The role of protein L-isoaspartyl/D-aspartyl O-methyltransferase (PIMT) in intracellular signal transduction.

Under physiological conditions, L-aspartyl (L-Asp) and L-asparaginyl residues in proteins are spontaneously isomerized or racemized to D-aspartyl (D-Asp) or D,L-isoaspartyl (D,L-isoAsp) residue. These atypical Asp residues can interfere with protein activity and lead to disruption of cellular function. Protein L-isoaspartyl/D-aspartyl O-methyltransferase (PIMT) is a repair enzyme that initiates the conversion of L-isoAsp (or D-Asp) residues to L-Asp residues. PIMT-Deficient mice exhibit accumulation of L-isoAsp in several tissues and die from progressive epileptic seizures at a mean age of 42 days. However, the biological roles of PIMT are still largely unknown. To further our understanding of the function of this protein, we developed an assay to measure PIMT activity in cell lysates. Additionally, we generated PIMT-knockdown cells by stable transfection of HEK293 cells with PIMT small interfering (si) RNA. Northern blotting and immunoblot analysis revealed that PIMT mRNA and protein levels were significantly decreased in the knockdown cells. In addition, significant levels of proteins that contained isoAsp residues accumulated in these cells, and immunoblot analysis revealed that Raf-1, MEK, and ERK were hyperphosphorylated upon EGF stimulation compared to control cells. These results indicate that the ability to repair atypical Asp residues is important for normal MAP kinase signaling.

The role of isoaspartate in fibrillation and its prevention by Protein-L-isoaspartyl methyltransferase.

BACKGROUND: Isomerization of aspartate to isoaspartate (isoAsp) on aging causes protein damage and malfunction. Protein-L-isoaspartyl methyltransferase (PIMT) performs a neuroprotective role by repairing such residues. A hexapeptide, Val-Tyr-Pro-(isoAsp)-His-Ala (VA6), a substrate of PIMT, is shown to form fibrils, while the normal Asp-containing peptide does not. Considering the role of PIMT against epileptic seizure, the combined effect of PIMT and two antiepileptic drugs (AEDs) (valproic acid and stiripentol) was investigated for anti-fibrillation activity. METHODS: Structural/functional modulations due to the binding of AEDs to PIMT were investigated using biophysical techniques. Thioflavin T (ThT) fluorescence assay and microscopic methods were employed to study fibril formation by VA6. In vitro experiments with PC12 cells were carried out with PIMT/AEDs. RESULTS: ThT assay indicated reduction of fibrillation of VA6 by PIMT. AEDs stabilize PIMT, bind close to the cofactor binding site, possibly exerting allosteric effect, increase the enzymatic activity, and anti-fibrillation efficacy. Furthermore, Aß42, implicated in Alzheimer's disease, undergoes ß-sheet to α-helix transition in presence of PIMT. Studies with PC12 derived neurons showed that PIMT and PIMT/AEDs exerted neuroprotective effect against anti-NGF induced neurotoxicity. This was further validated against neurotoxicity induced by Aß42 in primary rat cortical neurons. CONCLUSIONS: The study provides a new perspective to the role isoAsp in protein fibrillation, PIMT in its prevention and AEDs in enhancing the activity of the enzyme. GENERAL SIGNIFICANCE: IsoAsp, with an additional C atom in the main-chain of polypeptide chain, may make it more susceptible to fibrillation. PIMT alone, or in association with AEDs prevents this.

[Role of isomerized protein repair enzyme, PIMT, in cellular functions].

