Protein L-isoaspartate O-methyltransferase Research Articles

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.

Natural isoaspartyl protein modification of ZAP70 alters T cell responses in lupus

Protein posttranslational modifications (PTMs) arise in a number of normal cellular biological pathways and in response to pathology caused by inflammation and/or infection. Indeed, a number of PTMs have been identified and linked to specific autoimmune responses and metabolic pathways. One particular PTM, termed isoaspartyl (isoAsp or isoD) modification, is among the most common spontaneous PTM occurring at physiological pH and temperature. Herein, we demonstrate that isoAsp modifications arise within the ZAP70 protein tyrosine kinase upon T-cell antigen receptor (TCR) engagement. The enzyme protein L-isoaspartate O-methyltransferase (PCMT1, or PIMT, EC 2.1.1.77) evolved to repair isoaspartyl modifications in cells. In this regard, we observe that increased levels of isoAsp modification that arise under oxidative stress are correlated with reduced PIMT activity in patients with systemic lupus erythematosus (SLE). PIMT deficiency leads to T cell hyper-proliferation and hyper-phosphorylation through ZAP70 signaling. We demonstrate that inducing the overexpression of PIMT can correct the hyper-responsive phenotype in lupus T cells. Our studies reveal a phenotypic role of isoAsp modification and phosphorylation of ZAP70 in lupus T cell autoimmunity and provide a potential therapeutic target through the repair of isoAsp modification.

Isomerization of Asp is essential for assembly of amyloid-like fibrils of αA-crystallin-derived peptide.

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66–80 peptide, corresponding to the 66–80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66–80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66–80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact β-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.

Isolation and Identification of Protein L-Isoaspartate-O-Methyltransferase (PIMT) Interacting Proteins in Salmonella Typhimurium.

Protein L-isoaspartate-O-methyltransferase (PIMT) plays an important role in restoration of covalently damaged Asn/Asp residues. It repairs the racemized forms of these amino acids in protein by forming a labile isoAsp methyl ester which readily converts back to the succinimide intermediate. Spontaneous hydrolysis of the intermediate further restores a minor portion to the normal Asp residues. While significant numbers of PIMT targets have been identified in eukaryotes, very few are documented from prokaryotes. Temperature (42°C) induced elevation in PIMT expression level has been recently shown in a poultry isolate of Salmonella Typhimurium (ST). The enzyme was also found to be crucial for survival, virulence and colonization of ST in poultry. In the present study, co-immunoprecipitation (Co-IP) approach was used (for isolation) followed by LC-MS analysis to identify the PIMT interacting proteins of ST. Four different proteins were identified among which cytochrome C biogenesis protein A (CcmA) was further expressed in recombinant form and analysed for interaction with recombinant PIMT (rPIMT) by microtiter plate assay. Additionally, the findings were supported by alterations in secondary structure of the proteins upon co-incubation.

Protein L-Isoaspartate O-Methyltransferase (PCMT1): A Key Player of Spontaneously Arisen Protein Damage Repair

Protein L-Isoaspartate O-Methyltransferase (PCMT1): A Key Player of Spontaneously Arisen Protein Damage Repair

Methylation of histone H4 at aspartate 24 by protein L-isoaspartate O-methyltransferase (PCMT1) links histone modifications with protein homeostasis.

Histone modifications play crucial roles in modulating chromatin function and transcriptional activity. Due to their long half-life, histones can, in addition to post-translational modifications, also accumulate spontaneous chemical alterations, which can affect their functionality and require either protein repair or degradation. One of the major sources of such protein damage or ageing is the conversion of aspartate into isoaspartate residues that can then be methylated. Here, we characterize a novel histone modification, the methylation of histone H4 at aspartate 24 (H4D24me). We generated H4D24me specific antibodies and showed that H4D24me is ubiquitously present in different mouse and human cells. Our in vitro and in vivo data identified PCMT1 (Protein L-isoaspartate O-methyltransferase), an enzyme involved in protein repair, as a novel H4D24 specific histone methyltransferase. Furthermore, we demonstrated that VprBP (HIV-1 viral protein R (Vpr)-binding protein), a chromo domain-containing protein, specifically recognizes H4D24me potentially implicating H4D24me in H4 degradation. Thus, this work links for the first time a histone modification with histone protein aging and histone homeostasis, suggesting novel functions for histone modifications beyond transcriptional regulation.

