Gluconeogenesis, a pathway for glucose synthesis from non-carbohydrate substances, begins with the synthesis of oxaloacetate (OA) from pyruvate and intermediates of citric acid cycle in hepatocyte mitochondria. The traditional view is that OA does not cross the mitochondrial membrane and must be shuttled to the cytosol, where most enzymes involved in gluconeogenesis are compartmentalized, in the form of malate. Thus, the possibility of transporting OA in the form of aspartate has been ignored. In the article is shown that malate supply to the cytosol increases only when fatty acid oxidation in the liver is activated, such as during starvation or untreated diabetes. Alternatively, aspartate synthesized from OA by mitochondrial aspartate aminotransferase (AST) is transported to the cytosol in exchange for glutamate via the aspartate-glutamate carrier 2 (AGC2). If the main substrate for gluconeogenesis is an amino acid, aspartate is converted to OA via urea cycle, therefore, ammonia detoxification and gluconeogenesis are simultaneously activated. If the main substrate is lactate, OA is synthesized by cytosolic AST, glutamate is transported to the mitochondria through AGC2, and nitrogen is not lost. It is concluded that, compared to malate, aspartate is a more suitable form of OA transport from the mitochondria for gluconeogenesis.
To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.
- MeSH
- buněčný cyklus fyziologie MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- fosfoproteiny chemie metabolismus MeSH
- fosforylace MeSH
- glutamáty metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- izotopové značení metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- peptidy metabolismus MeSH
- peroxiredoxin VI chemie metabolismus MeSH
- proteolýza * MeSH
- proteom genetika metabolismus MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- sestřihové faktory chemie metabolismus MeSH
- signální transdukce genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Fertilization is a multiple step process leading to the fusion of female and male gametes and the formation of a zygote. Besides direct gamete membrane interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves gamete communication via chemical molecules triggering various signaling pathways. This work focuses on a mouse taste receptor, mTAS1R3, encoded by the Tas1r3 gene, as a potential receptor mediating chemical communication between gametes using the C57BL/6J lab mouse strain. In order to specify the role of mTAS1R3, we aimed to characterize its precise localization in testis and sperm using super resolution microscopy. The testis cryo-section, acrosome-intact sperm released from cauda epididymis and sperm which underwent the acrosome reaction (AR) were evaluated. The mTAS1R3 receptor was detected in late spermatids where the acrosome was being formed and in the acrosomal cap of acrosome intact sperm. AR is triggered in mice during sperm maturation in the female reproductive tract and by passing through the egg surroundings such as cumulus oophorus cells. This AR onset is independent of the extracellular matrix of the oocyte called zona pellucida. After AR, the relocation of mTAS1R3 to the equatorial segment was observed and the receptor remained exposed to the outer surroundings of the female reproductive tract, where its physiological ligand, the amino acid L-glutamate, naturally occurs. Therefore, we targeted the possible interaction in vitro between the mTAS1R3 and L-glutamate as a part of chemical communication between sperm and egg and used an anti-mTAS1R3-specific antibody to block it. We detected that the acrosome reacted spermatozoa showed a chemotactic response in the presence of L-glutamate during and after the AR, and it is likely that mTAS1R3 acted as its mediator.
