Abstract
Micronutrient sensing is critical for cellular growth and differentiation. Deficiencies in essential nutrients such as iron strongly affect neuronal cell development and may lead to defects in neuronal function that cannot be remedied by subsequent iron supplementation. To understand the adaptive intracellular responses to iron deficiency in neuronal cells, we developed and utilized a Stable Isotopic Labeling of Amino acids in Cell culture (SILAC)-based quantitative phosphoproteomics workflow. Our integrated approach was designed to comprehensively elucidate the changes in phosphorylation signaling under both acute and chronic iron-deficient cell models. In addition, we analyzed the differential cellular responses between iron deficiency and hypoxia (oxygen-deprived) in neuronal cells. Our analysis identified nearly 16,000 phosphorylation sites in HT-22 cells, a hippocampal-derived neuronal cell line, more than ten percent of which showed at least 2-fold changes in response to either hypoxia or acute/chronic iron deficiency. Bioinformatic analysis revealed that iron deficiency altered key metabolic and epigenetic pathways including the phosphorylation of proteins involved in iron sequestration, glutamate metabolism, and histone methylation. In particular, iron deficiency increased glutamine-fructose-6-phosphate transaminase (GFPT1) phosphorylation, which is a key enzyme in the glucosamine biosynthesis pathway and a target of 5′ AMP-activated protein kinase (AMPK), leading to reduced GFPT1 enzymatic activity and consequently lower global O-GlcNAc modification in neuronal cells. Taken together, our analysis of the phosphoproteome dynamics in response to iron and oxygen deprivation demonstrated an adaptive cellular response by mounting post-translational modifications that are critical for intracellular signaling and epigenetic programming in neuronal cells.
Highlights
Iron deficiency (ID) is one of the most prevalent micronutrient deficiencies, affecting approximately 30% of pregnant women and pre-school age children worldwide, and it causes poor long-term neurodevelopment outcomes and increased risks of psychiatric disorders in later life [1,2]
We identified diverse signaling mechanisms that were differentially regulated by ID and hypoxia in hippocampal neuronal cells
To perform kinase activity analysis of our dataset, we developed and utilized a sampling-based statistical approach, termed Kinase Activity Profiling Analysis (KAPA), to evaluate the site-specific changes in the abundance of kinase targets that can be applied to any species with available kinasesubstrate databases
Summary
Iron deficiency (ID) is one of the most prevalent micronutrient deficiencies, affecting approximately 30% of pregnant women and pre-school age children worldwide, and it causes poor long-term neurodevelopment outcomes and increased risks of psychiatric disorders in later life [1,2]. Iron is an essential nutrient for cell development and function [3]. Chronic ID leads to anemia and affects brain development, during the rapid growth period that spans the late third trimester of fetal life and early childhood. Early-life (fetal and early postnatal) ID anemia causes neurodevelopment deficits, including in learning and memory, which are not fully rescued by the subsequent iron supplementation and resolution of ID anemia [4,5,6]. Insufficient iron uptake from the cellular microenvironment can lead to ID and thereby abnormal growth and development [12,13]. Elucidating the iron-sensitive targets that regulate intracellular signaling constitutes an important step toward the development of more effective therapeutic approaches
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