Dataset Information

Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif.

ABSTRACT: Under ambient air conditions, NO inhibits NMDAR activity by reacting with the NR2A subunit C399 along with two additional cysteine pairs if their disulfide bonds are reduced to free thiol groups [NR1(C744,C798); NR2(C87,C320)]. Here we demonstrate that relative hypoxia enhances S-nitrosylation of NMDARs by a unique mechanism involving an "NO-reactive oxygen sensor motif" whose determinants include C744 and C798 of the NR1 subunit. Redox reactions involving these two thiol groups sensitize other NMDAR sites to S-nitrosylation and consequent receptor inhibition, while their own nitrosylation has little effect on NMDAR activity. The crystal structure of the ligand-binding domain of NR1 reveals a flexible disulfide bond (C744-C798), which may account for its susceptibility to reduction and subsequent reaction with NO that is observed with biochemical techniques. These thiols may be nitrosylated preferentially during increasing hypoxia or stroke conditions, thus preventing excessive activity associated with cytotoxicity while avoiding blockade of physiologically active NMDARs.

SUBMITTER: Takahashi H

PROVIDER: S-EPMC1855274 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif.

Takahashi Hiroto H Shin Yeonsook Y Cho Seung-Je SJ Zago Wagner M WM Nakamura Tomohiro T Gu Zezong Z Ma Yuliang Y Furukawa Hiroyasu H Liddington Robert R Zhang Dongxian D Tong Gary G Chen Huei-Sheng Vincent HS Lipton Stuart A SA

Neuron 20070101 1

Under ambient air conditions, NO inhibits NMDAR activity by reacting with the NR2A subunit C399 along with two additional cysteine pairs if their disulfide bonds are reduced to free thiol groups [NR1(C744,C798); NR2(C87,C320)]. Here we demonstrate that relative hypoxia enhances S-nitrosylation of NMDARs by a unique mechanism involving an "NO-reactive oxygen sensor motif" whose determinants include C744 and C798 of the NR1 subunit. Redox reactions involving these two thiol groups sensitize other ...[more]

PMID: 17196530

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Project description:The activity of the heterodimeric transcription factor hypoxia inducible factor (HIF) is regulated by the post-translational, oxygen-dependent hydroxylation of its ?-subunit by members of the prolyl hydroxylase domain (PHD or EGLN)-family and by factor inhibiting HIF (FIH). PHD-dependent hydroxylation targets HIF? for rapid proteasomal degradation; FIH-catalysed asparaginyl-hydroxylation of the C-terminal transactivation domain (CAD) of HIF? suppresses the CAD-dependent subset of the extensive transcriptional responses induced by HIF. FIH can also hydroxylate ankyrin-repeat domain (ARD) proteins, a large group of proteins which are functionally unrelated but share common structural features. Competition by ARD proteins for FIH is hypothesised to affect FIH activity towards HIF?; however the extent of this competition and its effect on the HIF-dependent hypoxic response are unknown.To analyse if and in which way the FIH/ARD protein interaction affects HIF-activity, we created a rate equation model. Our model predicts that an oxygen-regulated sequestration of FIH by ARD proteins significantly shapes the input/output characteristics of the HIF system. The FIH/ARD protein interaction is predicted to create an oxygen threshold for HIF? CAD-hydroxylation and to significantly sharpen the signal/response curves, which not only focuses HIF? CAD-hydroxylation into a defined range of oxygen tensions, but also makes the response ultrasensitive to varying oxygen tensions. Our model further suggests that the hydroxylation status of the ARD protein pool can encode the strength and the duration of a hypoxic episode, which may allow cells to memorise these features for a certain time period after reoxygenation.The FIH/ARD protein interaction has the potential to contribute to oxygen-range finding, can sensitise the response to changes in oxygen levels, and can provide a memory of the strength and the duration of a hypoxic episode. These emergent properties are predicted to significantly shape the characteristics of HIF activity in animal cells. We argue that the FIH/ARD interaction should be taken into account in studies of the effect of pharmacological inhibition of the HIF-hydroxylases and propose that the interaction of a signalling sensor with a large group of proteins might be a general mechanism for the regulation of signalling pathways.

Project description:BackgroundHypoxia-based cell culture experiments are routine and essential components of in vitro cancer research. Most laboratories use low-cost portable modular chambers to achieve hypoxic conditions for cell cultures, where the sealed chambers are purged with a gas mixture of preset O2 concentration. Studies are conducted under the assumption that hypoxia remains unaltered throughout the 48 to 72 hour duration of such experiments. Since these chambers lack any sensor or detection system to monitor gas-phase O2, the cell-based data tend to be non-uniform due to the ad hoc nature of the experimental setup.MethodologyWith the availability of low-cost open-source microcontroller-based electronic project kits, it is now possible for researchers to program these with easy-to-use software, link them to sensors, and place them in basic scientific apparatus to monitor and record experimental parameters. We report here the design and construction of a small-footprint kit for continuous measurement and recording of O2 concentration in modular hypoxia chambers. The low-cost assembly (US$135) consists of an Arduino-based microcontroller, data-logging freeware, and a factory pre-calibrated miniature O2 sensor. A small, intuitive software program was written by the authors to control the data input and output. The basic nature of the kit will enable any student in biology with minimal experience in hobby-electronics to assemble the system and edit the program parameters to suit individual experimental conditions.Results/conclusionsWe show the kit's utility and stability of data output via a series of hypoxia experiments. The studies also demonstrated the critical need to monitor and adjust gas-phase O2 concentration during hypoxia-based experiments to prevent experimental errors or failure due to partial loss of hypoxia. Thus, incorporating the sensor-microcontroller module to a portable hypoxia chamber provides a researcher a capability that was previously available only to labs with access to sophisticated (and expensive) cell culture incubators.

Dataset Information

Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif.

Publications

Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif.

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