Protein L-isoaspartyl O-methyltransferase Research Articles

IsoAsp-Quest: Workflow development for isoAsp identification using database searches.

A recent study reported that isomerization of aspartyl residues (Asp) occurs in various tissues and proteins in vivo. For a comprehensive analysis of post-translational modifications, the MS-based proteomic approach is a straightforward method; however, the isomerization of Asp does not alter its molecular weight. Therefore, a unique method is required to analyze Asp isomers using mass spectrometry. Herein, we present a novel strategy, isoAsp-Quest, which is a database search-oriented isoAsp identification method. isoAsp is specifically converted to 18O-labeled Lα-Asp by the enzymatic reaction of protein L-isoaspartyl-O-methyltransferase (PIMT) in 18O water with a mass shift of 2 Da, which, in principle, enables us to distinguish Asp isomers. However, in practice, a labeled Lα-Asp signal overlaps with that of endogenous Lα-Asp, making detection challenging. Therefore, degradation of the endogenous Lα-Asp peptide by AspN and subsequent removal of AspN were performed prior to the PIMT reaction. This strategy was applied to bovine lens α-crystallin. Consequently, several Asp isomerization sites, consistent with human αA-crystallin, were identified in bovine αA-crystallin, indicating that this strategy is also effective for biological proteins. Therefore, isoAsp-Quest enables the analysis of Lβ-Asp in a straightforward and rapid workflow, which may be useful for the quality control of protein products and biomarker discovery.

Maize PIMT2 repairs damaged 3-METHYLCROTONYL COA CARBOXYLASE in mitochondria, affecting seed vigor.

PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) affects seed vigor by repairing damaged proteins. While PIMT is capable of isoaspartyl (isoAsp) repair in all proteins, those proteins most susceptible to isoAsp formation have not been well characterized, and the mechanisms by which PIMT affects seed vigor remain largely unknown. Using co-immunoprecipitation and LC-MS/MS, we found that maize (Zea mays) PIMT2 (ZmPIMT2) interacted predominantly with both subunits of maize 3-METHYLCROTONYL COA CARBOXYLASE (ZmMCC). ZmPIMT2 is specifically expressed in the maize embryo. Both mRNA and protein levels of ZmPIMT2 increased during seed maturation and declined during imbibition. Maize seed vigor was decreased in the zmpimt2 mutant line, while overexpression of ZmPIMT2 in maize and Arabidopsis thaliana increased seed vigor upon artificial aging. ZmPIMT2 was localized in the mitochondria, as determined by subcellular localization assays using maize protoplasts. ZmPIMT2 binding to ZmMCCα was confirmed by luciferase complementation tests in both tobacco (Nicotiana benthamiana) leaves and maize protoplasts. Knockdown of ZmMCCα decreased maize seed aging tolerance. Furthermore, overexpression of ZmPIMT2 decreased the accumulation of isoAsp of ZmMCCα protein in seed embryos that underwent accelerated aging treatment. Taken together, our results demonstrate that ZmPIMT2 binds ZmMCCα in mitochondria, repairs isoAsp damage, and positively affects maize seed vigor.

PIMT is a novel and potent suppressor of endothelial activation.

Proinflammatory agonists provoke the expression of cell surface adhesion molecules on endothelium in order to facilitate leukocyte infiltration into tissues. Rigorous control over this process is important to prevent unwanted inflammation and organ damage. Protein L-isoaspartyl O-methyltransferase (PIMT) converts isoaspartyl residues to conventional methylated forms in cells undergoing stress-induced protein damage. The purpose of this study was to determine the role of PIMT in vascular homeostasis. PIMT is abundantly expressed in mouse lung endothelium and PIMT deficiency in mice exacerbated pulmonary inflammation and vascular leakage to LPS(lipopolysaccharide). Furthermore, we found that PIMT inhibited LPS-induced toll-like receptor signaling through its interaction with TNF receptor-associated factor 6 (TRAF6) and its ability to methylate asparagine residues in the coiled-coil domain. This interaction was found to inhibit TRAF6 oligomerization and autoubiquitination, which prevented NF-κB transactivation and subsequent expression of endothelial adhesion molecules. Separately, PIMT also suppressed ICAM-1 expression by inhibiting its N-glycosylation, causing effects on protein stability that ultimately translated into reduced EC(endothelial cell)-leukocyte interactions. Our study has identified PIMT as a novel and potent suppressor of endothelial activation. Taken together, these findings suggest that therapeutic targeting of PIMT may be effective in limiting organ injury in inflammatory vascular diseases.

