Protein L-isoaspartyl Methyltransferase Research Articles

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.

Protective role of madecassoside from Centella asiatica against protein L-isoaspartyl methyltransferase deficiency-induced neurodegeneration

Protein L-isoaspartyl methyltransferase (PIMT/PCMT1) could repair l-isoaspartate (L-isoAsp) residues formed by deamidation of asparaginyl (Asn) residues or isomerization of aspartyl (Asp) residues in peptides and proteins during aging. Aside from abnormal accumulation of L-isoAsp, PIMT knockout (KO) mice mirrors some neuropathological hallmarks such as anxiety-like behaviors, impaired spatial memory and aberrant synaptic plasticity in the hippocampus of neurodegenerative diseases (NDs), including Alzheimer's disease (AD) and related dementias, and Parkinson's disease (PD). While some reports indicate the neuroprotective effect of madecassoside (MA) as a triterpenoid saponin component of Centella asiatica, its role against NDs-related anxiety and cognitive impairment remains unclear. Therefore, we investigated the effect of MA against anxiety-related behaviors in PIMT deficiency-induced mouse model of NDs. Results obtained from the elevated plus maze (EPM) test revealed that MA treatment alleviated anxiety-like behaviors in PIMT knockout mice. Furthermore, Real-time PCR, electroencephalogram (EEG) recordings, transmission electron microscopy analysis and ELISA were carried out to evaluate the expression of clock genes, sleep and synaptic function, respectively. The PIMT knockout mice were characterized by abnormal clock patterns, sleep disturbance and synaptic dysfunction, which could be improved by MA administration. Collectively, these findings suggest that MA exhibits neuroprotective effects associated with improved circadian rhythms sleep-wake cycle and synaptic plasticity in PIMT deficient mice, which could be translated to ameliorate anxiety-related symptoms and cognitive impairments in NDs.

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 Protein-L-isoaspartyl Methyltransferase (PIMT) in the Suppression of Toxicity of the Oligomeric Form of Aβ42, in Addition to the Inhibition of Its Fibrillization.

The oligomeric form of amyloid-β peptide (Aβ42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD) and is responsible for cognitive deficits. The soluble oligomers are believed to be more toxic compared to the fibril form. Protein-L-isoaspartyl methyltransferase (PIMT) is a repair enzyme that converts aberrant isoAsp residues, formed spontaneously on isomerization of normal Asp and Asn residues, back to typical Asp. It was shown to inhibit the fibrillization of Aβ42 (containing three Asp residues), and here, we investigate its effect on the size, conformation, and toxicity of Aβ42 oligomers (AβO). Far-UV CD indicated a shift in the conformational feature of AβOs from the random coil to β-sheet in the presence of PIMT. Binding of bis-ANS to different AβOs (obtained using different concentrations of Aβ42 monomer) indicated the correlation of size of oligomers to hydrophobicity: the smallest AβO having the highest hydrophobicity is the most toxic. Dynamic light scattering showed an increase in size of AβO with the addition of PIMT, a contrasting role to that on Aβ fibril. Assays using PC12-derived neurons showed the neuroprotective role of PIMT against AβO-induced toxicity. Furthermore, we have elaborated on the molecular mechanism of the antifibrillar action of PIMT and how this function is correlated with its enzymatic activity. PIMT has a more pronounced effect on AβO as compared to a small heat shock protein, pointing to its importance for the amelioration of the adverse effect of both Aβ42 oligomers and fibrils.

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.

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.

Oryza coarctata PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs isoaspartyl modification to antioxidative enzymes and is implicated in seed traits in rice

