首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
We investigated whether alterations in glutamate metabolising glutamine synthetase activity occur in human epileptic neocortex, as shown previously for human epileptic hippocampus [Eid, T., Thomas, M.J., Spencer, D.D., Rundén-Pran, E., Lai, J.C.K., Malthankar, G.V., Kim, J.H., Danbolt, N.C., Ottersen, O.P., de Lanerolle, N.C., 2004. Loss of glutamine synthetase in the human epileptic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy. Lancet 363, 28-37]. Glutamine synthetase activity was equivalent in both non-epileptic and epileptic human neocortex. Epileptic tissue, however, was characterised by a 37% increase in the density of synaptosomal NMDA receptor sites compared to non-epileptic tissue, as revealed by a radioligand binding assay (B max(non-epileptic) 1.45 pmol/mg protein and B max(epileptic) 1.99 pmol/mg protein, P < 0.05). Our findings shed some doubts on a role of glutamine synthetase in the pathophysiology of epilepsy in the neocortex. However, the detection of a significantly reduced enzymatic activity in the epileptic amygdala supports the assumption that the enzyme defect is localized to the epileptic mesial temporal lobe of corresponding patients.  相似文献   

2.
The enzyme L-isoaspartyl methyltransferase (PIMT) is known to repair damaged proteins that have accumulated abnormal aspartyl residues during cell aging. However, little is known about the mechanisms involved in the regulation of PIMT expression. Here we report that PIMT expression in bovine aortic endothelial cells is regulated by cell detachment and readhesion to a substratum. During cell detachment, the PIMT level was rapidly and strongly increased and correlated with a stimulation of protein synthesis. Aside from endothelial cells, PIMT levels were also regulated by cell adhesion in various cancer cell lines. The upregulation of PIMT expression could be prevented by an anti-alphavbeta3 antibody (LM609) or by a cyclic RGD peptide (XJ735) specific to integrin alphavbeta3, indicating that this integrin was likely involved in PIMT regulation. Moreover, we found that PIMT expression returned to the basal level when cells were replated on a substratum after detachment, though downregulation of PIMT expression could be partly prevented by the PI3K inhibitors LY294002 and wortmannin, as well as by the proteasome inhibitors MG-132, lactacystin, and beta-lactone. These findings support the assumption that the PIMT level was downregulated by proteasomal degradation, involving the PI3K pathway, during cell attachment. This study reports new insights on the molecular mechanisms responsible for the regulation of PIMT expression in cells. The regulation of PIMT level upon cell-substratum contact suggests a potential role for PIMT in biological processes such as wound healing, cell migration, and tumor metastasis dissemination.  相似文献   

3.
Abnormal aspartyl residue formation such as L-isoaspartates occurs frequently during aging in long-lived proteins, resulting in the alteration of their structures and biological functions. In this study, we investigated the alteration of aspartyl residues in extracellular matrix (ECM) proteins, type-I collagen and fibronectin, and in integrin- and ECM-binding motifs during aging, as well as the resulting effects on cell biological functions such as migration and attachment. Using protein L-isoaspartyl methyltransferase (PIMT) to monitor the presence of L-isoaspartyl residues, we showed their accumulation during in vivo aging in type-I collagen from rats. In vitro aging of fibronectin as well as of peptides containing an integrin- or ECM-binding motif such as RGDSR, KDGEA and KDDL also resulted in the formation of L-isoaspartyl residues. While aged fibronectin does not alter cell adhesion and migration, type-I collagen aged 20 months reduced by 65% cell motility, but not adhesion, when compared to 3-month-aged type-I collagen. Finally, by repairing 20-month-old type-I collagen with recombinant PIMT (rPIMT), cell migration was recovered by 72%. These results strongly suggest that L-isoaspartyl residue formation in ECM proteins such as type-I collagen could play an important role in reducing cell migration and that PIMT could be a therapeutic tool to restore normal cell migration in pathological conditions where cell motility is crucial.  相似文献   