- MeSH
- buněčná diferenciace MeSH
- chemotaxe MeSH
- exprese genu MeSH
- glutamáty metabolismus MeSH
- interakce spermie a vajíčka * MeSH
- messenger RNA genetika MeSH
- mezibuněčná komunikace * MeSH
- myši MeSH
- receptory spřažené s G-proteiny genetika metabolismus MeSH
- spermie cytologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Bordetella pertussis is the causative agent of human whooping cough, a highly contagious respiratory disease which despite vaccination programs remains the major cause of infant morbidity and mortality. The requirement of the RNA chaperone Hfq for virulence of B. pertussis suggested that Hfq-dependent small regulatory RNAs are involved in the modulation of gene expression. High-throughput RNA sequencing revealed hundreds of putative noncoding RNAs including the RgtA sRNA. Abundance of RgtA is strongly decreased in the absence of the Hfq protein and its expression is modulated by the activities of the two-component regulatory system BvgAS and another response regulator RisA. Whereas RgtA levels were elevated under modulatory conditions or in the absence of bvg genes, deletion of the risA gene completely abolished RgtA expression. Profiling of the ΔrgtA mutant in the ΔbvgA genetic background identified the BP3831 gene encoding a periplasmic amino acid-binding protein of an ABC transporter as a possible target gene. The results of site-directed mutagenesis and in silico analysis indicate that RgtA base-pairs with the region upstream of the start codon of the BP3831 mRNA and thereby weakens the BP3831 protein production. Furthermore, our data suggest that the function of the BP3831 protein is related to transport of glutamate, an important metabolite in the B. pertussis physiology. We propose that the BvgAS/RisA interplay regulates the expression of RgtA which upon infection, when glutamate might be scarce, attenuates translation of the glutamate transporter and thereby assists in adaptation of the pathogen to other sources of energy.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Bordetella pertussis genetika metabolismus MeSH
- glutamáty metabolismus MeSH
- lidé MeSH
- malá nekódující RNA genetika MeSH
- regulace genové exprese u bakterií MeSH
- signální transdukce * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave β-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body. DATABASE: The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.
- MeSH
- antigeny povrchové chemie metabolismus MeSH
- glutamátkarboxypeptidasa II chemie metabolismus MeSH
- glutamáty chemie metabolismus MeSH
- karboxypeptidasy chemie metabolismus MeSH
- katalytická doména MeSH
- lidé MeSH
- molekulární struktura MeSH
- substrátová specifita MeSH
- termodynamika MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
In humans, aging is accompanied by the deterioration of the hearing function--presbycusis. The major etiology for presbycusis is the loss of hair cells in the inner ear; less well known are changes in the central auditory system. Therefore, we used 1H magnetic resonance spectroscopy at 3T tomograph to examine metabolite levels in the auditory cortex of three groups of subjects: young healthy subjects less than 30 years old and subjects older than 65 years either with mild presbycusis corresponding to their age or with expressed presbycusis. Hearing function in all subjects was examined by pure tone audiometry (125-16,000 Hz). Significant differences were found in the concentrations of glutamate and N-acetylaspartate, with lower levels in aged subjects. Lactate was particularly increased in subjects with expressed presbycusis. Significant differences were not found in other metabolites, including GABA, between young and elderly subjects. The results demonstrate that the age-related changes of the inner ear are accompanied by a decrease in the excitatory neurotransmitter glutamate as well as a lactate increase in the auditory cortex that is more expressed in elderly subjects with large hearing threshold shifts.
- MeSH
- audiometrie čistými tóny MeSH
- dospělí MeSH
- glutamáty metabolismus MeSH
- kyselina aspartová analogy a deriváty metabolismus MeSH
- laktáty metabolismus MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie * MeSH
- presbyakuze metabolismus MeSH
- senioři MeSH
- sluchové korové centrum metabolismus MeSH
- stárnutí metabolismus MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- MeSH
- buněčná smrt MeSH
- financování organizované MeSH
- glutamátové receptory metabolismus MeSH
- glutamáty metabolismus toxicita MeSH
- ischemie mozku MeSH
- lidé MeSH
- mozková hypoxie MeSH
- nervová tkáň patofyziologie MeSH
- neurony patologie MeSH
- neurotoxiny toxicita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Přes rozsáhlý výzkum zůstává etiopatogeneze poruch autistického spektra (PAS) nevysvětlená. Náš přehled přináší doklady o tom, že většina heterogenních symptomů PAS by mohla mít společné patofyziologické poruchy na buněčné úrovni spojené s dysregulací glutamátergní neurotransmise v mozku a excitotoxicitou. Autoři se zamýšlejí nad možnostmi prevence a léčení PAS.
