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Inactivation of eukaryotic initiation factor 2B in vitro by heat shock.

ABSTRACT: Protein synthesis in rat H35 Reuber hepatoma cells is rapidly inhibited on heat shock. At mild heat-shock temperatures the main cause for inhibition is the inactivation of the guanine nucleotide exchange factor eukaryotic initiation factor 2B (eIF2B); under more severe heat-shock conditions the activity of several initiation factors is compromised. eIF2B is required for GDP/GTP exchange on eIF2, which delivers methionyl-tRNA to the 40 S ribosomal subunit. We have tried to elucidate the mechanism underlying the inactivation of eIF2B by assays in vitro. Incubation of cell extracts at 41 degreesC or higher led to the inactivation of eIF2B. In agreement with observations in cells exposed to mild heat shocks, the thermal inactivation of eIF2B could be ascribed to neither eIF2alpha phosphorylation nor the induction of another inhibitor. With the use of glycerol gradients we show that eIF2B forms aggregates in heat-treated extracts. Furthermore eIF2B activity is protected against heat shock in thermotolerant cells. Taken together, these results suggest a role for chaperones in the control of eIF2B activity.

SUBMITTER: Scheper GC

PROVIDER: S-EPMC1219710 | biostudies-other | 1998 Sep

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:In recent years, the phenotypes of leukodystrophies linked to mutations in the eukaryotic initiation factor 2B genes have been extended, classically called CACH/VWM (Childhood ataxia with cntral hypomyélination/vanishing white matter disorder). The large clinical spectrum observed from the more severe antenatal forms responsible for fetal death to milder adult forms with an onset after 16 years old and restricted to slow cognitive impairment have lead to the concept of eIF2B-related disorders. The typical MRI pattern with a diffuse CSF-like aspect of the cerebral white matter can lack particularly in the adult forms whereas an increasing number of patients with clinical and MRI criteria for CACH/VWM disease but without eIF2B mutations are found. Then we propose the use of biochemical markers to help in this difficult diagnosis. The biochemical diagnosis of eIF2B-related disorder is difficult as no marker, except the recently described asialotransferrin/transferrin ratio measured in cerebrospinal fluid, has been proposed and validated until now. Decreased eIF2B GEF activity has been previously reported in lymphoblastoid cell lines from 30 eIF2B-mutated patients. Our objective was to evaluate further the utility of this marker and to validate eIF2B GEF activity in a larger cohort as a specific diagnostic test for eIF2B-related disorders. METHODOLOGY/PRINCIPAL FINDINGS:We performed eIF2B GEF activity assays in cells from 63 patients presenting with different clinical forms and eIF2B mutations in comparison to controls but also to patients with defined leukodystrophies or CACH/VWM-like diseases without eIF2B mutations. We found a significant decrease of GEF activity in cells from eIF2B-mutated patients with 100% specificity and 89% sensitivity when the activity threshold was set at < or =77.5%. CONCLUSION:These results validate the measurement of eIF2B GEF activity in patients' transformed-lymphocytes as an important tool for the diagnosis of eIF2B-related disorders.

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Inactivation of eukaryotic initiation factor 2B in vitro by heat shock.

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