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Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast.

ABSTRACT: To investigate the mechanism of action of volatile anesthetics, we are studying mutants of the yeast Saccharomyces cerevisiae that have altered sensitivity to isoflurane, a widely used clinical anesthetic. Several lines of evidence from these studies implicate a role for ubiquitin metabolism in cellular response to volatile anesthetics: (i) mutations in the ZZZ1 gene render cells resistant to isoflurane, and the ZZZ1 gene is identical to BUL1 (binds ubiquitin ligase), which appears to be involved in the ubiquitination pathway; (ii) ZZZ4, which we previously found is involved in anesthetic response, is identical to the DOA1/UFD3 gene, which was identified based on altered degradation of ubiquitinated proteins; (iii) analysis of zzz1Delta zzz4Delta double mutants suggests that these genes encode products involved in the same pathway for anesthetic response since the double mutant is no more resistant to anesthetic than either of the single mutant parents; (iv) ubiquitin ligase (MDP1/RSP5) mutants are altered in their response to isoflurane; and (v) mutants with decreased proteasome activity are resistant to isoflurane. The ZZZ1 and MDP1/RSP5 gene products appear to play important roles in determining effective anesthetic dose in yeast since increased levels of either gene increases isoflurane sensitivity whereas decreased activity decreases sensitivity. Like zzz4 strains, zzz1 mutants are resistant to all five volatile anesthetics tested, suggesting there are similarities in the mechanisms of action of a variety of volatile anesthetics in yeast and that ubiquitin metabolism affects response to all the agents examined.

SUBMITTER: Wolfe D

PROVIDER: S-EPMC84909 | biostudies-literature | 1999 Dec

REPOSITORIES: biostudies-literature

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Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast.

Wolfe D D Reiner T T Keeley J L JL Pizzini M M Keil R L RL

Molecular and cellular biology 19991201 12

To investigate the mechanism of action of volatile anesthetics, we are studying mutants of the yeast Saccharomyces cerevisiae that have altered sensitivity to isoflurane, a widely used clinical anesthetic. Several lines of evidence from these studies implicate a role for ubiquitin metabolism in cellular response to volatile anesthetics: (i) mutations in the ZZZ1 gene render cells resistant to isoflurane, and the ZZZ1 gene is identical to BUL1 (binds ubiquitin ligase), which appears to be involve ...[more]

PMID: 10567550

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Project description:Macrophage-1 antigen (Mac-1, ?M?2) is a leukocyte adhesion molecule that plays a significant role in leukocyte crawling and phagocytosis, and is homologous to its sister protein leukocyte function-associated antigen-1 (LFA-1, ?L?2). The authors have previously demonstrated that volatile anesthetics isoflurane and sevoflurane bound to and inhibited LFA-1. Here, the hypothesis tested was that isoflurane and sevoflurane would inhibit Mac-1. A binding assay of Mac-1 to its ligand inter-cellular adhesion molecule-1 (ICAM-1) using V-bottom plates was established. The effect of isoflurane and sevoflurane on Mac-1 was examined using this ICAM-1 binding assay and by probing exposure of activation-sensitive epitopes. The docking program Glide was used to predict anesthetic binding site(s) on Mac-1. The functional role of this predicted binding site was then assessed by introducing point mutations in this region. Lastly, the effect of anesthetic on activating mutants was evaluated. The results indicated that isoflurane inhibited binding of Mac-1 to ICAM-1, but sevoflurane did not. Isoflurane also attenuated the exposure of the activation-sensitive epitopes. The docking simulation predicted the isoflurane binding site to be at the cavity underneath the ?7 helix of the ligand binding domain (the ?M I domain). Point mutants at this predicted binding site contained both activating and deactivating mutants, suggesting its functional significance. The binding of activating mutants ?M-Y267A ?2-WT and ?M-L312A ?2-WT to ICAM-1 was not affected by isoflurane, but binding of another activating mutant ?M-WT ?2-L132A was inhibited supporting the binding of isoflurane to this cavity. The conclusion reached from these findings was that isoflurane inhibited Mac-1 binding to ICAM-1 by binding to the cavity underneath the ?7 helix of the ?M I domain, but sevoflurane did not. Thus, because these common clinical volatile anesthetics demonstrated different effects on Mac-1, this implied their effects on the immune system might differ.

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Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast.

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Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast.

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