Isoaspartyl Protein Damage and Repair in Mouse Retina

To determine the propensity of retinal proteins for spontaneous damage via formation of isoaspartyl sites, a common type of protein damage that could contribute to retinal disease. Tissue extracts were obtained from retinas and brains of control mice and from mice in which the gene for protein L-isoaspartate O-methyltransferase (PIMT; an enzyme that repairs isoaspartyl protein damage) was knocked out. PIMT expression in these extracts was measured by Western blot, and its specific activity was assayed by monitoring the rate of [(3)H]methyl transfer from S-adenosyl-[methyl-(3)H]L-methionine to γ-globulin. Isoaspartate levels in extracts were measured by their capacity to accept [(3)H]methyl groups via the PIMT-catalyzed methylation reaction. To compare molecular weight distributions of isoaspartyl-rich proteins in retina versus brain, proteins from PIMT knockout (KO) and control mice were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). Isoaspartyl proteins were (3)H-labeled on-blot using a PIMT overlay and imaged by autoradiography. When normalized to the β-actin content of each tissue, retina was found to be nearly identical to brain with regard to expression and activity of PIMT and its propensity to accumulate isoaspartyl sites when PIMT is absent. The two tissues show distinct differences in the molecular weight distribution of isoaspartyl proteins. The retina is rich in PIMT activity and contains a wide range of proteins that are highly susceptible to this type of protein damage. Recoverin may be one such protein. Isoaspartate formation, along with oxidation, should be considered as a potential source of protein dysfunction and autoimmunity in retinal disease.

Integrated Proteomic Analysis of Major Isoaspartyl-Containing Proteins in the Urine of Wild Type and Protein l-Isoaspartate O-Methyltransferase-Deficient Mice

The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and nonenzymatic process, ubiquitous both in vivo and in nonbiological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC2.1.1.77) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. A total of 38 putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.

The V119I polymorphism in protein L-isoaspartate O-methyltransferase alters the substrate-binding interface

Protein L-isoaspartate O-methyltransferase (PIMT) repairs isoaspartate residues in damaged proteins, and it contains a Val-Ile polymorphismin in alpha5, approximately 13 A from its active site. Val119 has lower activity and thermal stability but increased affinity for endogenous substrates. Studies suggest that heterozygosity for Val/Ile favors efficient isoaspartate repair. We have performed multiple molecular dynamics simulations of 119I and 119V PIMT. Both V119 and I119 interact with the same residues throughout all of the simulations. However, the larger Ile altered the orientations of alpha5 and beta5, both of which have co-substrate binding residues on their distal ends. I119 increases the flexibility of several residues, loosening up the S-adenosylmethionine (SAM)-binding site. These subtle changes are propagated towards the isoaspartate-docking site via residues common to both active sites. The increased mobility in 119I PIMT reorients alpha3, resulting in a salt-bridge network at the substrate-binding interface that disrupts several key side-chain interactions in the isoaspartate site. In contrast, 119V PIMT remains quite rigid with little change to the co-substrate binding site, which could hinder SAM's binding and release, accounting for the decreased activity. These results shed light on the molecular basis behind the decreased activity and increased specificity for endogenous substrates of 119V PIMT relative to the 119I variant. 119I PIMT catalyzes the methylation reaction but may have difficulties recognizing and orienting specific substrates due to its distorted substrate-binding site. Heterozygosity for both the Ile and Val alleles may provide the best of both worlds, allowing the fast and specific methylation of damaged proteins.

Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1(-/-)) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1(-/-) mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-(3)H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1(-/-) animals compared with Pcmt1(+/+) littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, beta-synuclein, and alpha-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1(-/-) brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

Intracellular Protein Modification Associated with Altered T Cell Functions in Autoimmunity

Posttranslational protein modifications influence a number of immunologic responses ranging from intracellular signaling to protein processing and presentation. One such modification, termed isoaspartyl (isoAsp), is the spontaneous nonenzymatic modification of aspartic acid residues occurring at physiologic pH and temperature. In this study, we have examined the intracellular levels of isoAsp residues in self-proteins from MRL(+/+), MRL/lpr, and NZB/W F(1) mouse strains compared with nonautoimmune B10.BR mice. In contrast to control B10.BR or NZB/W mice, the isoAsp content in MRL autoimmune mice increased and accumulated with age in erythrocytes, brain, kidney, and T lymphocytes. Moreover, T cells that hyperproliferate to antigenic stimulation in MRL mice also have elevated intracellular isoAsp protein content. Protein l-isoaspartate O-methyltransferase activity, a repair enzyme for isoAsp residues in vivo, remains stable with age in all strains of mice. These studies demonstrate a role for the accumulation of intracellular isoAsp proteins associated with T cell proliferative defects of MRL autoimmune mice.

Increase in intracellular protein modification associated with altered lymphocyte functions in autoimmunity

Lymphocytes that react with ubiquitous self-antigens are typically eliminated within the thymus and bone marrow to prevent autoimmune response. However, not all self-antigens exist during thymic selection, which results in the escape of autoreactive T and B cells to the peripheral circulation. Posttranslational modification of protein usually generates neo-epitopes from self-proteins, causing a break in immune tolerance. One such modification, termed isoaspartyl (isoAsp), is the spontaneous non-enzymatic modification of aspartate or asparagine residues occurring at physiologic pH and temperature. The widely distributed protein L-isoaspartate-O-methyl-transferase (PIMT) enzyme is the repair system for abnormal isoaspartyl residues in vivo. Our laboratory has previously reported that PIMT deficient mice exhibit T cell hyperproliferation and lupus-like autoimmunity. Recently, we found that in contrast to control B10.BR mice, the isoAsp content in MRL autoimmune mice increased and accumulated with age in erythrocytes, brain, kidney, and in T lymphocytes. The lupus-CD4+ T cells exhibit hyperproliferative response to antigen stimulation and increase in isoaspartyl content both in vitro and in vivo as T cells are activated. These studies demonstrate a role for the accumulation of intracellular isoaspartyl proteins associated with T cell proliferative defects in autoimmune. (These studies were supported by NIH grant AI-48120 to MJM.)

Structural Integrity of Histone H2B in Vivo Requires the Activity of Protein l-IsoaspartateO-Methyltransferase, a Putative Protein Repair Enzyme

Protein L-isoaspartate O-methyltransferase (PIMT) is postulated to repair beta-aspartyl linkages (isoaspartyl (isoAsp)) that accumulate at certain Asp-Xaa and Asn-Xaa sites in association with protein aging and deamidation. To identify major targets of PIMT action we cultured rat PC12 cells with adenosine dialdehyde (AdOx), a methyltransferase inhibitor that promotes accumulation of isoAsp in vivo. Subcellular fractionation of AdOx-treated cells revealed marked accumulation of isoAsp in a 14-kDa nuclear protein. Gel electrophoresis and chromatography of nuclei (3)H-methylated in vitro by PIMT revealed this protein to be histone H2B. The isoAsp content of H2B in AdOx-treated cells was approximately 18 times that in control cells, although no isoAsp was seen in other core histones, regardless of treatment. To confirm the relevance and specificity of this effect, we measured isoAsp levels in histones from brains of PIMT knockout mice. IsoAsp was found at near stoichiometric levels in H2B extracted from knockout brains and was at least 80 times greater than that in H2B from normal mice. Little or no isoAsp was detected in H2A, H3, or H4 from mice of either genotype. Accumulation of isoAsp in histone H2B may disrupt normal gene regulation and contribute to the reduced life span that characterizes PIMT knockouts. In addition to disrupting protein function, isoAsp has been shown to trigger immunity against self-proteins. The propensity of H2B to generate isoAsp in vivo may help explain why this histone in particular is found as a major antigen in autoimmune diseases such as lupus erythematosus.