4.
l-Aspartyl (l-Asp) and l-asparaginyl residues in proteins isomerize or racemize to d,l-isoaspartyl (d,l-isoAsp) or d-aspartyl (d-Asp) residues during protein aging. These atypical aspartyl residues can interfere with the biological function of the protein and lead to cellular dysfunction. Protein l-isoaspartyl (d-aspartyl) methyltransferase (PIMT) is a repair enzyme that facilitates conversion of l-isoAsp and d-Asp to l-Asp. PIMT deficient mice exhibit accumulation of l-isoAsp in several tissues and die, on average, 12 days after birth from progressive epileptic seizures with grand mal and myoclonus features. However, little is known about the molecular mechanisms by which accumulation of the aberrant residues leads to cellular abnormalities. In this study, we established PIMT-knockdown cells using a short interfering RNA expression system and characterized the resultant molecular abnormalities in intracellular signaling pathways. PIMT-knockdown cells showed significant accumulation of proteins with isomerized residues, compared to control cells. In the PIMT-knockdown cells, Raf-1, MEK, and ERK, members of the MAPK cascade, were hyperphosphorylated after EGF stimulation compared to control cells. These results suggest that PIMT repair of abnormal proteins is necessary to maintain normal MAPK signaling.  相似文献   

5.
M B Mudgett  J D Lowenson    S Clarke 《Plant physiology》1997,115(4):1481-1489
Protein L-isoaspartate (D-aspartate) O-methyltransferases (MTs; EC 2.1.1.77) can initiate the conversion of detrimental L-isoaspartyl residues in spontaneously damaged proteins to normal L-aspartyl residues. We detected this enzyme in 45 species from 23 families representing most of the divisions of the plant kingdom. MT activity is often localized in seeds, suggesting that it has a role in their maturation, quiescence, and germination. The relationship among MT activity, the accumulation of abnormal protein L-isoaspartyl residues, and seed viability was explored in barley (Hordeum vulgare cultivar Himalaya) seeds, which contain high levels of MT. Natural aging of barley seeds for 17 years resulted in a significant reduction in MT activity and in seed viability, coupled with increased levels of "unrepaired" L-isoaspartyl residues. In seeds heated to accelerate aging, we found no reduction of MT activity, but we did observe decreased seed viability and the accumulation of isoaspartyl residues. Among populations of accelerated aged seed, those possessing the highest levels of L-isoaspartyl-containing proteins had the lowest germination percentages. These results suggest that the MT present in seeds cannot efficiently repair all spontaneously damaged proteins containing altered aspartyl residues, and their accumulation during aging may contribute to the loss of seed viability.  相似文献   

6.
Injury to rat blood vessels in vivo was found to release intracellular pools of protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) into the extracellular milieu, where it becomes trapped. This trapped cohort of PIMT is able to utilize radiolabeled S-adenosyl-L-methionine (AdoMet) introduced into the circulation to methylate blood vessel proteins containing altered aspartyl residues. As further shown in this study, methylated substrates are detected only at the specific site of injury. In vitro studies more fully characterized this endogenous PIMT activity in thoracic aorta and inferior vena cava. Methylation kinetics, immunoblotting, and the lability of methylated substrates at mild alkaline pH were used to demonstrate that both types of blood vessel contain an endogeneous protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT). At least 50% of the PIMT activity is resistant to nonionic detergent extraction, suggesting that the enzyme activity becomes trapped within or behind the extracellular matrix (ECM). Quantities of lactate dehydrogenase (LDH), another soluble enzyme of presumed intracellular origin, were found to be similarly trapped in the extracellular space of blood vessels.  相似文献   

