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

The 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.

ABI transcription factors and PROTEIN L-ISOASPARTYL METHYLTRANSFERASE module mediate seed desiccation tolerance and longevity in Oryza sativa.

In contrast to desiccation-tolerant orthodox seeds, recalcitrant seeds are desiccation sensitive and are unable to survive for a prolonged time. Here, our analyses of Oryza species with contrasting seed desiccation tolerance reveals that PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT), an enzyme that repairs abnormal isoaspartyl (isoAsp) residues in proteins, acts as a key player that governs seed desiccation tolerance to orthodox seeds but is ineffective in recalcitrant seeds. We observe that, unlike the orthodox seed of Oryza sativa, desiccation intolerance of the recalcitrant seeds of Oryza coarctata are linked to reduced PIMT activity and increased isoAsp accumulation due to the lack of coordinated action of ABA and ABI transcription factors to upregulate PIMT during maturation. We show that suppression of PIMT reduces, and its overexpression increases, seed desiccation tolerance and seed longevity in O. sativa. Our analyses further reveal that the ABI transcription factors undergo isoAsp formation that affect their functional competence; however, PIMT interacts with and repairs isoAsp residues and facilitates their functions. Our results thus illustrate a new insight into the mechanisms of acquisition of seed desiccation tolerance and longevity by ABI transcription factors and the PIMT module.

Kinetics of Aspartimide Formation and Hydrolysis in Lasso Peptide Lihuanodin.

Aspartimides are notorious as undesired side products in solid-phase peptide synthesis and in pharmaceutical formulations. However, we have discovered several ribosomally synthesized and post-translationally modified peptides (RiPPs) in which aspartimide is installed intentionally via enzymatic activity of protein l-isoaspartyl methyltransferase (PIMT) homologues. In the case of the lasso peptide lihuanodin, the methyltransferase LihM recognizes the lassoed substrate pre-lihuanodin, specifically methylating the side chain of an l-Asp residue in the ring portion of the lasso peptide. The subsequent nucleophilic attack from the adjacent amide leads to the formation of an aspartimide. The resulting aspartimide hydrolyzes regioselectively to l-Asp in buffers above pH 7. Here we report the first Michaelis-Menten kinetic measurements of such a RiPP-associated PIMT homologue, LihM, acting on its cognate substrate pre-lihuanodin. Additionally, we measured the rate of aspartimide hydrolysis, which allowed us to deduce the kinetics of the entire reaction network. The relative magnitudes of these rates explain the accumulation and relative stability of aspartimide-containing lihuanodin. We also demonstrate that the residue C-terminal to the aspartimide controls the regioselectivity of hydrolysis and thus the threadedness of the peptide.

Adenosine dialdehyde, a methyltransferase inhibitor, induces colorectal cancer cells apoptosis by regulating PIMT:p53 interaction.

Post-translational modification (PTM) is important in controlling many biological processes by changing the structure and function of a protein. Protein methylation is an important PTM, and the role of methyltransferases has been implicated in numerous cellular functions. Protein L-isoaspartyl methyltransferase (PIMT) is ubiquitously expressed in almost all organisms and govern important cellular processes including apoptosis. Among other functions, PIMT has also been identified as a potent oncogene because it destabilizes the structure of the tumor suppressor p53 via methylation at the transactivation domain. In the present study we identified that out of the three methyltransferase inhibitors tested, namely, S-adenosyl-l-homocysteine (AdoHcy), adenosine and adenosine dialdehyde (AdOx), only AdOx augments p53 expression by destabilizing PIMT structure, as evident from far-UV CD. The effect of the inhibitors, AdOx in particular, to the structure of PIMT, and the binding of PIMT to the p53 transactivation domain have been investigated by docking and molecular dynamics simulations. AdOx significantly increases p53 accumulation and nuclear translocation in colon cancer cells, triggering the p53-mediated apoptotic pathway. To better understand the molecular mechanisms underlying p53 accumulation in colon cancer cells, we observed that the level of PIMT is considerably lower in AdOx-treated cells, reducing its association with p53, which stabilized p53. p53 then transactivated BAX, increasing the BAX: BCL-2 ratio and causing colon cancer cell death.

The role of PIMT in Alzheimer's disease pathogenesis: A novel hypothesis.

There are multiple theories of Alzheimer's disease pathogenesis. One major theory is that oxidation of amyloid beta (Aß) promotes plaque deposition that directly contributes to pathology. A competing theory is that hypomethylation of DNA (due to altered one carbon metabolism) results in pathology through altered gene regulation. Herein, we propose a novel hypothesis involving L-isoaspartyl methyltransferase (PIMT) that unifies the Aß and DNA hypomethylation hypotheses into a single model. Importantly, the proposed model allows bidirectional regulation of Aß oxidation and DNA hypomethylation. The proposed hypothesis does not exclude simultaneous contributions by other mechanisms (e.g., neurofibrillary tangles). The new hypothesis is formulated to encompass oxidative stress, fibrillation, DNA hypomethylation, and metabolic perturbations in one carbon metabolism (i.e., methionine and folate cycles). In addition, deductive predictions of the hypothesis are presented both to guide empirical testing of the hypothesis and to provide candidate strategies for therapeutic intervention and/or nutritional modification. HIGHLIGHTS: PIMT repairs L-isoaspartyl groups on amyloid beta and decreases fibrillation. SAM is a common methyl donor for PIMT and DNA methyltransferases. Increased PIMT activity competes with DNA methylation and vice versa. The PIMT hypothesis bridges a gap between plaque and DNA methylation hypotheses.

