation.

Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.

The protein l-isoaspartyl (d-aspartyl) methyltransferase protects against dopamine-induced apoptosis in neuroblastoma SH-SY5Y cells

Parkinson’s disease (PD) is the most common neurodegenerative motor disorder in the world. The main causes of neurodegeneration in PD are mitochondrial impairment and oxidative stress promoted by dopamine (DA) metabolism in the cytosol. Protein l-isoaspartyl (d-aspartyl) methyltransferase (PIMT) is a protein repair enzyme with anti-apoptotic properties. We previously reported that PIMT was downregulated at both gene and protein levels by DA-induced oxidative stresses in SH-SY5Y neuroblastoma cells. The purpose of the current study was to investigate the anti-apoptotic function of PIMT toward DA-induced cell death to better understand its specific neuroprotective role. Overexpression of wild-type PIMT, in contrast to its inactive mutant, protected SH-SY5Y cells from cell death and caspase 3 activation upon DA treatments. The intrinsic pathway of apoptosis as measured by caspase 9 activity was triggered by reactive oxygen species produced from DA metabolism, and overexpression of wild-type PIMT completely prevented caspase 9 activity stimulated by DA. In addition, cells overexpressing wild-type PIMT produced significantly less reactive oxygen species despite DA treatment compared to cells that do not overexpress PIMT. Together, these data indicate that DA-associated PIMT downregulation is an important event contributing to neuronal cell death. More importantly, the PIMT anti-apoptotic capacity seems to be dependent on its involvement in the cellular antioxidant machinery.

Methionine sulfoxide reductase B3 deficiency causes hearing loss due to stereocilia degeneration and apoptotic cell death in cochlear hair cells

Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically reduces methionine-R-sulfoxide to methionine. A recent genetic study showed that the MSRB3 gene is associated with autosomal recessive hearing loss in human deafness DFNB74. However, the precise role of MSRB3 in the auditory system and the pathogenesis of hearing loss have not yet been determined. This work is the first to generate MsrB3 knockout mice to elucidate the possible pathological mechanisms of hearing loss observed in DFNB74 patients. We found that homozygous MsrB3(-/-) mice were profoundly deaf and had largely unaffected vestibular function, whereas heterozygous MsrB3(+/-) mice exhibited normal hearing similar to that of wild-type mice. The MsrB3 protein is expressed in the sensory epithelia of the cochlear and vestibular tissues, beginning at E15.5 and E13.5, respectively. Interestingly, MsrB3 is densely localized at the base of stereocilia on the apical surface of auditory hair cells. MsrB3 deficiency led to progressive degeneration of stereociliary bundles starting at P8, followed by a loss of hair cells, resulting in profound deafness in MsrB3(-/-) mice. The hair cell loss appeared to be mediated by apoptotic cell death, which was measured using TUNEL and caspase 3 immunocytochemistry. Taken together, our data suggest that MsrB3 plays an essential role in maintaining the integrity of hair cells, possibly explaining the pathogenesis of DFNB74 deafness in humans caused by MSRB3 deficiency.