Abstract 12458: Protein L-isoaspartyl O-methyltransferase Attenuates Endothelial Activation Through Inhibitory Interaction With TRAF6 and ICAM1

Introduction: Endothelial activation provokes expression of cell surface adhesion molecules to facilitate circulating leukocyte diapedesis, which demands rigorous surveillance to prevent excessive inflammation response and host damage. As a repair enzyme, protein L-isoaspartyl O-methyltransferase (PIMT) methylates and converts isoaspartyl (isoAsp) residuals back to conventional form to avoid protein damage and aging. However, the role of PIMT in vascular homeostasis remains to be delineated. Methods and Results: Murine acute lung injury (ALI) model was deployed to investigate the significance of PIMT in endothelial activation and vascular inflammation. Results showed that mice harboring heterozygous deletion of PIMT displayed exacerbated pulmonary vascular inflammation. Ectopic overexpression of PIMT protected endothelial cells against endotoxin stimulated endothelial activation, as characterized by attenuated expression of inflammatory cytokines, chemokines, cell adhesion molecules and monocytic adherence to activated endothelial cells. Mechanistically, we found that PIMT inhibits lipopolysaccharide (LPS)-induced endothelial NF-κB transactivation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) was identified as a dynamic binding partner and substrate of PIMT in regulating NF-κB signaling. PIMT catalyzed methylation at an asparagine derived isoAsp residual of TRAF6 directly prevented its oligomerization and auto-ubiquitination. Strikingly, we also found that PIMT specifically impeded N-glycosylation at multiple sites of endothelial intercellular adhesion molecule 1 (ICAM1). The hypo-glycosylated ICAM1 elicited impaired interaction with cell skeletal and reduced stability, which further diminished endothelial activation. Conclusions: PIMT negatively modulates endothelial activation through restricting activation of TRAF6 and inhibiting N-glycosylation of adhesion molecules. Our study provides a novel insight into the role of protein O-methylation in regulating endothelial inflammation and suggest that targeting PIMT may have potential therapeutic applications in treating inflammatory vascular disorders.

A DFT calculation on nonenzymatic degradation of isoaspartic residue.

βAsp is an isomer of Asp that can be formed by either deamidation of Asn or isomerization of Asp and known as biological clock. The presence of βAsp affects the proteolytic stability of the protein. Formation of the isomerized Asp plays a diverse and crucial role in aging, cancer, autoimmune, neurodegenerative, and other diseases. A number of methods have been developed to detect βAsp, and they are usually used in conjunction. Because of identical mass, differentiation of βAsp and Asp residues is challenged. Degradation of βAsp is still unclear and needed to be explored. The energetics and mechanism of five possible pathways for cleavages at βAsp in peptide model have been investigated by DFT/B3LYP/6-311 + + G(d,p) level of the theory. The calculations show that peptide bond cleavage at α-chain (amino side) due to αOC → αCN ring closure is the most favorable reaction. The result is in agreement with experiment utilizing PSD/CRF method. The second most favorable pathway is due to αOC → βC ring closure results in β-chain cleavage. The cleavage products βAsp and Asp fragments can be used to signify an abundance of βAsp residue in nonenzymatic condition. Other three cyclizations initiated by either α- or β-amino nitrogen result in various cleavages, isomerization to Asp, and reconversion to original βAsp. These three cyclization pathways are obstructed because they require mostly high activation barriers and their intermediates are quite less thermodynamically stable. Thus, computational results also confirm that βAsp → Asp is prohibited in case of nonenzymatic condition which means that protein L-isoaspartyl O-methyl transferase (PIMT) is needed for this modification.