Despite the great array of observations, the etiopathogenesis of the autism spectrum disorders (ASD) is poorly defined. Our review offers evidence that most heterogeneous symptoms of ASD might have a common set of pathophysiological events at cellular level closely connected with dysregulation of gluta-matergic neurotransmission in the brain with excitotoxicity as a common underlying mechanism. The suggested unifying hypothesis offers the means for efficient prevention and amelioration of ASD.
- MeSH
- autistická porucha MeSH
- centrální nervový systém růst a vývoj účinky záření MeSH
- epilepsie etiologie patofyziologie MeSH
- financování organizované MeSH
- fluoridy toxicita MeSH
- glutamáty fyziologie metabolismus škodlivé účinky MeSH
- hliník toxicita MeSH
- kyselina aspartová fyziologie metabolismus škodlivé účinky MeSH
- neurotransmiterové látky fyziologie metabolismus MeSH
- receptory N-methyl-D-aspartátu fyziologie MeSH
- zpožděný efekt prenatální expozice MeSH
- Publikační typ
- přehledy MeSH
Dysfunkce genu pro NRl podjednotku NMDA receptoru se podílí na patogenezi schizofrenie. Tuto hypotézu podporuje psychóze podobné chování SOdenních potkaních samců a pokles specifické vazby (3H)lutamátu do NMDA receptoru v různých oblastech jejich mozku. Kvantitativní RT-PCR s vnitřní standardizací však neodhalila významné změny v hladinách mRNA pro NRl podjednotku v levém a pravém hipokampu naivních a kontrolních potkanů a zvířat ovlivněných kyselinou chinolinovou (QUIN; 250 nmol/0.25 ^iL pufru do každé z postranních mozkových komor) 12. den po narození. Naopak kvantitativní western bloty odhalily zvýšenou expresi NRl proteinu v levém hipokampu vzhledem ke kontralaterálnímu hipokampu u naivních zvířat. Tato lateralita zmizela u zvířat neonatálně ovlivněných QUIN, u kterých se v dospělosti objevilo chování podobné psychóze, a současně byl v jejich levém hipokampu prokázán statisticky významný pokles exprese proteinu NRl podjednotky. Tyto výsledky ukazují, že exprese této podjednotky vykazuje změny, které jsou podobné změnám, které byly popsány u pacientů trpících schizofrenií.
Dysfunction of the gene for the NR1 subunit of NMDA receptor has been implicated in the pathogenesis of schizophrenia. In suppo rt of this hypothesis are psychosis-like behavioral abnormalities in 50-day-old rat males with attenuated specific synaptosomal binding of [ 3 H]glutamate to NMDA receptor in their brains. Quantitative internally standardized RT-PCR assay did not reveal any significant changes in m RNA levels of NR1 subunit in left and right hippocampi of naïve rats and controls as well as in young adult males treated with quinolinic acid (QUIN; 250 nmol/0.25 μ L buffered saline into each lateral cerebral ventricle) on postnatal day 12. In contrast, quantitative Western blotting disclos ed increased protein levels of NR1 subunit in the left hippocampus of naïve young adults. The laterality disappeared in animals wi th psychosis- -like behavior together with significant decrease of NR1 protein in their left hippocampi. These results suggest that protein e xpression of NR1 subunit exhibits changes comparable to those observed in patients with schizophrenia.
- Klíčová slova
- NMDA receptor, exprese NR1 podjednotky, animální model, prepulzní inhibice akustického úleku,
- MeSH
- exprese genu genetika účinky léků MeSH
- glutamáty biosyntéza metabolismus MeSH
- hipokampus patofyziologie MeSH
- kyselina chinolinová MeSH
- lidé MeSH
- messenger RNA MeSH
- modely u zvířat MeSH
- mozková kůra metabolismus účinky léků MeSH
- potkani Wistar MeSH
- receptory N-methyl-D-aspartátu biosyntéza fyziologie nedostatek MeSH
- schizofrenie * etiologie farmakoterapie genetika chemicky indukované patofyziologie MeSH
- úleková reakce fyziologie MeSH
- western blotting využití MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- glutamáty metabolismus MeSH
- ischemie mozku krev MeSH
- leucin metabolismus MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
