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1.
Cell Biol Int ; 45(11): 2316-2330, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34314072

ABSTRACT

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.


Subject(s)
Diabetic Nephropathies/genetics , MicroRNAs/genetics , Neurodegenerative Diseases/genetics , Animals , China , Gene Expression/genetics , Gene Expression Profiling/methods , Male , Mice , Mice, Knockout , MicroRNAs/analysis , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Transcriptome/genetics
2.
PLoS One ; 9(6): e100622, 2014.
Article in English | MEDLINE | ID: mdl-24955845

ABSTRACT

Isoaspartate (isoAsp) formation is a common type of spontaneous protein damage that is normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). PIMT-KO (knockout) mice exhibit a pronounced neuropathology highlighted by death from an epileptic seizure at 30 to 60 days after birth. The mechanisms by which isoaspartyl damage disrupts normal brain function are incompletely understood. Proteomic analysis of the PIMT-KO mouse brain has shown that a number of key neuronal proteins accumulate high levels of isoAsp, but the extent to which their cellular functions is altered has yet to be determined. One of the major neuronal targets of PIMT is creatine kinase B (CKB), a well-characterized enzyme whose activity is relatively easy to assay. We show here that (1) the specific activity of CKB is significantly reduced in the brains of PIMT-deficient mice, (2) that in vitro aging of recombinant CKB results in significant accumulation of isoAsp sites with concomitant loss of enzymatic activity, and (3) that incubation of in vitro aged CKB with PIMT and its methyl donor S-adenosyl-L-methionine substantially repairs the aged CKB with regard to both its isoAsp content and its enzymatic activity. These results, combined with similarity in phenotypes of PIMT-KO and CKB-KO mice, suggests that loss of normal CKB structure and function contributes to the mechanisms by which isoAsp accumulation leads to CNS dysfunction in the PIMT-KO mouse.


Subject(s)
Brain/enzymology , Brain/physiopathology , Creatine Kinase/metabolism , Isoaspartic Acid/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Amino Acid Sequence , Animals , Biocatalysis , Brain/pathology , Creatine Kinase/chemistry , Heterozygote , Humans , Isoaspartic Acid/chemistry , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Phenotype , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Recombinant Proteins/metabolism , Sequence Alignment , Tissue Extracts
3.
J Biol Chem ; 289(24): 16936-53, 2014 Jun 13.
Article in English | MEDLINE | ID: mdl-24764295

ABSTRACT

The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50-300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.


Subject(s)
Isoaspartic Acid/metabolism , Metalloproteases/metabolism , Peptides/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Animals , Humans , Metalloproteases/genetics , Phylogeny , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Proteolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
4.
J Proteome Res ; 12(10): 4566-76, 2013 Oct 04.
Article in English | MEDLINE | ID: mdl-23947766

ABSTRACT

Protein l-isoaspartyl methyltransferase (PIMT) repairs the isoaspartyl residues (isoAsp) that originate from asparagine deamidation and aspartic acid (Asp) isomerization to Asp residues. Deletion of the gene encoding PIMT in mice (Pcmt1) leads to isoAsp accumulation in all tissues measured, especially in the brain. These PIMT-knockout (PIMT-KO) mice have perturbed glutamate metabolism and die prematurely of epileptic seizures. To elucidate the role of PIMT further, brain proteomes of PIMT-KO mice and controls were analyzed. The isoAsp levels from two of the detected 67 isoAsp sites (residue 98 from calmodulin and 68 from glyceraldehyde-3-phosphate dehydrogenase) were quantified and found to be significantly increased in PIMT-KO mice (p < 0.01). Additionally, the abundance of at least 151 out of the 1017 quantified proteins was found to be altered in PIMT-KO mouse brains. Gene ontology analysis revealed that many down-regulated proteins are involved in cellular amino acid biosynthesis. For example, the serine synthesis pathway was suppressed, possibly leading to reduced serine production in PIMT-KO mice. Additionally, the abundances of enzymes in the glutamate-glutamine cycle were altered toward the accumulation of glutamate. These findings support the involvement of PIMT in glutamate metabolism and suggest that the absence of PIMT also affects other processes involving amino acid synthesis and metabolism.


Subject(s)
Brain/metabolism , Glutamic Acid/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Proteome/metabolism , Amino Acid Sequence , Animals , Female , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Principal Component Analysis , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Proteome/chemistry , Proteomics
5.
Anal Chem ; 85(4): 2423-30, 2013 Feb 19.
Article in English | MEDLINE | ID: mdl-23327623

ABSTRACT

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.