Proteins are subject to various types of spontaneous modifications that can disrupt their structures with sometimes adverse affects on biological activity. The formation of L-isoaspartyl (or D-aspartyl) residues, through either the deamidation of asparagine or dehydration of aspartate, is one of the most frequent types of deterioration occurring under physiological conditions. Protein L-isoaspartate/D-aspartate o-methyltransferase (PIMT) is a conserved and ubiquitous enzyme that participates in the repair of various isomerized proteins. PIMT catalyzes the transfer of the methyl group of S-adenosyl-L-methionine onto the alpha-carboxyl group of an L-isoaspartyl (or the beta-carboxyl group of an D-aspartyl) residue, which initiates the conversion of this residue to an L-aspartyl residue. PIMT-deficient mice have been shown to die at a mean age of 42 days from progressive epileptic seizures with grand mal and myoclonus. Although PIMT-deficiency clearly leads to the accumulation of isomerized proteins, it is currently unclear how this causes progressive epilepsy in PIMT-deficient mice. As a first step towards understanding this, we developed a new assay to measure PIMT activity in cell lysates. Additionally, we isolated PIMT knockdown cells from HEK293 cells that were stably transfected with a PIMT small interfering RNA expression vector. PIMT activities were significantly decreased in the PIMT knockdown cells, and analysis of the transfectants revealed that MEK and ERK were hyperactivated after cell stimulation with epidermal growth factor (EGF). These results indicate that the ability to repair L-isoaspartyl-(or D-aspartyl-) containing proteins is important for the maintenance of normal MEK-ERK signaling.

Accelerated protein damage in brains of PIMT+/- mice; a possible model for the variability of cognitive decline in human aging.

Isoaspartate formation is a common type of protein damage normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). Mice with a knockout of the gene (Pcmt1) for this enzyme (KO, -/-) exhibit a pronounced neuropathology with fatal epileptic seizures at 30-60 days. Heterozygous (HZ, +/-) mice have 50% of the PIMT activity found in wild-type (WT, +/+) mice, but appear normal. To see if HZ mice exhibit accelerated aging at the molecular level, we compared brain extracts from HZ and WT mice at 8 months and 2 years with regard to PIMT activity, isoaspartate levels, and activity of an endogenous PIMT substrate, creatine kinase B. PIMT activity declined modestly with age in both genotypes. Isoaspartate was significantly higher in HZ than WT mice at 8 months and more so at 2 years, rising 5× faster in HZ males and 3× faster in females. Creatine kinase activity decreased with age and was always lower in the HZ mice. These findings suggest the individual variation of human PIMT levels may significantly influence the course of age-related central nervous system dysfunction.

Aging as war between chemical and biochemical processes: protein methylation and the recognition of age-damaged proteins for repair.

Deamidated, isomerized, and racemized aspartyl and asparaginyl residues represent a significant part of the spontaneous damage to proteins that results from the aging process. The accumulation of these altered residues can lead to the loss of protein function and the consequent loss of cellular function. However, almost all cells in nature contain a methyltransferase that can recognize the major damaged form of the L-isoaspartyl residue, and some of these enzymes can also recognize the racemized D-aspartyl residue. The methyl esterification reaction can initiate the conversion of these altered residues to the normal L-aspartyl form, although there is no evidence yet that the L-asparaginyl form can be regenerated. This enzyme, the protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77), thus functions as a protein repair enzyme. The importance of this enzyme in attenuating age-related protein damage can be seen by the phenotypes of organisms where the gene encoding has been disrupted, or where its expression has been augmented.

Improved rotorod performance and hyperactivity in mice deficient in a protein repair methyltransferase.

The protein L-isoaspartate (D-aspartate)-O-methyltransferase participates in the repair of age-induced protein damage by initiating the conversion of abnormal aspartyl residues within proteins to normal L-aspartyl residues. Previous studies have shown that mice deficient in the gene encoding this enzyme (Pcmt1-/-) accumulate damaged proteins, have altered levels of brain S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy), and suffer from epileptic seizures that result in death at an average age of about 42 days. In this study, we found that the behavior of Pcmt1-/- mice is abnormal in comparison to their wild-type (Pcmt1+/+) and heterozygous (Pcmt1+/-) littermates in two standard quantitative behavioral assays - the accelerating rotorod and the open-field test. On the accelerating rotorod, we found Pcmt1-/- mice actually perform significantly better than their heterozygous and wild-type littermates, a situation that has only been infrequently described in the literature and has not been described to date for epilepsy-prone mice. The Pcmt1-/- mice show, however, hyperactivity in the open-field test that becomes more pronounced with age, with a partial habituation with time in the chamber. Additionally, these mice demonstrate a strong thigmotaxic movement pattern. We present evidence that these phenotypes are not related to the alterations of the AdoMet/AdoHcy ratio in the brain and thus may be a function of the accumulation of damaged proteins. These results implicate a role for this enzyme in motor coordination and cerebellum development and suggest the importance of the function of the repair methyltransferase in hippocampal-dependent spatial learning.