The protective role of protein L-isoaspartyl (D-aspartate) O-methyltransferase for maintenance of mitochondrial morphology in A549 cell

ABSTRACTPurpose: The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells. Materials and Methods: We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1. Results: We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content. Conclusions: The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.

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.

Crystal structure and activity of protein L-isoaspartyl-O-methyltransferase from Vibrio cholerae, and the effect of AdoHcy binding

The repair enzyme Protein L-isoaspartyl-O-methyltransferase (PIMT) is widely distributed in various organisms. PIMT catalyzes S-adenosylmethionine (AdoMet) dependent methylation of abnormal L-isoaspartyl residues, formed by the deamidation of asparagines and isomerization of aspartates. We report the crystal structure of PIMT of Vibrio cholerae (VcPIMT), the aetiological agent for cholera, complexed with the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy) to 2.05 Å resolution. A stretch of residues (39–58), lining the substrate-binding site, is disordered. Urea-induced unfolding free energy for apo and VcPIMT-AdoHcy complex reveals greater stability for the cofactor-bound protein. The kinetic parameters for the methyltransferase activity of the recombinant VcPIMT was determined using a continuous spectrophotometric color-based assay using the peptide substrate [VYP(L-isoD)HA]. The enzyme exhibited activity higher than the Escherichia coli enzyme and closer to those from thermophilic bacteria and the mammalian source. The association constant for substrate binding is 2.29 × 106 M−1, quite similar to that for AdoHcy. The crystal structure and the model of the peptide-bound structure indicate that the majority of the interactions used for cofactor/substrate binding are provided by the main-chain atoms. Evolutionary relationships derived based on a phylogenetic tree constructed using the PIMT sequences are in conformity with the crystal structures of nine AdoHcy-bound PIMTs.

Prediction of binding modes between protein l-isoaspartyl (d-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening

Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, d-α-Asp and d-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of d-β-Asp is the largest and the proportions of l-β-Asp and d-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and d-α-Asp is frequently considered. The protein l-isoaspartyl (d-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and d-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and d-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or d-α-Asp residues were investigated using computational protein–ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and d-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and d-α-Asp peptides. In contrast, for peptides containing l-α-Asp or d-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods.

Cloning and sequencing of protein L-isoaspartyl O-methyl transferase of Salmonella Typhimurium isolated from poultry

Aim: To clone the Salmonella Typhimurium protein L-isoaspartyl O-methyl transferase (PIMT) enzyme and to analyze the sequence with PIMT gene of other pathogenic serovars of Salmonella. Materials and Methods: Salmonella Typhimurium strain E-2375 was procured from the National Salmonella Center, IVRI. The genomic DNA was isolated from Salmonella Typhimurium. Polymerase chain reaction (PCR) was carried out to amplify PIMT gene using the designed primers. The PCR product was cloned into pET28c plasmid vector and transformed into Escherichia coli DH5α cells. The plasmid was isolated from E. coli and was sequenced. The sequence was analyzed and submitted in Genbank. Results: The PCR product revealed a distinct amplicon of 627 bp. The clone was confirmed by PCR. Sequencing data revealed 100% homology between PIMT sequences from Salmonella Typhimurium strain E-2375 (used in the current study) and PIMT sequences of standard reported strain (Salmonella Typhimurium str. LT2) in NCBI data base. This submitted sequence in Genbank having accession no. KJ575536. Conclusions: PIMT gene of Salmonella is highly conserved in most of the pathogenic Salmonella serovars. The PIMT clone can be used to isolate PIMT protein. This PIMT protein will be helpful to identify isoaspartate containing proteins thus can help in study Salmonella virulence.