7.
8.
As a result of blood vessel injury, protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT), a normally intracellular enzyme, becomes trapped within the meshwork of the vascular extracellular matrix where it can methylate substrate proteins. In this investigation we examined the distribution of such altered aspartyl-containing substrate proteins in the vascular wall. Nearly 90% of all the altered aspartyl residues were inaccessible to intracellular PIMT. Proteins of the extracellular matrix were found to be the major repository of altered aspartyl-containing polypeptides in the blood vessel wall, accounting for 70% of the total amount. Proteolytic cleavage of extracellular matrix proteins with cyanogen bromide (CNBr) revealed that collagens account for most of the altered aspartyl-containing proteins of the ECM. As a consequence of blood vessel injury, both type I and type III collagen along with other proteins were found to become methylated by injury-released PIMT. It is estimated that 1 cm of vein contains on the order of 5×1014 altered aspartyl residues involving between 1% and 5% of the total extracellular protein.  相似文献   

9.
Among the different forms of epilepsies, mesiotemporal lobe epilepsy (MTLE) is one of the most common and represents the main pharmaco-resistant form of epilepsy. There is therefore an urgent need to better understand this form of epilepsy to develop better anti-epileptic drugs. Many rodent models are mimicking some aspects of the human temporal lobe epilepsy but only few are addressing most of the human mesiotemporal lobe epilepsy. In this article, we describe the main characteristics of a mouse of model of mesial temporal lobe epilepsy. This model is generated by a single injection of kainic acid into the dorsal hippocampus which reproduces most of the morphological and electrophysiological features of human MTLE in a mouse. This model may help to better understand mesial temporal lobe epilepsy and the development of new therapeutic drugs.  相似文献   

10.
Protein L-isoaspartyl methyltransferases (PIMT; EC 2.1.1.77) catalyze the S-adenosylmethionine-dependent methylation of L-isoaspartyl residues that arise spontaneously in proteins with age, thereby initiating a repair process that restores the normal backbone configuration to the damaged polypeptide. In Drosophila melanogaster, overexpression of PIMT in transgenic flies extends the normal life span, suggesting that protein damage can be a limiting factor in longevity. To understand structural features of the Drosophila PIMT (dPIMT) important for catalysis, the crystal structure of dPIMT was determined at a resolution of 2.2 A, and site-directed mutagenesis was used to identify the role of Ser-60 in catalysis. The core structure of dPIMT is similar to the modified nucleotide-binding fold observed in PIMTs from extreme thermophiles and humans. A striking difference of the dPIMT structure is the rotation of the C-terminal residues by 90 degrees relative to the homologous structures. Effectively, this displacement generates a more open conformation that allows greater solvent access to S-adenosylhomocysteine, which is almost completely buried in other PIMT structures. The enzyme may alternate between the open conformation found for dPIMT and the more closed conformations described for other PIMTs during its catalytic cycle, thereby allowing the exchange of substrates and products. Catalysis by dPIMT requires the side chain of the conserved, active site residue Ser-60, since substitution of this residue with Thr, Gln, or Ala reduces or abolishes the methylation of both protein and isoaspartyl peptide substrates.  相似文献   

11.
Spontaneous isoaspartyl formation from aspartyl dehydration or asparaginyl deamidation is a major source of modifications in protein structures. In cells, these conformational changes could be reverted by the protein L-isoaspartyl methyltransferase (PIMT) repair enzyme that converts the isoaspartyl residues into aspartyl. The physiological importance of this metabolism has been recently illustrated in plants. Recent developments allowing peptide isomer identification and quantification at the proteome scale are portrayed. The relevance of these new proteomic approaches based on 2-D electrophoresis or electron capture dissociation analysis methods was initially documented in mammals. Extended use to Arabidopsis model systems is promising for the discovery of controlling mechanisms induced by these particular post-translational modifications and their biological role in plants.  相似文献   

