Phosphoinositide-calcium Signaling In Cell Regulation

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Every biochemical process that happens in a eukaryotic cell relies upon a molecular information flow that leads from cell surface receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. A proper information transmission requires a high degree of organization where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases even if the affected molecular pathways differ according to the kind of disease, such as cancer, diabetes or neurodegenerative diseases just to name a few. Research described in this report has focused on the question of how cells organize their internal membranes to provide a structural framework on which molecular signaling complexes assemble to ensure proper information processing. Lipid composition of cellular membranes is a major determinant of their biophysical properties and is unique to the different cellular organelles. How cells achieve and maintain the proper lipid composition of their membranes is poorly understood. Cellular processes that affect membrane lipid composition of organelles are often targeted by cellular pathogens such as viruses to force the cells to produce the pathogen instead of performing the cells normal functions. Better understanding of these processes not only can provide new strategies to fight various human diseases but also to intercept the life cycle of cellular pathogens offering an alternative to antimicrobial drugs. Metabolic routing maintains the unique fatty acid composition of phosphoinositides A unique feature of phosphatidylinositol (PI) and its phosphorylated PPIn derivatives is that they are highly enriched in poly-unsaturated arachidonic acid at the sn-2 position of the glycerol backbone, such that the stearoyl (C18:0)-arachidonoyl (C20:4) species is the predominant cellular form of PI. The metabolic processes responsible for regulating this enrichment are not fully understood, nor is it known what importance this unique side chain composition has for normal cell physiology. In this series of studies, we investigated the question whether PI synthesis uses a specific dedicated pool of the lipid precursor phosphatidic acid (PA) in the endoplasmic reticulum (ER). Since the PA pools in the ER originate from different metabolic pathways such as de novo synthesis or phospholipase C (PLC)- and phospholipase D (PLD)-mediated PA generation it was important to determine whether PA coming from all these sources are equally available for PI synthesis. We used a combination of approaches, all applied to a single cell line, to gain comprehensive information on the metabolic fates of PI precursors by following their side chain signature and matching their kinetics with bioluminescent resonance energy transfer (BRET)-based lipid measurements specifically within the PM. We also performed Lipidomics analyses and combined it with isotope labeling and pharmacological studies to identify differences in the handling of lipid intermediates with a specific fatty acid composition that are involved in PPIn homeostasis. These studies concluded that metabolic routing of PA occurs at the ER and shows a clear preference for the stearoyl-arachidonoyl (38:4) species of DAG as well as PA for conversion into PI, especially during the rapid recycling of breakdown products generated through PI(4,5)P2 hydrolysis upon PLC activation within the PM. The significance of these studies is that by better understanding the principles by which lipid precursors are segregated in the ER to serve various lipid synthetic routs, it will be possible to selectively alter cellular lipid metabolism to interfere with lipid storage without affecting the membrane architecture of eukaryotic cells and organisms. Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha Exocytosis is one of the most important membrane remodeling events by which bioactive molecules, such as hormones, neurotransmitters are rapidly secreted from cells to alter the function of other cells. The release of cargo from secretory vesicles requires their fusion with the plasma membrane. Rapid Ca2+ elevation in the cytoplasm is the most commonly used signal to trigger this process both in synaptic transmission and hormone release from endocrine cells. Phosphoinositide lipids (PPIns), in particular PI(4,5)P2 enriched in the plasma membrane is critical for membrane fusion event. In this collaborative study, led by the group of Dr. Stojilkovic at the NICHD, it was tested which PPIns control the exocytosis process in the cells of the pituitary gland. Single cell RNA sequencing in cells obtained from rat pituitaries revealed the expression of several PI lipid kinases such as Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in at least 10% of lactotrophs cells that are responsible for the secretion of Prolactin (PRL). Using a pharmacological approach to specifically inhibit these enzymes it was possible to show that PI4P made in the plasma membrane by PI4KA is critical for exocytosis without affecting the calcium signals that trigger secretion. They also showed that inhibition of the PI4KB enzyme that generates PI4P in the Golgi is dispensable for the exocytic step. These experiments revealed a key role of PI4KA-derived PI4P in the plasma membrane in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4,5)P2-dependent calcium signaling. These studies identified a new type of regulation in the exocytic process that is currently being further investigated. PI4K2A mutations in human causes innate error in intracellular trafficking with developmental and epileptic-dyskinetic encephalopathy PI4K2A is one of the four PI 4-kinase enzymes that generates the lipid, phosphatidylinositol 4-phosphate (PI4P) in cellular membranes. PI4K2A is located at the trans-Golgi network as well as on the surface of endosomes providing PI4P primarily in the late endosomal/lysosomal compartment. Recent studies from our laboratory showed that PI4K2A in late endosomes is required for efficient fusion of autophagosomes and lysosomes. Other studies showed a critical role of PI4K2A in the repair of damaged lysosomes. Our group was approached by a clinical genetic group working in the Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, UK. They identified two patients presented with developmental and epileptic-dyskinetic encephalopathy associated with corpus callosum dysgenesis, diffuse white matter volume loss, and hypoplastic vermis as shown by neuroimaging. In addition, these patients showed neurodevelopmental delay and recurrent infections with one of them dying at toddler age. Whole exome sequencing revealed mutations in the PI4K2A gene. Our group has performed functional studies recreating these mutations in PI4K2A and testing the properties of the protein in cells in which the PI4K2A gene was inactivated by CRISPR/Cas9 gene editing. These studies showed that the mutant enzyme lost its ability to localize to endomembranes and to catalyze the formation of PI4P in late endosomes. Understanding the consequences of PI4K2A defects in human and their link to the clinical presentation will help us better understand the complexities of brain development and the identification of new means by which such disease conditions can be improved.