Subject(s)
Isoaspartic Acid/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Proteins/analysis , Proteomics , Tandem Mass Spectrometry , Animals , Chromatography, High Pressure Liquid , Deamination , Mice , Mice, Knockout , Peptides/analysis , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Proteins/metabolism
6.
PLoS One ; 7(10): e46719, 2012.
Article in English | MEDLINE | ID: mdl-23071621

ABSTRACT

L-isoaspartyl (D-aspartyl) O-methyltransferase deficient mice (Pcmt1(-/-)) accumulate isomerized aspartyl residues in intracellular proteins until their death due to seizures at approximately 45 days. Previous studies have shown that these mice have constitutively activated insulin signaling in their brains, and that these brains are 20-30% larger than those from age-matched wild-type animals. To determine whether insulin pathway activation and brain enlargement is responsible for the fatal seizures, we administered wortmannin, an inhibitor of the phosphoinositide 3-kinase that catalyzes an early step in the insulin pathway. Oral wortmannin reduced the average brain size in the Pcmt1(-/-) animals to within 6% of the wild-type DMSO administered controls, and nearly doubled the lifespan of Pcmt1(-/-) at 60% survival of the original population. Immunoblotting revealed significant decreases in phosphorylation of Akt, PDK1, and mTOR in Pcmt1(-/-) mice and Akt and PDK1 in wild-type animals upon treatment with wortmannin. These data suggest activation of the insulin pathway and its resulting brain enlargement contributes to the early death of Pcmt1-/- mice, but is not solely responsible for the early death observed in these animals.


Subject(s)
Androstadienes/administration & dosage , Insulin/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Seizures/drug therapy , Administration, Oral , Androstadienes/pharmacology , Animals , Asparagine/metabolism , Aspartic Acid/metabolism , Brain/drug effects , Brain/metabolism , Brain/pathology , Female , Insulin/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Knockout , Organ Size/drug effects , Phosphoinositide-3 Kinase Inhibitors , Phosphoproteins/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein Processing, Post-Translational/drug effects , Seizures/metabolism , Signal Transduction , Wortmannin
7.
J Biol Chem ; 281(44): 33802-13, 2006 Nov 03.
Article in English | MEDLINE | ID: mdl-16959769

ABSTRACT

Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-(3)H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 (3)H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases.


Subject(s)
Brain/enzymology , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Animals , Isoaspartic Acid/biosynthesis , Isoaspartic Acid/chemistry , Mass Spectrometry , Mice , Mice, Knockout , Molecular Structure , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Proteomics , Rats , Substrate Specificity
8.
J Biol Chem ; 281(43): 32619-29, 2006 Oct 27.
Article in English | MEDLINE | ID: mdl-16923807

ABSTRACT

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.


Subject(s)
Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Proteomics , Animals , Autoradiography , Brain Chemistry , Cell Fractionation , Methylation , Mice , Mice, Knockout , Peptide Mapping , Precipitin Tests , Protein D-Aspartate-L-Isoaspartate Methyltransferase/analysis , Protein D-Aspartate-L-Isoaspartate Methyltransferase/chemistry , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Subcellular Fractions , Substrate Specificity
9.
J Biol Chem ; 281(13): 8389-98, 2006 Mar 31.
Article in English | MEDLINE | ID: mdl-16443604

ABSTRACT

The accumulation of potentially deleterious L-isoaspartyl linkages in proteins is prevented by the action of protein L-isoaspartyl O-methyltransferase, a widely distributed enzyme that is particularly active in mammalian brain. Methyltransferase-deficient (knock-out) mice exhibit greatly increased levels of isoaspartate and typically succumb to fatal epileptic seizures at 4-10 weeks of age. The link between isoaspartate accumulation and the neurological abnormalities of these mice is poorly understood. Here, we demonstrate that synapsin I from knock-out mice contains 0.9 +/- 0.3 mol of isoaspartate/mol of synapsin, whereas the levels in wild-type and heterozygous mice are undetectable. Transgenic mice that selectively express methyltransferase only in neurons show reduced levels of synapsin damage, and the degree of reduction correlates with the phenotype of these mice. Isoaspartate levels in synapsin from the knock-out mice are five to seven times greater than those in the average protein from brain cytosol or from a synaptic vesicle-enriched fraction. The isoaspartyl sites in synapsin from knock-out mice are efficiently repaired in vitro by incubation with purified methyltransferase and S-adenosyl-L-methionine. These findings demonstrate that synapsin I is a major substrate for the isoaspartyl methyltransferase in neurons and suggest that isoaspartate-related alterations in the function of presynaptic proteins may contribute to the neurological abnormalities of mice deficient in this enzyme.


