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. Creation of a molecular toolset to manipulate and monitor subcellular phosphatidylinositol 3,5-bisphosphate. Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a minor phospholipid component of cellular membranes. Based on studies using inhibitors of PIKfyve, the enzyme that produces PI(3,5)P2, this regulatory lipid is critical for the control of several important cellular functions linked to the endo-lysosomal system. Despite its well-recognized importance, the precise sites of subcellular enrichment and molecular targets of PI(3,5)P2 are poorly understood due to the lack of molecular tools to identify the lipid in cells or to manipulate its level in specific cellular organelles. In our most recent studies, in collaboration with the group of Dr. John Burke in Canada, we have designed, generated and characterized a short engineered catalytic fragment of the human PIKfyve enzyme, which potently converts PI 3-phosphate (PI3P) to PI(3,5)P2. This novel tool allowed for the evaluation of reported PI(3,5)P2-sensitive biosensors and showed that the recently identified phox homology domain (PX) of the Dictyostelium discoideum (Dd) protein, SNXA, can be used to monitor the production of PI(3,5)P2 in live cells. Moreover, we have shown that using these tools, we could substantially increase PI(3,5)P2 levels in the membranes of specific organelles, allowing assessment of the cellular processes that change when more of this lipid is present in the membrane. Furthermore, we have developed the DdSNXA-PX-based probe into bioluminescence resonance energy transfer (BRET)-based biosensors for real-time monitoring of PI(3,5)P2 generation within specific endocytic compartments of entire cell populations. The importance of these studies is that they have now provided the scientific community a molecular tool-set, which will help identify the molecular targets and biological functions of the hitherto enigmatic lipid, PI(3,5)P2. Given the recent developments describing beneficial effects of interfering with PI(3,5)P2 generation in mouse models of Lou Gehrig disease, better understanding of PI(3,5)P2 functions could facilitate the development of strategies to fight this devastating disease. Sorting nexin 10 regulates lysosomal ionic homeostasis via ClC-7 by controlling PI(3,5)P2 Mutations or elimination of the protein, sorting nexin 10 (Snx10) are associated with neurodegeneration, blindness, and osteopetrosis. The latter condition has been traced to impaired function of osteoclasts, the bone-resorbing cells in SNX10 mutant patients. Our group has been approached by the group of Dr. Spencer Freeman and Sergio Grinstein, in Canada, who found that macrophages (phagocytic cells that share many features with osteoclasts) deleted in SNX10 show impaired resolution of their phagosomes due to defective chloride accumulation in their lysosomes. It was found that depletion of SNX10 diminished the activity of the lysosomal chloride/proton antiporter, ClC-7, a protein that is under the negative control by the regulatory lipid, PI(3,5)P2. Using the molecular tools that we have developed, the Freeman group was able to show that normal SNX10 limits the formation of PI(3,5)P2 at the lysosome and releases the activity of the ClC-7 transporter from its PI(3,5)P2-mediated inhibition, ultimately enabling the transporter to transport chloride to the lysosomes, a requirement for the activity of the proteolytic enzymes that digest the content of phagosomes. These studies helped better understand the processes that are impaired in patients that suffer from either SNX10 or ClC-7 mutations and will facilitate the development of treatments. Molecular basis for plasma membrane recruitment of PI4KA by EFR3. Phosphatidylinositol 4-kinase type-III alpha (PI4KA) is a critically important lipid kinase enzyme that plays central roles in eukaryotic cells in controlling signal transduction at the plasma membrane (PM) and orchestrating the entire cellular lipid metabolic network. PI4KA also serves as an essential host factor for the replication of many picornaviruses that cause human disease. PI4KA functions as part of a tetrameric complex comprising of three additional proteins, TTC7, Fam126, and EFR3 (all three proteins existing in A and B forms in mammals), the latter being an anchor to keep the complex at the PM. There is an increasing number of patients with identified causative mutations in PI4KA presenting with severe neuro-developmental, immunological and gastrointestinal conditions. Some of these mutations impair the interaction of the proteins with its protein binding partners, rather than the enzymatic activity of the isolated enzyme, therefore, understanding the structural basis of these molecular interactions is important to better understand the impact of disease-causing mutations. In these studies, our group has collaborated with the group of Dr. John Burke in Canada, who has been a pioneer in structural studies on inositol lipid kinases, to identify the structural elements that provide the interaction between EFR3 and the rest of the PI4KA-TTC7-FAM126 complex. This interaction has proven to be essential for the recruitment of the PI4KA complex to the PM as proven by mutational analysis, live cell imaging and bioluminescence energy transfer (BRET)-based measurements in intact cells. Since the PI4KA complex and its membrane recruitment is also essential for the replication of many picornaviruses, these structural studies may help design new therapeutical strategies to combat these infections.