Isoaspartate accumulation in creatine kinase B in vivo and in vitro is associated with loss of enzymatic activity (949.1)

Formation of abnormal isoaspartyl (isoAsp) residues occurs frequently in proteins and peptides under physiological conditions, often resulting in the alteration of their structure and function. IsoAsp residues can be efficiently repaired by the action of protein L‐isoaspartyl O‐methyltransferase (PIMT), a cellular repair enzyme that catalyzes the conversion of isoAsp to normal Asp residues. The brain‐enriched isoform of creatine kinase (CK‐B) plays a key role in intracellular energy metabolism and is highly susceptible to isoAsp formation. We investigated the effect of isoAsp formation on CK‐B activity, both in vivo in PIMT‐deficient mice, and in vitro using aged recombinant CK‐B. Accumulation of isoAsp residues in vivo was associated with a significant decrease in CK‐B activity. In vitro aging of recombinant human CK‐B also resulted in isoAsp formation and loss of CK‐B activity. IsoAsp sites in aged, recombinant CK‐B were efficiently repaired in vitro by the action of PIMT. These results (1) corroborate previous finding that CK‐B is a major target of PIMT action in vivo, (2) demonstrate that isoAsp accumulation in CK‐B is associated with significant loss of function, and (3) suggest that loss of CK‐B function in vivo may contribute to the neuropathology and susceptibility to epileptic seizures that are characteristic of PIMT‐deficient mice.Grant Funding Source: Supported by NIH grant NS‐17269

Molecular Ageing of Alpha- and Beta-Synucleins: Protein Damage and Repair Mechanisms

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[3H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated 3H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of 3H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.

Protein l-isoaspartyl-O-methyltransferase of Vibrio cholerae: Interaction with cofactors and effect of osmolytes on unfolding

Protein l-isoaspartyl-O-methyltransferase (PIMT) is an ubiquitous enzyme widely distributed in cells and plays a role in the repair of deamidated and isomerized proteins. In this study, we show that this enzyme is present in cytosolic extract of Vibrio cholerae, an enteric pathogenic Gram-negative bacterium and is enzymatically active. Additionally, we focus on the detailed biophysical characterization of the recombinant PIMT from V. cholerae to gain insight into its structure, stability and the cofactor binding. The equilibrium denaturation of PIMT has been studied using tryptophan fluorescence and CD spectroscopy. The far- and near-UV CD, as well as fluorescence experiments reveal the presence of a non-native intermediate in the folding pathway. Binding of the hydrophobic fluorescent probe, bis-ANS, to the intermediate occurs with high affinity because of the exposure of the hydrophobic clusters during the unfolding process. The existence of the probable intermediate has also been confirmed from limited tryptic digestion and DLS experiments. The protein shows higher binding affinity for AdoHcy, in comparison to AdoMet, and the binding increases the midpoint of thermal unfolding by 6 and 5 °C, respectively. Modeling and molecular dynamics simulations also support the higher stability of the protein in presence of AdoHcy.

Requirement of protein l-isoaspartyl O-methyltransferase for transcriptional activation of trefoil factor 1 (TFF1) gene by estrogen receptor alpha

Lysine- and arginine-specific methyltransferases have been shown to act as either direct or secondary transcriptional co-activator of the estrogen receptor (ERα). However, little is known about the role of protein l-isoaspartyl O-methyltransferase (PIMT) on transcriptional regulation. Here, we show that PIMT acts as a co-activator for ERα-mediated transcription. Activation of the estrogen response element (ERE) promoter by β-estradiol (E2) was suppressed by knockdown of PIMT, and enhanced by overexpression of wild-type PIMT. However, the ERE promoter activity was resistant to E2 stimulation in cells overexpressing a catalytically inactive PIMT mutant, G88A. Consistent with these results, the expression of the endogenous ERα response gene trefoil factor 1 (TFF1) by E2 was completely abrogated by PIMT depletion and decreased to approximately 50% when PIMT mutant G88A was expressed. In addition, over-expression of PIMT significantly increased the levels of TFF1 mRNA in the presence or absence of E2. Interestingly, PIMT interacted with ERα and was distributed to the cytosol and the nucleus. The chromatin immunoprecipitation analysis revealed that PIMT was recruited to the promoter of TFF1 gene together with ERα in an E2-dependent manner, which was accompanied by uploading of RNA polymerase II on the promoter. Taken together, the results suggest that PIMT may act as a co-activator in ERα-mediated transcription through its recruitment to the promoter via interacting with ERα.

