Open Access Dissertation
Date of Award
Miriam L. Greenberg
Inositol is a six-carbon cyclitol that is ubiquitous in biological systems. It is a precursor for the synthesis of numerous biologically important compounds, including inositol phosphates and phosphoinositides that are essential for cell function and viability. Inositol compounds play a role in membrane formation, gene regulation, signaling, regulation of ion channels, and membrane trafficking. Furthermore, inositol regulates hundreds of genes, including those involved in the biosynthesis of inositol and phospholipids. While transcriptional regulation of inositol biosynthesis has been extensively studied and well characterized, regulation of inositol biosynthesis at the enzymatic level has not been addressed. The current study shows that myo-inositol 3-phosphate synthase (MIPS), the enzyme that catalyzes the rate-limiting step in inositol biosynthesis, is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in yeast and human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site-mutants of both yeast and human MIPS indicated that the three conserved sites affect MIPS activity. Two of the phosphosites are inhibitory, and one is critical for activity.
Previous studies have shown that valproate (VPA), a branched chain fatty acid that has been successfully used for the treatment of bipolar disorder, epilepsy, and migraine, causes inositol depletion by inhibiting MIPS in vivo but not in vitro, which suggests that inhibition is indirect. Elimination of the two inhibitory phosphosites caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to VPA, suggesting that VPA-mediated inositol depletion may result from phosphorylation of MIPS.
Decreased IP3 signaling caused by Inositol depletion has been proposed as a mechanism that underlies the therapeutic effect of VPA. However, no direct correlation between altered IP3 signaling and the therapeutic effect has been established. Because of the versatility of inositol compounds, inositol depletion may have more far-reaching consequences that may account for the effect of VPA. This study showed that inositol depletion caused by VPA or by starvation of ino1Δ cells, which cannot synthesize inositol, perturbs vacuolar structure, decreases vacuolar ATPase (V-ATPase) proton pumping, and causes partial un-coupling of the V-ATPase. These perturbations were rescued by inositol supplementation. Furthermore, VPA compromised the synthesis of PI3,5P2, which is necessary for stabilization of the V-ATPase complex. Osmotic stress, known to increase PI3,5P2 levels, did not rescue the compromised PI3,5P2 levels, nor did it induce vacuolar fragmentation in VPA-treated cells, suggesting that perturbation of the V-ATPase is a consequence of altered PI3,5P2 homeostasis under inositol-limiting conditions. These findings identify novel consequences of inositol depletion and provide evidence for a previously unidentified link between inositol levels and the V-ATPase.
To identify novel pathways and processes that are altered in response to VPA, a yeast cDNA library was screened for genes that increase sensitivity to VPA when overexpressed. One of the major categories identified was endocytic trafficking genes, which led to the hypothesis that VPA perturbs endocytosis. The study showed that VPA perturbs endocytosis in yeast and human cells. Evidence showed that the likely mechanism underlying decreased endocytosis by VPA is decreased PI4,5P2 levels.
Taken together, my studies led to three major novel findings. First, I identified a regulatory mechanism of inositol biosynthesis characterized by phosphorylation of the rate-limiting enzyme myo-inositol 3-phosphate synthase (MIPS). Second, I demonstrated that the highly conserved vacuolar ATPase (V-ATPase) is a target of VPA. Third, my studies indicated that VPA-mediated inositol depletion perturbs endocytosis in both yeast and mammalian cells. These findings suggest new mechanisms that may underlie the therapeutic action of VPA, and identify potential targets that may be used for the development of more effective and safer drugs.
Deranieh, Rania M., "Regulation Of Inositol Biosynthesis And Cellular Consequences Of Inositol Depletion: Implications For The Mechanism Of Action Of Valproate" (2014). Wayne State University Dissertations. 1045.