Damaged proteins bearing L-isoaspartyl residues and aging: a dynamic equilibrium between generation of isomerized forms and repair by PIMT.

Proteins are susceptible to numerous non-enzymatic post-translational modifications which occur both during normal aging and in neurodegenerative states. For instance, formation of abnormal L-isoaspartyl residues arising from both the deamidation of L-asparaginyl residues and the isomerization of L-aspartyl residues is a frequent chemical modification that affects proteins. The formation of L- isoaspartyl residues in proteins alters their three-dimensional structure leading usually to a loss of function. Notably, accumulation of isomerized proteins could contribute to metabolic dysfunctions in neuronal cells during aging reducing cognitive functions in elderly patients and would eventually promote the development of neurodegenerative diseases. The protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) is an enzyme that recognizes and repairs the abnormal L-isoaspartyl residues in proteins. Its expression appears to decline during aging which could partially explain the build up of damaged proteins in old age. In this review, we summarize recent findings, based mostly on proteomic data, regarding the formation and accumulation of proteins bearing atypical L-isoaspartyl residues as well as PIMT functions during normal aging and in some neurodegenerative diseases. The emphasis is on possible molecular mechanisms controlling PIMT expression and the ability of PIMT to repair isomerized substrates during aging. Investigation of processes regulating age-related accumulation of isomerized proteins is a promising avenue to a better understanding of aging at the protein level.

[Seed aging and survival mechanisms]. / Vieillissement des semences et mécanismes de survie.

Aging and death are universal to living systems. In temperate climate latitudes the mature seeds of higher plants are exposed to aging and have developed resistance mechanisms allowing survival and plant propagation. In addition to the physicochemical properties of the seed that confer stress resistance, the protein metabolism contributes importantly to longevity mechanisms. Recently, genetic studies have demonstrated the occurrence of the Protein L-isoaspartyl methyltransferase repair enzyme in controlling age-related protein damages and seed survival. These protective mechanisms by protein repair are widespread in all kingdoms, so that the use of seeds as models to study these controlling processes offers the prospect of understanding longevity mechanisms better.

Accumulation of proteins bearing atypical isoaspartyl residues in livers of alcohol-fed rats is prevented by betaine administration: effects on protein-L-isoaspartyl methyltransferase activity.

BACKGROUND/AIMS: Protein-L-isoaspartyl methyltransferase (PIMT) is a methyltransferase that plays a crucial role in the repair of damaged proteins. In this study, we investigated whether ethanol exposure causes an accumulation of modified proteins bearing atypical isoaspartyl residues that may be related to impaired PIMT activity. We further sought to determine whether betaine administration could prevent the accumulation of these types of damaged proteins. METHODS: Livers of male Wistar rats, fed the Lieber DeCarli control, ethanol or 1% betaine-supplemented diets for 4 weeks, were processed for PIMT-related analyses. RESULTS: We observed a significant increase in the accumulation of modified proteins bearing isoaspartyl residues, i.e. the substrates for PIMT, in homogenate samples and various subcellular fractions of livers from ethanol-fed rats. Betaine supplementation prevented this accumulation of damaged proteins. In contrast, ethanol exposure induced no changes in the PIMT enzyme activity levels as compared to controls. The accumulation of damaged proteins negatively correlated with hepatic S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratios. CONCLUSIONS: Ethanol consumption results in the accumulation of modified proteins bearing atypical isoaspartyl residues via impaired in vivo PIMT activity. Betaine administration prevents the ethanol-induced accumulation of isoaspartyl-containing proteins by restoring the PIMT-catalyzed protein repair reaction through normalizing the hepatocellular SAM:SAH ratios.