Protein methylation is mainly observed in lysine, arginine, and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing ...Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18, and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.
Summary
γ‐Enolase is a neurotrophic‐like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which ...cleaves the C‐terminal end of the molecule. We have investigated the expression and colocalization of γ‐enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ‐enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C‐terminally cleaved form of γ‐enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ‐enolase in microglial cells in response to amyloid‐β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ‐enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ‐enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid‐β‐related neurodegeneration.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Enolase, a multifunctional protein with diverse isoforms, has generally been recognized for its primary roles in glycolysis and gluconeogenesis. The shift in isoform expression from α-enolase to ...neuron-specific γ-enolase extends beyond its enzymatic role. Enolase is essential for neuronal survival, differentiation, and the maturation of neurons and glial cells in the central nervous system. Neuron-specific γ-enolase is a critical biomarker for neurodegenerative pathologies and neurological conditions, not only indicating disease but also participating in nerve cell formation and neuroprotection and exhibiting neurotrophic-like properties. These properties are precisely regulated by cysteine peptidase cathepsin X and scaffold protein γ
-syntrophin. Our findings suggest that γ-enolase, specifically its C-terminal part, may offer neuroprotective benefits against neurotoxicity seen in Alzheimer's and Parkinson's disease. Furthermore, although the therapeutic potential of γ-enolase seems promising, the effectiveness of enolase inhibitors is under debate. This paper reviews the research on the roles of γ-enolase in the central nervous system, especially in pathophysiological events and the regulation of neurodegenerative diseases.
Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. ...Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and
-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
γ-Enolase, a glycolytic enzyme, is expressed specifically in neurons. It exerts neurotrophic activity and has been suggested to regulate growth, differentiation, survival and regeneration of neurons. ...In the present study, we investigated the involvement of γ-enolase in PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) signalling, the two pathways triggered predominantly by neurotrophic factors. Whereas the PI3K/Akt pathway, rather than the MAPK/ERK pathway, is involved in γ-enolase-enhanced cell survival, γ-enolase-stimulated neurite outgrowth requires both pathways, i.e. the activation of both PI3K and ERK1/2, leading to subsequent expression of the growth-cone-specific protein GAP-43 (growth-associated protein of 43 kDa). MEK (MAPK/ERK kinase) and PI3K inhibition blocked or attenuated the neurite outgrowth associated with dynamic remodelling of the actin-based cytoskeleton. We show that γ-enolase-mediated PI3K activation regulates RhoA kinase, a key regulator of actin cytoskeleton organization. Moreover, the inhibition of RhoA downstream effector ROCK (Rho-associated kinase) results in enhanced γ-enolase-induced neurite outgrowth, accompanied by actin polymerization and its redistribution to growth cones. Our results show that γ-enolase controls neuronal survival, differentiation and neurite regeneration by activating the PI3K/Akt and MAPK/ERK signalling pathways, resulting in downstream regulation of the molecular and cellular processes of cytoskeleton reorganization and cell remodelling, activation of transcriptional factors and regulation of the cell cycle.
Inflammation plays a central role in the processes associated with neurodegeneration. The inflammatory response is mediated by activated microglia that release inflammatory mediators to the neuronal ...environment. Microglia-derived lysosomal cathepsins, including cathepsin X, are increasingly recognized as important mediators of the inflammation involved in lipopolysaccharide (LPS)-induced neuroinflammation. The current study was undertaken to investigate the role of cathepsin X and its molecular target, γ-enolase, in neuroinflammation and to elucidate the underlying mechanism. We determined that the exposure of activated BV2 and EOC 13.31 cells to LPS led to increased levels of cathepsin X protein and activity in the culture supernatants in a concentration- and time-dependent manner. In contrast, LPS stimulation of these two cells reduced the release of active γ-enolase in a manner regulated by the cathepsin X activity. Cathepsin X inhibitor AMS36 significantly reduced LPS-induced production of nitric oxide, reactive oxygen species and the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α from BV2 cells. Inhibition of cathepsin X suppressed microglial activation through the reduced caspase-3 activity, together with diminished microglial cell death and apoptosis, and also through inhibition of the activity of the mitogen-activated protein kinases. Further, SH-SY5Y treatment with culture supernatants of activated microglial cells showed that cathepsin X inhibition reduces microglia-mediated neurotoxicity. These results indicate that up-regulated expression and increased release and activity of microglial cathepsin X leads to microglia activation-mediated neurodegeneration. Cathepsin X inhibitor caused neuroprotection via its inhibition of the activation of microglia. Cathepsin X could thus be a potential therapeutic target for neuroinflammatory disorders.
•Microglia activation induces cathepsin X expression and its activity.•Cathepsin X inhibition suppresses microglia activation.•Cathepsin X mediates microglia activation-mediated neurodegeneration.•Cathepsin X inhibition exerts neuroprotection.
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GEOZS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Neurotrophins can activate multiple signalling pathways in neuronal cells through binding to their cognate receptors, leading to neurotrophic processes such as cell survival and differentiation. ...γ-Enolase has been shown to have a neurotrophic activity that depends on its translocation towards the plasma membrane by the scaffold protein γ1-syntrophin. The association of γ-enolase with its membrane receptor or other binding partners at the plasma membrane remains unknown.
