Phosphoinositide Messengers in Cellular Signaling and Trafficking

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, given 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 John Burke, we 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 us to evaluate 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 showed 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 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 provide the scientific community with a molecular tool-set that 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 amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig disease, better understanding of PI(3,5)P₂ functions could facilitate the development of strategies to fight this devastating disease.

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 was approached by the group of Spencer Freeman and Sergio Grinstein, 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 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 who suffer from either SNX10 or ClC-7 mutations, and will facilitate the development of treatments.

Phosphatidylinositol 4-kinase type-III alpha (PI4KA) is a critically important lipid kinase enzyme, which 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 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. In an increasing number of patients presenting with severe neurodevelopmental, immunological, and gastrointestinal conditions, causative mutations in PI4KA have been identified. Some of these mutations impair the interaction of the proteins with their 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 John Burke, 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. Given that the PI4KA complex and its membrane recruitment are also essential for the replication of many picornaviruses, these structural studies may help design new therapeutical strategies to combat these infections.