PROTEIN L -ISOASPARTYL METHYLTRANSFERASE (PIMT) is a protein repairing enzyme, which is highly abundant in orthodox seeds, and plays an important role in seed vigor and longevity. PIMT essentially repairs isoaspartyl modification in proteins. Despite PIMT has been characterized from several orthodox seed producing plant species, role and regulation of PIMTs in recalcitrant seed producing plants are still limited. In the present study, PIMT from Oryza coarctata, which produces recalcitrant seeds and possess both enzymatically active ( Oc PIMT1–1 and Oc PIMT2–1) and inactive ( Oc PIMT1–2 and Oc PIMT2–2) PIMT isoforms , are functionally characterized through biochemical and genetic approach. We show that PIMT isoforms are differentially localized in Oryza sativa and Oryza coarctata. We also report that enzymatically active OcPIMTs isoforms, but not enzymatically inactive OcPIMTs isoforms, could impart seed vigor, viability and longevity in A. thaliana . Likewise, rice transgenic lines were also generated, and ectopic overexpression of enzymatically active OcPIMT isoforms resulted in increased seed length and weight with improved seed vigor and longevity. Subsequent analysis revealed that antioxidant enzymes ( Os APX and Os CAT) are susceptible to isoAsp modification, which negatively influences their biological functions; however, OcPIMT s physically interact, repairs and protect their function from harmful isoAsp modification, and thereby modulate ROS homeostasis in seeds during aging. Collectively, our results highlight the mechanisms and importance of ectopic expression of OcPIMT isoforms in seed desiccation tolerance and subsequent vigor, viability and longevity in rice. • O. coarctata encodes functionally active and inactive PIMT isoforms. • Ectopic over-expression of functionally active PIMT isoforms improves seed vigor and longevity in rice. • Antioxidative enzymes are susceptible to isoAsp damage during aging and seed storage. • OcPIMT isoforms interacts and repairs antioxidant enzymes from isoAsp damage and maintains its enzymatic properties. • Overall PIMT mediated repair is important to maintain seed vigor and viability.

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.

Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide.

Microviridins and other ω-ester-linked peptides, collectively known as graspetides, are characterized by side-chain-side-chain linkages installed by ATP-grasp enzymes. Here we report the discovery of a family of graspetides, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase. Using heterologous expression, we produced fuscimiditide, a ribosomally synthesized and post-translationally modified peptide (RiPP). NMR analysis of fuscimiditide revealed that the peptide contains two ester cross-links forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We fully reconstituted fuscimiditide biosynthesis in vitro including formation of the ester and aspartimide moieties. The aspartimide moiety embedded in fuscimiditide hydrolyses regioselectively to isoaspartate. Surprisingly, this isoaspartate-containing peptide is also a substrate for the L-isoaspartyl methyltransferase homologue, thus driving any hydrolysis products back to the aspartimide form. Whereas an aspartimide is often considered a nuisance product in protein formulations, our data suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.

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.

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.

The Protein L-Isoaspartyl (D-Aspartyl) Methyltransferase Regulates Glial-to-Mesenchymal Transition and Migration Induced by TGF-β1 in Human U-87 MG Glioma Cells.

The enzyme PIMT methylates abnormal aspartyl residues in proteins. U-87 MG cells are commonly used to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT isoform I possessed oncogenic features when overexpressed in U-87 MG and U-251 MG glioma cells. Higher levels of wild-type PIMT stimulated migration and invasion in both glioma cell lines. Conversely, PIMT silencing reduced these migratory abilities of both cell lines. These results indicate that PIMT could play a critical role in glioblastoma growth. Here, we investigated for the first time, molecular mechanisms involving PIMT in the regulation of epithelial to mesenchymal transition (EMT) upon TGF-β1 treatments. Gene array analyses indicated that EMT genes but not PIMT gene were regulated in U-87 MG cells treated with TGF-β1. Importantly, PIMT silencing by siRNA inhibited in vitro migration in U-87 MG cells induced by TGF-β1. In contrast, overexpressed wild-type PIMT and TGF-β1 had additive effects on cell migration. When PIMT was inhibited by siRNA, this prevented Slug induction by TGF-β1, while Snail stimulation by TGF-β1 was increased. Indeed, overexpression of wild-type PIMT led to the opposite effects on Slug and Snail expression dependent on TGF-β1. These data highlighted the importance of PIMT in the EMT response dependent on TGF-β1 in U-87 MG glioma cells by an antagonist regulation in the expression of transcription factors Slug and Snail, which are critical players in EMT.

PIMT-Mediated Labeling of l-Isoaspartic Acid with Tris Facilitates Identification of Isomerization Sites in Long-Lived Proteins.

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate formed during reaction of l-isoAsp-containing peptides with PIMT and S-adenosyl methionine (SAM) is reactive with Tris base and results in a Tris-modified aspartic acid residue with a mass shift of +103 Da. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that Tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.

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.

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.