Subject(s)
Brain/enzymology , Brain/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Synapsins/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Binding Sites , Blotting, Western , Calmodulin/analysis , Calmodulin/isolation & purification , Cattle , Cell Fractionation , Cluster Analysis , Electrophoresis, Polyacrylamide Gel , Heterozygote , Isoenzymes/genetics , Isoenzymes/metabolism , Methylation , Mice , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein D-Aspartate-L-Isoaspartate Methyltransferase/analysis , Protein D-Aspartate-L-Isoaspartate Methyltransferase/chemistry , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Sequence Homology, Amino Acid , Subcellular Fractions , Substrate Specificity , Synapsins/isolation & purification , Trypsin/pharmacology
10.
J Comp Neurol ; 493(4): 524-37, 2005 Dec 26.
Article in English | MEDLINE | ID: mdl-16304629

ABSTRACT

Recent studies have demonstrated that mice lacking protein L-isoaspartate (D-aspartate) O-methyltransferase (Pcmt1-/- mice) have alterations in the insulin-like growth factor-I (IGF-I) and insulin receptor pathways within the hippocampal formation as well as other brain regions. However, the cellular localization of these changes and whether the alterations might be associated with an increase in cell number within proliferative regions, such as the dentate gyrus, were unknown. In this study, stereological methods were used to demonstrate that these mice have an increased number of granule cells in the granule cell layer and hilus of the dentate gyrus. The higher number of granule cells was accompanied by a greater number of cells undergoing mitosis in the dentate gyrus, suggesting that an increase in neuronal cell proliferation occurs in this neurogenic zone of adult Pcmt1-/- mice. In support of this, increased doublecortin labeling of immature neurons was detected in the subgranular zone of the dentate gyrus. In addition, double immunofluorescence studies demonstrated that phosphorylated IGF-I/insulin receptors in the subgranular zone were localized on immature neurons, suggesting that the increased activation of one or both of these receptors in Pcmt1-/- mice could contribute to the growth and survival of these cells. We propose that deficits in the repair of isoaspartyl protein damage leads to alterations in metabolic and growth-receptor pathways, and that this model may be particularly relevant for studies of neurogenesis that is stimulated by cellular damage.


Subject(s)
Cell Proliferation , Dentate Gyrus/enzymology , Neurons/enzymology , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Receptor, Insulin/metabolism , Animals , Cell Differentiation/physiology , Dentate Gyrus/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/cytology , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Stem Cells/cytology , Stem Cells/enzymology , Tissue Distribution
11.
Biol Pharm Bull ; 28(9): 1590-6, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16141521

ABSTRACT

Isomerization of L-aspartate and deamidation of L-asparagine in proteins or peptides dominantly give rise to L-isoaspartate by a non-enzymatic reaction via succinimide as a intermediate under physiological conditions. Isoaspartates have been identified in a variety of cellular proteins in vivo as well as pathologically deposited proteins in neurodegenerative brain tissue. We described here that the formation of isoaspartate is enhanced in amyloid-beta (Abeta) peptides in Alzheimer's disease (AD). Specific antibodies recognizing isoaspartate of Abeta revealed that isomerized Abeta peptides were deposited in senile plaques as well as amyloid-bearing vessels. Moreover, it was revealed that Abeta peptides, isomerized at position 7 or 23, were differentially deposited in senile plaques and vascular amyloids in AD brains. In vitro experiments showed that the modification at position 23 greatly enhanced the aggregation of Abeta. Furthermore, systematic proline substitution analyses revealed that the beta-turn structure at positions 22 and 23 of Abeta42 plays a crucial role in the aggregation and neurotoxicity of Abeta peptides. It is suggested that spontaneous isomerization at position 23 induces the conformational change to form a beta-turn at position 23, which plays a pathogenic role in the deposition of Abeta peptides in sporadic AD. Protein L-isoaspartyl methyltransferase (PIMT) is a putative protein repair enzyme, which converts L-isoaspartyl residues in damaged proteins to normal L-aspartyl residues. PIMT-deficient mice manifested neurodegenerative changes concomitant with the accumulation of L-isoaspartate in the brain. We discuss here the pathological implications of the formation of isoaspartate in damaged proteins during neurodegeneration in model mice and AD.


Subject(s)
Isoaspartic Acid/physiology , Alzheimer Disease/enzymology , Alzheimer Disease/metabolism , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Animals , Brain Chemistry/physiology , Humans , Isoaspartic Acid/metabolism , Mice , Mice, Knockout , Molecular Sequence Data , Plaque, Amyloid/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Proteins/chemistry , Proteins/metabolism
12.
Behav Brain Res ; 153(1): 129-41, 2004 Aug 12.
Article in English | MEDLINE | ID: mdl-15219714

ABSTRACT

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.