In the present study, we used immunoprecipitation and immunofluorescence to show that γ-enolase associates with the intracellular domain of the tropomyosin receptor kinase (Trk) family of tyrosine kinase receptors at the plasma membrane of differentiated SH-SY5Y cells.
In differentiated SH-SY5Y cells with reduced expression of γ1-syntrophin, the association of γ-enolase with the Trk receptor was diminished due to impaired translocation of γ-enolase towards the plasma membrane or impaired Trk activity. Treatment of differentiated SH-SY5Y cells with a γ-Eno peptide that mimics γ-enolase neurotrophic activity promoted Trk receptor internalisation and endosomal trafficking, as defined by reduced levels of Trk in clathrin-coated vesicles and increased levels in late endosomes. In this way, γ-enolase triggers Rap1 activation, which is required for neurotrophic activity of γ-enolase. Additionally, the inhibition of Trk kinase activity by K252a revealed that increased SH-SY5Y cell survival and neurite outgrowth mediated by the γ-Eno peptide through activation of signalling cascade depends on Trk kinase activity.
These data therefore establish the Trk receptor as a binding partner of γ-enolase, whereby Trk endosomal trafficking is promoted by γ-Eno peptide to mediate its neurotrophic signalling. Video abstract.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Curcumin exerts a protective effect in cerebral ischemia through its anti-oxidant and anti-inflammatory activities. gamma-enolase is a glycolytic enzyme expressed in neurons that is known to exerts a ...neuroprotective effect. We investigated whether curcumin regulates gamma-enolase expression in focal cerebral ischemic injury in rats. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia. Adult male rats were injected intraperitoneally with either vehicle or curcumin (50 mg/kg) 1 h after MCAO and cerebral cortex tissues were isolated 24 h after MCAO. We found that MCAO-induced injury resulted in a reduction in gamma-enolase expression in vehicle-treated animals using a proteomics approach. However, this reduction was attenuated in animals with MCAO treated with curcumin. Reverse-transcription PCR and Western blot analyses also showed that curcumin treatment prevented the MCAO injury-induced reduction in gamma-enolase expression. The results of this study suggest that curcumin exerts its neuroprotective function in focal cerebral ischemia by regulating the expression of gamma-enolase.
γ-Enolase acts as a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. It is shown in this study to exert a protective effect against amyloid-β-peptide ...(Aβ)-induced neurotoxicity in rat pheochromocytoma PC12 cells. Aβ-induced toxicity was abolished in the presence of the active C-terminal peptide of γ-enolase (γ-Eno) as measured by cell viability, lactate dehydrogenase release, sub-G1 cell population, intracellular reactive oxygen species, mitochondrial functions and apoptotic morphology. γ-Eno caused downregulation of the pro-apoptotic protein Bax and upregulation of the anti-apoptotic protein Bcl-2, as well as reduced caspase-3 activation. Exposure to Aβ increased surface expression of p75 neurotrophin receptor (p75(NTR)), and the increase was abolished in the presence of γ-Eno peptide. Further, pretreatment with γ-Eno suppressed the activation of mitogen-activated protein kinases p38 and Jun-N-terminal kinase, which are p75(NTR) downstream effectors in apoptotic signaling. Moreover, Aβ triggered γ-enolase co-immunoprecipitation with p75(NTR) as well as their strong association in the perimembrane region as shown by confocal microscopy, which further supports the interaction between these two proteins in cells insulted by Aβ peptide. Our results indicate the possible use of γ-enolase C-terminal peptide for treating or preventing Alzheimer's disease.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Previous experiments indicated that 1-bromopropane (1-BP), an alternative to chloroflurocarbons, is neurotoxic and inhibits spermiation in the testis. Here we investigated the reversibility of the ...toxic effects of 1-BP in rats. Male Wistar rats were divided into three equal groups of 24 each and exposed by inhalation to 0, 400 or 1000 ppm of 1-BP for 6 weeks (8 hrs/day, 7 days/week). Eight rats from each group were sacrificed at the end of 6 weeks exposure, and at 4 and 14 weeks after the end of exposure, to assess the recovery processes. We studied sperm count, motility, morphology and testicular histopathology, as well as blood pressure, skin temperature and hindlimb muscle strength. At the end of 6 weeks of exposure to 1000 ppm (0 week recovery), testicular weight, epididymal weight, sperm count and motility were low, morphologically abnormal sperm were increased and spermatogenic cells showed diffuse degeneration. These changes did not show full recovery at 14 weeks recovery, with the exception of the prostate and seminal vesicular weights, which recovered back to control values. At 400 ppm, increased retained spermatids at 0 week recovery returned to normal at 4 weeks recovery. Exposure to 1000 ppm produced sustained reduction of hindlimb muscle strength at 14 weeks recovery, whereas normalization of the skin temperature and blood pressure was noted after transient changes. Our study showed that the effect of 1-BP on spermatogenesis is dose-dependent; low exposure inhibited spermiation and hormone-dependent organ weight reduction and these changes were transient, while a higher dose of 1000 ppm 1-BP caused persistent depletion of spermatogenic cells.
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