Human Protein-l-isoaspartate O-Methyltransferase Domain-Containing Protein 1 (PCMTD1) Associates with Cullin-RING Ligase Proteins.

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

Methionine sulfoxide reductase B5 plays a key role in preserving seed vigor and longevity in rice (Oryza sativa).

Oxidation of methionine leads to the formation of methionine S-sulfoxide and methionine R-sulfoxide, which can be reverted by two types of methionine sulfoxide reductase (MSR): MSRA and MSRB. Though the role of MSR enzymes has been elucidated in various physiological processes, the regulation and role of MSR in seeds remains poorly understood. In this study, through molecular, biochemical, and genetic studies using seed-specific overexpression and RNAi lines of OsMSRB5 in Oryza sativa, we demonstrate the role of OsMSRB5 in maintaining seed vigor and longevity. We show that an age-induced reduction in the vigor and viability of seeds is correlated with reduced MSR activity and increased methionine sulfoxide (MetSO) formation. OsMSRB5 expression increases during seed maturation and is predominantly localized to the embryo. Further analyses on transgenic lines reveal the role of OsMSRB5 in modulating reactive oxygen species (ROS) homeostasis to preserve seed vigor and longevity. We show that ascorbate peroxidase and PROTEIN l-ISOASPARTYL METHYLTRANSFERASE undergo MetSO modification in seeds that affects their functional competence. OsMSRB5 physically interacts with these proteins and reverts this modification to facilitate their functions and preserve seed vigor and longevity. Our results thus illustrate the role of OsMSRB5 in preserving seed vigor and longevity by modulating ROS homeostasis in seeds.

Arabidopsis protein l-ISOASPARTYL METHYLTRANSFERASE repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance.

Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification.

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

Protein-L-isoaspartate O-methyltransferase is required for in vivo control of oxidative damage in red blood cells.

Red blood cells have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which red blood cells neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how red blood cells repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to red blood cells is direct repair of the damaged molecules, as red blood cells cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. Red blood cells express an L-Isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters red blood cell metabolism in a healthy state, but does not impair the circulatory lifespan of red blood cells. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, red blood cells from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.

Differential expression of microRNAs associated with neurodegenerative diseases and diabetic nephropathy in protein l-isoaspartyl methyltransferase-deficient mice.

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), an enzyme repairing isoaspartate residues in peptides and proteins that result from the spontaneous decomposition of normal l-aspartyl and l-asparaginyl residues during aging, has been revealed to be involved in neurodegenerative diseases (NDDs) and diabetes. However, the molecular mechanisms for a putative association of PIMT dysfunction with these diseases have not been clarified. Our study aimed to identify differentially expressed microRNAs (miRNAs) in the brain and kidneys of PIMT-deficient mice and uncover the epigenetic mechanism of PIMT-involved NDDs and diabetic nephropathy (DN). Differentially expressed miRNAs by sequencing underwent target prediction and enrichment analysis in the brain and kidney of PIMT knockout (KO) mice and age-matched wild-type (WT) littermates. Sequence analysis revealed 40 differentially expressed miRNAs in the PIMT KO mouse brain including 25 upregulated miRNAs and 15 downregulated miRNAs. In the PIMT KO mouse kidney, there were 80 differentially expressed miRNAs including 40 upregulated miRNAs and 40 downregulated miRNAs. Enrichment analysis and a systematic literature review of differentially expressed miRNAs indicated the involvement of PIMT deficiency in the pathogenesis in NDDs and DN. Some overlapped differentially expressed miRNAs between the brain and kidney were quantitatively assessed in the brain, kidney, and serum-derived exosomes, respectively. Despite being preliminary, these results may aid in investigating the pathological hallmarks and identify the potential therapeutic targets and biomarkers for PIMT dysfunction-related NDDs and DN.

Protein Kinase C Theta Modulates PCMT1 through hnRNPL to Regulate FOXP3 Stability in Regulatory T Cells.

T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype.

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.

Functional divergence of annotated l-isoaspartate O-methyltransferases in an α-proteobacterium.