12.
Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal l-isoaspartyl residues (IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence of isopeptide bonds impairs protein structure/function. To minimize the damage, IsoAsp can be “repaired” by the protein l-isoaspartyl/d-aspartyl O-methyltransferase (PIMT) and S-adenosylmethionine (AdoMet) is the methyl donor of this reaction. PIMT is a repair enzyme that initiates the conversion of l-isoAsp (or d-Asp) residues to l-Asp residues. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease principally affecting motor neurons. The condition of oxidative stress reported in familial and sporadic forms of ALS prompted us to investigate Asn deamidation in ALS tissue. Erythrocytes (RBCs) were selected as a model system since they are unable to replace damaged proteins and protein methylesterification is virtually the only AdoMet-consuming reaction operating in these cells. Our data show that, in vitro assay, abnormal IsoAsp residues were significantly higher in ALS patients erythrocyte membrane proteins with an increased methyl accepting capability relative to controls (p < 0.05). Moreover, we observed a reduction in AdoMet levels, while AdoHcy concentration was comparable to that detected in the control, resulting in a lower [AdoMet]/[AdoHcy] ratio. Then, the accumulation of altered aspartyl residues in ALS patients is probably related to a reduced efficiency of the S-adenosylmethionine (AdoMet)-dependent repair system causing increased protein instability at Asn sites. The increase of abnormal residues represents a new protein alteration that may be present not only in red blood cells but also in other cell types of patients suffering from ALS.  相似文献   

13.
Luo J  Zeng K  Zhang C  Fang M  Zhang X  Zhu Q  Wang L  Wang W  Wang X  Chen G 《Neurochemical research》2012,37(7):1381-1391
The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein identified as a signaling molecule of Semaphorin-3A and act as axon repellent guidance factor in nervous system. Recent studies indicated that axon guidance molecules may play a role in synaptic reorganization in the adult brain and thereby promote epileptogenesis. This study aimed to investigate expression pattern of CRMP-1 in epileptogenesis. Using double immunofluorescence labeling, immunohistochemistry and western blot analysis, we looked into the CRMP-1 expression in temporal neocortex from patients with temporal lobe epilepsy (TLE) and histological normal temporal neocortex from the controls. We also studied the expression pattern of CRMP-1 in hippocampus and adjacent cortex of a TLE rat model on 6, 24, 72 h, 1, 2 weeks, 1 month, and 2 months post-seizure, and from control rats. CRMP-1 was mainly expressed in the neuronal cytoplasm in the temporal lobe of intractable TLE patients, which was co-expressed with -2. CRMP-1 expression was downregulated in temporal neocortical of TLE patients. In addition, in pilocarpine-induced animal model of epilepsy, CRMP-1 dynamically decreased in a range of 2 months. Thus, our results indicate that CRMP-1 may be involved in the development of TLE.  相似文献   

14.
SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.  相似文献   

15.
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.  相似文献   

16.
Several studies have shown that serum- and glucocorticoid-induced protein kinase 1(SGK1) can regulate both glutamate receptors and glutamate transporters and may participate in the regulation of neuroexcitability in neuronal diseases. In our previous study, we analyzed differential gene expression in the anterior temporal neocortex of drug-refractory epilepsy patients relative to control patients using a complementary DNA microarray and found that the SGK1 gene was up-regulated more than twofold in the brain tissues of epileptic patients. In the current study, we measured SGK1 expression in the brain tissues of humans and in an experimental model of rat epilepsy in order to explore the relationship between SGK1 expression and epilepsy. The SGK1 expression was detected in thirty human brain tissues derived from patients undergoing operation for drug-refractory epilepsy and was also detected in eight samples from autopsies. Meanwhile, we investigated SGK1 expression during the epileptic process in rats using immunofluorescence, RT-PCR and western blot analysis. SGK1 expression was enhanced in the temporal neocortex of patients with drug-refractory epilepsy and was also highly expressed in the rat brain during different phases of the epileptic process. SGK1 expression was also related with the elevation of EAAT3, which expression reduced after knockdown SGK1. These results provide new insight into the potential role of SGK1 in the pathophysiology of epilepsy.  相似文献   

