Every biochemical process that happens in an eukaryotic cell relies upon a molecular information flow that leads from 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 with the structural framework on which molecular signaling complexes assemble to ensure proper information processing. These cellular processes 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. The first set of experiments focused on the question of how the beta-glucocerebrosidase enzyme (GBA) reaches the lysosomes. Defects in the function of this enzyme cause Gaucher disease, one of the most common human lysosomal storage diseases. GBA is synthesized in the endoplasmic reticulum (ER) and binds to a receptor protein, called LIMP2 that carries GBA all the way to the lysosome. In a proteomic analysis both GBA and LIMP2 was found in a complex with one of the phosphatidylinositol kinase enzymes, PI4KB. Inositol lipids are a small fraction of membrane phospholipids with important regulatory functions and we determined whether phosphatidylinositol 4-phosphate (PI4P), the lipid product of PI4KB, is important for LIMP2/GBA transport to the lysosome. We found that distinct phosphatidylinositol 4-kinases (PI4Ks) play important roles at multiple steps in the trafficking pathway of the LIMP-2/GBA complex. Acute depletion of phosphatidylinositol 4-phosphate in the Golgi caused accumulation of LIMP-2 in this compartment, and PI4KB was found to be responsible for controlling the exit of LIMP-2 from the Golgi. In contrast, depletion of PI4K2A, another PI4K, blocked trafficking at a post-Golgi compartment, leading to accumulation of LIMP-2 in enlarged endosomal vesicles. PI4K2A depletion also caused secretion of missorted GBA into the medium, which was attenuated by limiting LIMP-2/GBA exit from the Golgi by PI4KB inhibitors. These studies identified PI4KB and PI4K2A as important regulators of lysosomal delivery of GBA, revealing a new element of control to sphingolipid homeostasis by phosphoinositides. The importance of these studies is that PI4P and sphingolipids are not only critical for cellular functions, but also for replication of certain viruses within the cell. Several Pharma companies are developing PI4K inhibitors as potential antiviral agents. It is critically important that we understand what processes are controlled by these enzymes in the cell to properly evaluate the potential risks and benefits from the use of such inhibitors. In a second set of studies the role of plasma membrane phosphoinositides, PI4P and PI(4,5)P2 was studied in cellular regulation. PI4P is a metabolic precursor of PI(4,5)P2 in the plasma membrane and the two lipids often change in parallel during physiological stimulation of cell surface receptors. To be able to alter the levels of these two lipids separately, a novel molecular tool was developed, which consisted of a hybrid enzyme formed from a PI(4,5)P2 5-phosphatase and the yeast Sac1 phosphatase that hydrolyses PI4P. This hybrid enzyme, called pseudojanin (PJ), was acutely recruited to the plasma membrane by a drug-induced heterodimerizetion system developed in our laboratory. Versions of PJ with defective 5-phosphatase or 4-phosphatase activities (or lacking both activities) were generated and with these tools PI4P and PI(4,5)P2 levels could be selectively manipulated in a rapidly controlled manner. These studies showed that cells are able to maintain their PI(4,5)P2 pools at a wide range of PI4P levels provided that the PI4K enzyme(s) that generate PI4P at the plasma membrane are functional. However, PI4P in the plasma membrane turned out to play a very important role in supplying an electrostatic interaction that is required for proper functions of some ion channels and for the membrane targeting of a whole set of peripheral membrane proteins such as several small GTP binding proteins. These studies uncovered an important and hitherto unrecognized role of PI4P at the plasma membrane, namely its ability to fulfill functions that had been traditionally attributed to PI(4,5)P2. It is also notable that differences exist between proteins that selectively require PI(4,5)P2 (such as the cold-sensing menthol channels) and those that can use either lipids to support their functions (such as the heat-sensing vanilloid receptors). These studies were done in close collaboration with Dr. Robin Irvines group in Cambridge, UK. The importance of these studies is that they help us better understand how some of the most important regulators of cell proliferation, such as the Ras proteins that are mutated in a significant number of cancers bind to the plasma membrane, since membrane binding is absolutely critical for their normal and oncogenic activities. In related studies using similar approaches performed in collaboration with Dr. Peter Varnais group in Budapest, Hungary, the PI(4,5)P2 requirement of G protein-coupled receptor internalization was analyzed using AT1 angiotensin, beta-2 adrenergic and type-2C serotoninergic receptors. These studies showed that ligand-induced interaction of AT1, 5HT2C and beta2A receptors with beta-arrestin-2 was unaffected by PI(4,5)P2 depletion. However, trafficking of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PI(4,5)P2. Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PI(4,5)P2 depletion and here the stimulated AT1 receptors clustered along the plasma membrane without entering the cells. These data suggest that in the absence of PI(4,5)P2 these receptors move into clathrin-coated membrane structures that are not cleaved efficiently and hence cannot reach the early endosomal compartment. The significance of these studies is that G protein-coupled receptors regulate almost every process in the human body and they are targeted by a large fraction of human medicines. Cells regulate the sensitivity of these receptors by the very processes that are the focus of these studies and better understanding of these pathways will help us decipher disease mechanisms and design better drugs.

Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2012
Total Cost
$1,106,876
Indirect Cost
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State
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Alvarez-Prats, Alejandro; Bjelobaba, Ivana; Aldworth, Zane et al. (2018) Schwann-Cell-Specific Deletion of Phosphatidylinositol 4-Kinase Alpha Causes Aberrant Myelination. Cell Rep 23:2881-2890
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