Subject(s)
Exploratory Behavior/physiology , Hyperkinesis , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Rotarod Performance Test , Age Factors , Aging/physiology , Animals , Brain Chemistry , Chromatography, High Pressure Liquid/methods , Diet, Reducing/methods , Genotype , Homocysteine/blood , Mice , Mice, Knockout , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Protein D-Aspartate-L-Isoaspartate Methyltransferase/physiology , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/metabolism , Stereotyped Behavior/physiology , Survival Rate , Time Factors
13.
J Immunol ; 171(6): 2840-7, 2003 Sep 15.
Article in English | MEDLINE | ID: mdl-12960305

ABSTRACT

It is clear that many factors can perturb T cell homeostasis that is critical in the maintenance of immune tolerance. Defects in the molecules that regulate homeostasis can lead to autoimmune pathology. This simple immunologic concept is complicated by the fact that many self-proteins undergo spontaneous posttranslational modifications that affect their biological functions. This is the case in the spontaneous conversion of aspartyl residues to isoaspartyl residues, a modification occurring at physiological pH and under conditions of cell stress and aging. We have examined the effect of isoaspartyl modifications on the effector functions of T lymphocytes in vivo using mice lacking the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT). PCMT(-/-) CD4(+) T cells exhibit increased proliferation in response to mitogen and Ag receptor stimulation as compared with wild-type CD4(+) T cells. Hyperproliferation is marked by increased phosphorylation of members of both the TCR and CD28 signaling pathways. Wild-type mice reconstituted with PCMT(-/-) bone marrow develop high titers of anti-DNA autoantibodies and kidney pathology typical of that found in systemic lupus erythematosus. These observations, coupled with the fact that humans have polymorphisms in the pcmt gene, suggest that isoaspartyl self-proteins may alter the maintenance of peripheral immune tolerance.


Subject(s)
Autoantibodies/biosynthesis , Autoantigens/immunology , Autoantigens/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Animals , Bone Marrow Transplantation/immunology , CD28 Antigens/pharmacology , Cell Division/genetics , Cell Division/immunology , Immunophenotyping , Isoaspartic Acid/metabolism , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , Lymph Nodes/enzymology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymphocyte Activation/genetics , Lymphoid Tissue/enzymology , Lymphoid Tissue/metabolism , Lymphoid Tissue/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogens/pharmacology , Phosphorylation , Protein D-Aspartate-L-Isoaspartate Methyltransferase/physiology , Receptors, Antigen, T-Cell/physiology , Signal Transduction/genetics , Signal Transduction/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology
14.
J Neurosci Res ; 69(3): 341-52, 2002 Aug 01.
Article in English | MEDLINE | ID: mdl-12125075

ABSTRACT

Protein-L-isoaspartyl methyltransfearase (PIMT) plays a physiological role in the repair of damaged proteins containing isoaspartyl residues. In previous studies, we showed that PIMT-deficient mice developed a fatal epileptic seizure associated with the accumulation of damaged proteins in the brain. The mutant mice also showed a neurodegenerative pathology in hippocampi and impaired spatial memory. Still undefined, however, is how the accumulation of isoaspartates leads to the death of PIMT-deficient mice. In the present study, we generated PIMT transgenic (Tg) mice to investigate whether the exogenous expression of PIMT could improve the symptoms associated with PIMT deficiency. Rescue experiments showed that Tg expression of PIMT driven by a prion promoter effectively cured the PIMT-deficient mice. Biochemically, a higher expression level of transgene led to the effective repair of damaged proteins in vivo. Although a lower level of expression caused an accumulation of damaged proteins in a partially rescued line, the mice survived. Interestingly, synapsin I, which was extensively modified posttranslationally in PIMT-deficient mice, was specifically repaired in a partially rescued, but symptom-improved, Tg line. Our results suggest that an overall accumulation of damaged proteins does not necessarily lead to a fatal epileptic seizure, whereas certain modifications, such as changes in synapsin I, may play a pivotal pathological role in epilepsy.