17.
Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.  相似文献   

18.
Several studies have shown that serum- and glucocorticoid-induced protein kinase 1(SGK1) can regulate both glutamate receptors and glutamate transporters and may participate in the regulation of neuroexcitability in neuronal diseases. In our previous study, we analyzed differential gene expression in the anterior temporal neocortex of drug-refractory epilepsy patients relative to control patients using a complementary DNA microarray and found that the SGK1 gene was up-regulated more than twofold in the brain tissues of epileptic patients. In the current study, we measured SGK1 expression in the brain tissues of humans and in an experimental model of rat epilepsy in order to explore the relationship between SGK1 expression and epilepsy. The SGK1 expression was detected in thirty human brain tissues derived from patients undergoing operation for drug-refractory epilepsy and was also detected in eight samples from autopsies. Meanwhile, we investigated SGK1 expression during the epileptic process in rats using immunofluorescence, RT-PCR and western blot analysis. SGK1 expression was enhanced in the temporal neocortex of patients with drug-refractory epilepsy and was also highly expressed in the rat brain during different phases of the epileptic process. SGK1 expression was also related with the elevation of EAAT3, which expression reduced after knockdown SGK1. These results provide new insight into the potential role of SGK1 in the pathophysiology of epilepsy.  相似文献   

19.
A better understanding is needed of how glutamate metabolism is affected in mesial temporal lobe epilepsy (MTLE). Here we investigated glial–neuronal metabolism in the chronic phase of the kainate (KA) model of MTLE. Thirteen weeks following systemic KA, rats were injected i.p. with [1-13C]glucose. Brain extracts from hippocampal formation, entorhinal cortex, and neocortex, were analyzed by 13C and 1H magnetic resonance spectroscopy to quantify 13C labeling and concentrations of metabolites, respectively. The amount and 13C labeling of glutamate were reduced in the hippocampal formation and entorhinal cortex of epileptic rats. Together with the decreased concentration of NAA, these results indicate neuronal loss. Additionally, mitochondrial dysfunction was detected in surviving glutamatergic neurons in the hippocampal formation. In entorhinal cortex glutamine labeling and concentration were unchanged despite the reduced glutamate content and label, possibly due to decreased oxidative metabolism and conserved flux of glutamate through glutamine synthetase in astrocytes. This mechanism was not operative in the hippocampal formation, where glutamine labeling was decreased. In neocortex labeling and concentration of GABA were increased in epileptic rats, possibly representing a compensatory mechanism. The changes in the hippocampus might be of pathophysiological importance and merit further studies aiming at resolving metabolic causes and consequences of MTLE. Special issue article in honor of Dr. Frode Fonnum.  相似文献   

20.
目的探讨miR-134、CREB、pCREB在癫痫大鼠海马及难治性癫痫患者颞叶脑组织中的表达及意义。方法难治性癫痫患者及非癫痫对照组颞叶组织、氯化锂-匹罗卡品癫痫大鼠及空白对照组海马组织中,应用实时荧光定量PCR技术检测microRNA-134(miR-134)的表达,用Western blot方法检测CREB及p CREB的表达,用免疫组织化学方法检测人脑颞叶皮质及大鼠海马区CREB、p CREB的表达。结果与对照组相比miR-134表达在难治性癫痫患者中明显降低(P〈0.05),在癫痫模型组中点燃后3、7、14、60 d明显降低(P〈0.05),1 d与30 d表达降低较对照组差异无显著性(P〉0.05);癫痫模型组CREB在3、7、14、60 d时间点明显升高(P〈0.05)、pCREB各时间点表达均高于空白对照组(P〈0.05)。结论难治性癫痫患者颞叶皮质及癫痫动物海马中miR-134表达下降,CREB、pCREB表达升高,提示其可能在癫痫发生发展机制中起重要作用。  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号