Effect of gold nanoparticles on the structure and neuroprotective function of protein L-isoaspartyl methyltransferase (PIMT)

Fibrillation of peptides and proteins is implicated in various neurodegenerative diseases and is a global concern. Aging leads to the formation of abnormal isoaspartate (isoAsp) residues from isomerization of normal aspartates in proteins, triggering fibril formation that leads to neurodegenerative diseases. Protein L-isoaspartyl methyltransferase (PIMT) is a repair enzyme which recognizes and converts altered isoAsp residues back to normal aspartate. Here we report the effect of gold nanoparticles (AuNPs) of different sizes on the structure and function of PIMT. Spherical AuNPs, viz. AuNS5, AuNS50 and AuNS100 (the number indicating the diameter in nm) stabilize PIMT, with AuNS100 exhibiting the best efficacy, as evident from various biophysical experiments. Isothermal titration calorimetry (ITC) revealed endothermic, but entropy driven mode of binding of PIMT with all the three AuNSs. Methyltransferase activity assay showed enhanced activity of PIMT in presence of all AuNSs, the maximum being with AuNS100. The efficacy of PIMT in presence of AuNS100 was further demonstrated by the reduction of fibrillation of Aβ42, the peptide that is implicated in Alzheimer’s disease. The enhancement of anti-fibrillation activity of PIMT with AuNS100 was confirmed from cell survival assay with PC12 derived neuronal cells against Aβ42 induced neurotoxicity.

The enzyme L-isoaspartyl (D-aspartyl) methyltransferase promotes migration and invasion in human U-87 MG and U-251 MG glioblastoma cell lines

The protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) recognizes abnormal L-isoaspartyl and D-aspartyl residues in proteins. Among examined tissues, PIMT shows the highest level in the brain. The U-87 MG cell line is a commonly used cellular model to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT amount increased when U-87 MG cells were detached from the extracellular matrix. Recently, we also showed that PIMT possessed pro-angiogenic properties. Together, these PIMT features led us to postulate that PIMT could play a critical role in glioblastoma growth. Here, we investigate PIMT role in U-87 MG cell viability, adhesion, migration, invasion, and colony formation and in the reorganization of the actin and tubulin cytoskeleton. PIMT inhibition by siRNA significantly reduced in vitro cell migration and invasion in various assays, including wound-healing assay, Boyden chambers coated with gelatin and Matrigel invasion assay. Conversely, in stably transfected U-87 MG cells overexpressing wild-type PIMT, cell migration, invasive capacity and colony formation significantly increased. However, in stably transfected cells with the gene encoding for mutated PIMT(D83V), despite of its overexpression, migration and invasion remained similar to those observed in control cells. In all these conditions, cell viability was unaffected. Importantly, overexpressed wild-type PIMT and mutated PIMT(D83V) have opposite effects on the organization of microtubules and actin cytoskeleton and thus on morphology of U-87 cells. These data highlighted the importance of PIMT level and its catalytic activity in migration and invasion of U-87 glioma cells and its possible contribution in cancer invasion during glioma growth.

L-Isoaspartyl Methyltransferase Deficiency in Zebrafish Leads to Impaired Calcium Signaling in the Brain.

Isomerization of l-aspartyl and l-asparaginyl residues to l-isoaspartyl residues is one type of protein damage that can occur under physiological conditions and leads to conformational changes, loss of function, and enhanced protein degradation. Protein l-isoaspartyl methyltransferase (PCMT) is a repair enzyme whose action initiates the reconversion of abnormal l-isoaspartyl residues to normal l-aspartyl residues in proteins. Many lines of evidence support a crucial role for PCMT in the brain, but the mechanisms involved remain poorly understood. Here, we investigated PCMT activity and function in zebrafish, a vertebrate model that is particularly well-suited to analyze brain function using a variety of techniques. We characterized the expression products of the zebrafish PCMT homologous genes pcmt and pcmtl. Both zebrafish proteins showed a robust l-isoaspartyl methyltransferase activity and highest mRNA transcript levels were found in brain and testes. Zebrafish morphant larvae with a knockdown in both the pcmt and pcmtl genes showed pronounced morphological abnormalities, decreased survival, and increased isoaspartyl levels. Interestingly, we identified a profound perturbation of brain calcium homeostasis in these morphants. An abnormal calcium response upon ATP stimulation was also observed in mouse hippocampal HT22 cells knocked out for Pcmt1. This work shows that zebrafish is a promising model to unravel further facets of PCMT function and demonstrates, for the first time in vivo, that PCMT plays a pivotal role in the regulation of calcium fluxes.

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.

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.