Cross-regulation between protein L-isoaspartyl O-methyltransferase and ERK in epithelial mesenchymal transition of MDA-MB-231 cells

Protein L-isoaspartyl O-methyltransferase (PIMT) regulates cell adhesion in various cancer cell lines through activation of integrin αv and the PI3K pathway. The epithelial mesenchymal transition (EMT) enables epithelial cells to acquire the characteristics of mesenchymal cells, and to allow them to migrate for metastasis. Here, we examined the relationship between PIMT and EMT with attached or detached MDA-MB 231 cells. Human breast cancer cell line MDA-MB-231 cells were maintained in a suspension on poly-HEMA in the presence or absence of PIMT siRNA or ERK inhibitor PD98059. The mRNAs and proteins were analyzed using RT-PCR and immunoblotting, respectively. During cellular incubation under detached conditions, PIMT, integrin αv and EMT proteins, such as Snail, Slug and matrix metalloproteinase 2 (MMP-2), were significantly increased in correlation with the phosphorylation of ERK1/2. The ERK inhibitor PD98059 (25 μmol/L) strongly suppressed the expression of the proteins and PIMT. Interestingly, PIMT siRNA blocked the phosphorylation of ERK and the expression of the EMT proteins. Additionally, PIMT and ERK phosphorylation were both co-activated by treatment with TGF-β (10 ng/mL) and TNF-α (10 ng/mL). A tight cross-regulation exists between ERK and PIMT in regards to their activation and expression during the EMT.

Knock-down of protein L-isoaspartyl O-methyltransferase increases β-amyloid production by decreasing ADAM10 and ADAM17 levels

To examine the role of protein L-isoaspartyl O-methyltransferase (PIMT; EC 2.1.1.77) on the secretion of Aβ peptides. HEK293 APPsw cells were treated with PIMT siRNA or adenosine dialdehyde (AdOX), a broad-spectrum methyltransferase inhibitor. Under the conditions, the level of Aβ secretion and regulatory mechanism by PIMT were examined. Knock-down of PIMT and treatment with AdOX significantly increased Aβ(40) secretion. Reductions in levels of PIMT decreased the secretion of soluble amyloid precursor protein alpha (sAPPα) without altering the total expression of APP or its membrane-bound C83 fragment. However, the levels of the C99 fragment generated by β-secretase were enhanced. Moreover, the decreased secretion of sAPPα resulting from PIMT knock-down seemed to be linked with the suppression of the expression of α-secretase gene products, α-disintegrin and metalloprotease 10 (ADAM10) and ADAM17, as indicated by Western blot analysis. In contrast, ADAM10 was not down-regulated in response to treatment with the protein arginine methyltransferase (PRMT) inhibitor, AMI-1. This study demonstrates a novel role for PIMT, but not PRMT, as a negative regulator of Aβ peptide formation and a potential protective factor in the pathogenesis of AD.

Mass spectrometric analysis of asparagine deamidation and aspartate isomerization in polypeptides.

One of the most frequent modifications in proteins and peptides is the deamidation of asparagine, a spontaneous non-enzymatic reaction leading to a mixture of L,D-succinimidyl, L,D-aspartyl, and L,D-isoaspartyl forms, with L-isoaspartyl dominating. Spontaneous isomerization of L-Asp yields the same products. In vivo, these unusual forms of aspartate are repaired by the protein L-isoaspartyl O-methyltransferase enzyme, with the balance between isomerization and repair affecting the organism physiology. Mass spectrometric analysis of this balance involves isomer separation, iso-Asp/Asp quantification, and iso-Asp site identification. This review highlights the issues associated with these steps and discusses the prospects of high-throughput iso-Asp analysis.