Spontaneous formation of isoaspartates (isoDs) often causes protein damage. l-Isoaspartate O-methyltransferase (PIMT) repairs isoD residues by catalyzing the formation of an unstable l-isoaspartyl methyl ester that spontaneously converts to an l-aspartyl residue. PIMTs are widely distributed in all three domains of life and have been studied most intensively in connection with their role in protein repair and aging in plants and animals. Studies of bacterial PIMTs have been limited to Escherichia coli, which has one PIMT. The α-proteobacterium Rhodopseudomonas palustris has three annotated PIMT genes, one of which (rpa2580) has been found to be important for cellular longevity in a growth-arrested state. However, the biochemical activities of these three R. palustris PIMTs are unknown. Here, we expressed and characterized all three annotated PIMT proteins, finding that two of them, RPA0376 and RPA2838, had PIMT activity, whereas RPA2580 did not. RPA0376 and RPA2838 single- and double-deletion mutants did not differ in longevity from WT R. palustris and did not exhibit elevated levels of isoD residues in aged cells. Comparative sequence analyses revealed that RPA2580 belongs to a separate phylogenetic group of annotated PIMT proteins present in the α-proteobacteria. Our results suggest that this group of proteins is not involved in repair of protein isoD residues. In addition, the bona fide bacterial PIMT enzymes may play a different or subtler role in bacterial physiology than previously suggested.

The l-isoaspartate modification within protein fragments in the aging lens can promote protein aggregation.

Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.

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.

PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity.

Human carbonic anhydrase IX (CAIX), a unique member of the α carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function.

PIMT-Mediated Protein Repair: Mechanism and Implications.

Amino acids undergo many covalent modifications, but only few amino acid repair enzymes have been identified. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), also known as L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (PCMT), methylates covalently modified isoaspartate (isoAsp) residues accumulated in proteins via Asn deamidation and Asp hydrolysis. This cytoplasmic reaction occurs through the formation of succinimide cyclical intermediate and generates either isoAsp or Asp from succinimide. Succinimide conversion into Asp is spontaneous, while isoAsp is restored by PIMT using S-adenosylmethionine as a methyl donor. PIMT transforms isoAsp into succinimide, thereby creating an opportunity for the later to be converted into Asp. Apart from normal cell physiology, formation of isoAsp in proteins is promoted by various stress conditions. The resulting isoAsp can form a kink or bend in the protein backbone thus making the protein conformationally and functionally distorted. Many PIMT-interacting proteins (proteins with isoAsp residues) have been reported in eukaryotes, but only few of them have been found in prokaryotes. Extensive studies in mice have shown the importance of PIMT in neurodegeneration. Detail elucidation of PIMT function can create a platform for addressing various disorders such as Alzheimer's disease and cancer.

Polymorphic Variants of Human Protein l-Isoaspartyl Methyltransferase Affect Catalytic Activity, Aggregation, and Thermal Stability: IMPLICATIONS FOR THE ETIOLOGY OF NEUROLOGICAL DISORDERS AND COGNITIVE AGING.

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the human pcmt1 gene, catalyzes repair of abnormal l-isoaspartyl linkages in age-damaged proteins. Pcmt1 knock-out mice exhibit a profound neuropathology and die 30-60 days postnatal from an epileptic seizure. Here we express 15 reported variants of human PIMT and characterize them with regard to their enzymatic activity, thermal stability, and propensity to aggregation. One mutation, R36C, renders PIMT completely inactive, whereas two others, A7P and I58V, exhibit activity that is 80-100% higher than wild type. G175R is highly prone to aggregation and has greatly reduced activity. R17S and R17H show markedly enhanced sensitivity to thermal denaturation. Based on previous studies of moderate PIMT variation in humans and mice, we predict that heterozygosity for R36C, G175R, R17S, and R17H will prove detrimental to cognitive function and successful aging, whereas homozygosity (if it ever occurs) will lead to severe neurological problems in the young.

PCMT1 is an unfavorable predictor and functions as an oncogene in bladder cancer.

The role of protein l-isoaspartate (d-aspartate) O-methyltransferase (PCMT1) in human cancer was generally cognized. The clinical significance and biological function of PCMT1 in bladder cancer is still unknown. PCMT1 mRNA and protein expression levels in bladder cancer tissues and cell lines were detected by qRT-PCR, immunohistochemistry, or western blot. The correlation between PCMT1 expression and clinicopathological factors was analyzed through immunohistochemistry in 108 bladder cancer patients. Loss-of-function and gain-of-function studies were conducted to explore the biological function of PCMT1 in bladder cancer cell lines in regulating cell proliferation, migration, and invasion. In our results, we found that PCMT1 was overexpressed in bladder cancer tissues compared with normal urothelium tissues in microarray datasets (GSE3167). Then, we confirmed PCMT1 mRNA and protein expression were increased in bladder cancer tissues and cell lines compared with paired normal urothelium tissues and normal uroepithelial cell line. PCMT1 protein expression was obviously correlated with clinical stage, muscularis invasion, lymph node metastasis, and distant metastasis. Survival analysis showed that PCMT1 protein high-expression was an independent unfavorable prognostic factor for bladder cancer patients. The in vitro experiments showed PCMT1 regulated bladder cancer cells migration and invasion through modulating epithelial-mesenchymal transition (EMT)-associated genes expression including E-cadherin, vimentin, Snail and Slug, but had no effect on proliferation. In conclusion, PCMT1 is an unfavorable prognostic biomarker and involves in cells migration and invasion through regulating EMT-associated genes. © 2018 IUBMB Life, 70(4):291-299, 2018.