Novel Regulatory Mechanisms Of Inositol Biosynthesis In Saccharomyces Cerevisiae And Mammalian Cells, And Implications For The Mechanism Underlying Vpa-Induced Glucose 6-Phosphate Depletion

Publication Year:
2016
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Repository URL:
https://digitalcommons.wayne.edu/oa_dissertations/1610
Author(s):
Yu, Wenxi
Tags:
GSK3; inositol biosynthesis; inositol pyrophosphate; IP6K1; valproic acid; Biochemistry; Genetics; Molecular Biology
thesis / dissertation description
Myo-inositol is the precursor of all inositol containing molecules, including inositol phosphates,phosphoinositides and glycosylphosphatidylinositols, which are signaling molecules involved in many critical cellular functions. Perturbation of inositol metabolism has been linked to neurological disorders. Although several widely-used anticonvulsants and mood-stabilizing drugs have been shown to exert inositol depletion effects, the mechanisms of action of the drugs and the role of inositol in these diseases are not understood. Elucidation of the molecular control of inositol synthesis will shed light on the pathologies of inositol related illnesses.In Saccharomyces cerevisiae, deletion of the four glycogen synthase kinase-3 genes, MCK1, MRK1, MDS1, and YGK3, resulted in multiple features of inositol depletion. My studies demonstrated that the MCK1 gene is required for normal inositol homeostasis. mck1∆ and gsk3∆ (mck1∆mrk1∆mds1∆ygk3∆) cells exhibited similar features of inositol depletion. MCK1 ablation led to decreased myo-inositol-3-phosphate synthase (MIPS) activity and a decreased rate of inositol de novo synthesis. This is the first demonstration that Mck1 controls inositol synthesis by regulating MIPS activity.While elegant studies have revealed several inositol-regulating mechanisms in yeast, very little is known about regulation of inositol synthesis in mammals. My studies discovered that IP6K1, an inositol hexakisphosphate kinase that catalyzes the synthesis of inositol pyrophosphate, negatively regulates inositol synthesis in mammalian cells. Interestingly, IP6K1 preferentially bound to the phospholipid phosphatidic acid, and this binding was required for IP6K1 nuclear localization and the transcriptional regulation of Isyna1, which encodes mammalian MIPS. This is the first demonstration of the molecular control of de novo synthesis of inositol in mammalian cells.VPA depletes intracellular glucose 6-phosphate in yeast cells by an unidentified mechanism. My studies discovered that VPA inhibits expression of hexose transporter genes HXT2, HXT4, HXT6, and HXT7. Mig1, a DNA-binding transcription repressor that translocates to the nucleus to repress gene expression under high glucose conditions, is required to inhibit HXT2 and HXT4 expression. Interestingly, VPA triggered Mig1 nuclear localization under non-repressive conditions. Furthermore, ablation of REG1, which regulates Mig1 translocation, reversed VPA-induced inhibition of HXT4 expression. These findings suggest that VPA may inhibit glucose uptake by activating Mig1-mediated repression of hexose transporter genes.