Uncovering Novel Mechanisms for Limiting the Accumulation of Isoaspartyl‐Damaged Proteins

Although most organisms require protein L‐isoaspartyl O‐methyltransferase (PIMT) for the repair of isoaspartyl‐damaged proteins, recent studies have shown that Saccharomyces cerevisiae lacks any enzyme resembling PIMT. Using high‐performance liquid chromatography, matrix‐assisted laser desportion/ionization mass spectrometry, and a biochemical radiolabeling approach, the levels of isoaspartyl protein damage in S. cerevisiae were determined to be similar to other organisms with PIMT. Addition of 10 μM MG‐132 and/or 5 mM 3‐methyladenine proteasome and autophagy inhibitors, respectively, indicated that autophagy and the proteasomal degradation pathways both were important in the removal of isoaspartyl‐damaged proteins in specific environments and growth phases. Interestingly, combining various synthetic peptides with S. cerevisiae extract lysed without protease inhibitors resulted in the favorable cleavage of the isoaspartyl‐containing synthetic peptide KASAisoDLAKY, suggestive of an isoaspartyl‐specific protease. Understanding which proteins are most susceptible to damage, and the proteolytic pathways that prevent their accumulation, may aid in the development of therapeutic interventions that enhance repair pathways and thus the human healthspan. This research was supported by the UCLA Cellular and Molecular Training Grant.

Recognition of Age-damaged (R,S)-Adenosyl-L-methionine by Two Methyltransferases in the Yeast Saccharomyces cerevisiae

The biological methyl donor S-adenosylmethionine (AdoMet) can exist in two diastereoisomeric states with respect to its sulfonium ion. The S configuration, (S,S)-AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the R form, producing (R,S)-AdoMet. As of yet, (R,S)-AdoMet has no known physiological function and may inhibit cellular reactions. In this study, we found two Saccharomyces cerevisiae enzymes that are capable of recognizing (R,S)-AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, identified previously as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine, respectively. We found here that Sam4 recognizes both (S,S)- and (R,S)-AdoMet, but that its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet, whereas no activity is seen with the S,S form. R,S-Specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)-AdoMet in these organisms.

Diet-dependent survival of protein repair-deficient mice

Protein l-isoaspartyl ( d-aspartyl) O-methyltransferase (PCMT1) is a protein-repair enzyme, and mice lacking this enzyme accumulate damaged proteins in multiple tissues, die at an early age from progressive epilepsy and have an increased S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy) ratio in brain tissue. It has been proposed that the alteration of AdoMet and AdoHcy levels might contribute to the seizure phenotype, particularly as AdoHcy has anticonvulsant properties. To investigate whether altered AdoMet and AdoHcy levels might contribute to the seizures and thus the survivability of the repair-deficient mice, a folate-deficient amino acid-based diet was administered to the mice in place of a standard chow diet. We found that the low-folate diet significantly decreases the AdoMet/AdoHcy ratio in brain tissue and results in an almost threefold extension of mean life span in the protein repair-deficient mice. These results indicate that the increased AdoMet/AdoHcy ratio may contribute to the lowered seizure threshold in young PCMT1-deficient mice. However, mean survival was also extended almost twofold for mice on a control folate-replete amino acid-based diet compared to mice on the standard chow diet. Survival after 40 days was similar in the mice on the low- and high-folate amino acid-based diets, suggesting that the survival of older PCMT1-deficient mice is not affected by the higher brain AdoMet/AdoHcy ratio. Additionally, the surviving older repair-deficient mice have a significant increase in body weight when compared to age-matched normal mice, independent of the type of diet. This weight increase was not accompanied by an increase in consumption levels, indicating that the repair-deficient mice may also have an altered metabolic state.