Subject(s)
Brain/enzymology , Epilepsy/enzymology , Epilepsy/genetics , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/therapeutic use , Animals , Blotting, Western , Gene Expression Regulation, Enzymologic , Genetic Therapy , Immunohistochemistry , Mice , Mice, Transgenic , Phenotype , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Synapsins/metabolism
15.
J Neurosci Res ; 69(3): 353-61, 2002 Aug 01.
Article in English | MEDLINE | ID: mdl-12125076

ABSTRACT

Protein-L-isoaspartyl methyltransferase (PIMT) is a putative protein repair enzyme, which methylates the alpha-carboxyl group of atypical L-isoaspartyl residues in aged proteins and converts them to normal L-aspartyl residues. Two splicing variants, PIMT-I and PIMT-II, have been reported, although their biological functions and specific subcellular substrates are still to be defined. We and another group have previously showed that PIMT-deficient mice succumbed to fatal epileptic seizures associated with an abnormal accumulation of isoaspartate (IsoAsp) in the brain. In the present study, we prepared two recombinant adenovirus vectors that contained PIMT-I or PIMT-II, respectively, in order to investigate the differential biological roles of PIMT-I and PIMT-II. These recombinant viruses differentially conferred PIMT-I or PIMT-II expressions in cultured neurons. Biochemical analyses showed that either of PIMT-I or PIMT-II effectively repaired the damaged proteins in PIMT-deficient neurons, but the concomitant expression failed to show an additive effect in the repair of IsoAsp. These results suggested that PIMT-I and PIMT-II might share a common biological function and/or subcellular substrates. In addition, we administered an adeno-PIMT-I vector into the brain of PIMT-deficient mice at embryonic day 14.5 by an exo-utero method to assess the biological effects in vivo. The result showed that recombinant adeno-PIMT improved the symptoms of PIMT-deficient mice in vivo, but only partially repaired IsoAsp in damaged proteins. The gene therapy presented in this report provided a better prognosis for the survival of PIMT-deficient mice than the previously reported anti-epileptic drug therapy. The results suggested a new reagent for gene therapy applicable to ageing-associated neurodegenerative disorders.


Subject(s)
Brain/enzymology , Epilepsy/enzymology , Epilepsy/genetics , Genetic Therapy/methods , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/therapeutic use , Adenoviridae , Animals , Blotting, Western , Brain/embryology , Cell Culture Techniques , Fibroblasts , Genetic Vectors , Hippocampus/enzymology , Immunohistochemistry , Isoenzymes , Mice , Mice, Transgenic , Neurons/enzymology , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Recombinant Proteins/therapeutic use
16.
J Biol Chem ; 277(31): 27856-63, 2002 Aug 02.
Article in English | MEDLINE | ID: mdl-12023972

ABSTRACT

L-Isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) is a protein repair enzyme that initiates the conversion of abnormal D-aspartyl and L-isoaspartyl residues to the normal L-aspartyl form. In the course of this reaction, PCMT1 converts the methyl donor S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Due to the high level of activity of this enzyme, particularly in the brain, it seemed of interest to investigate whether the lack of PCMT1 activity might alter the concentrations of these small molecules. AdoMet and AdoHcy were measured in mice lacking PCMT1 (Pcmt1-/-), as well as in their heterozygous (Pcmt1+/-) and wild type (Pcmt1+/+) littermates. Higher levels of AdoMet and lower levels of AdoHcy were found in the brains of Pcmt1-/- mice, and to a lesser extent in Pcmt1+/- mice, when compared with Pcmt1+/+ mice. In addition, these levels appear to be most significantly altered in the hippocampus of the Pcmt1-/- mice. The changes in the AdoMet/AdoHcy ratio could not be attributed to increases in the activities of methionine adenosyltransferase II or S-adenosylhomocysteine hydrolase in the brain tissue of these mice. Because changes in the AdoMet/AdoHcy ratio could potentially alter the overall excitatory state of the brain, this effect may play a role in the progressive epilepsy seen in the Pcmt1-/- mice.


Subject(s)
Brain/metabolism , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/metabolism , Adenosylhomocysteinase , Animals , Aspartic Acid/metabolism , Hydrolases/metabolism , Kinetics , Methionine Adenosyltransferase/metabolism , Mice , Mice, Knockout , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Protein D-Aspartate-L-Isoaspartate Methyltransferase/genetics , Time Factors
17.
J Biol Chem ; 276(40): 37161-5, 2001 Oct 05.
Article in English | MEDLINE | ID: mdl-11479322

ABSTRACT

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.


Subject(s)
Adenosine/analogs & derivatives , Histones/chemistry , Protein D-Aspartate-L-Isoaspartate Methyltransferase/metabolism , Adenosine/pharmacology , Animals , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Histones/metabolism , Isoaspartic Acid/metabolism , Mice , PC12 Cells , Protein Conformation , Protein D-Aspartate-L-Isoaspartate Methyltransferase/deficiency , Rats , Substrate Specificity
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