OP55BIOCHEMICAL ANALYSIS COMBINED WITH 3D STRUCTURAL PROTEIN MODELLING IDENTIFIES THE ROLE OF SPECIFIC MITOCHONDRIAL MUTATIONS IN GBM

Abstract

INTRODUCTION: Altered mitochondrial metabolism is a hallmark of glioblastoma (GBM). Previous work in our group using next generation sequencing of GBM cell line mitochondrial DNAs (mtDNAs), combined with 3D structural protein analysis, identified 42 non-synonymous mutations, including 8 within the MT-CYB subunit of complex III (CIII) of the mitochondrial respiratory chain (MRC). Four MT-CYB mutations were predicted to impact on function and concomitant measurement of the MRC enzyme activities in a cohort of GBM cell lines revealed their potential structure-function relationships. METHOD: A cohort of 5 adult GBM cell lines were grown at 37°C and 5% CO2 and 25% O2 until 80% confluent. The cells were treated to allow access of ligands to the inner mitochondrial membrane so that relative activities of complex I, II, linked II/III and IV could be determined spectrophotometrically. RESULTS: Although diverse MRC activity profiles were seen between GBM cell lines, a relationship between CII/CIII activity and the MT-CYB mutation F18L was observed which correlated with structural predictions. GBM cell lines containing F18L, exhibited statistically elevated CII/CIII activity (p > 0.0001) relative to a non-neoplastic control cell line. CONCLUSION: We show a link between a single mtDNA mutation and altered mitochondrial metabolism in GBM which highlights the predictive power of our combined approach. F18L is a germ-line mutation occurring in ∼30% of GBM-patients, raising the possibility that some patients are predisposed to alterations in mitochondrial function. F18L thus provides a target for diagnostic/prognostic research and the development of patient-specific therapeutics.