Every biochemical process that happens in a 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 a 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. During this period we discovered that phosphatidylinositol 4-kinase alpha (PI4KA), one of the lipid kinases that phosphorylate the important lipid, phosphatidylinositol (PI) at the plasma membrane (PM) is essential for the transport and synthesis of a critical structural lipid, phosphatidylserine (PS). Prolonged (24-36h) treatment of cells with the PI4KA-specific inhibitor caused a 50% decrease in cellular PS content and the inhibitor acutely blocked PS synthesis. We have demonstrated that this inhibition was not due to a direct effect on the PS synthesizing enzymes, but it was due to a transport defect impeding transport of PS out of its site of synthesis in the ER to the PM. Since PS synthesis is under very strong feed-back inhibition, PS transport defects out of the ER lead to inhibition of PS synthesis. A recent study has identified mutations in the PS synthetic enzyme, PSS1 that rendered the enzyme insensitive to PS-mediated feed-back inhibition. These PSS1 mutations were found causative in a rare human disease, Lenz-Majewski Syndrome (LMS). We found that mutant PSS1 enzymes not only increased the level of PS to very high level, but they also reduced phosphatidylinositol 4-phosphate (PI4P) levels in several membrane compartments, including the Golgi and PM. This was due to the activation of the PI4P phosphatase enzyme in the ER by the accumulating PS. We postulated that there exists a mechanism that transports PS from the ER to the PM using the energy of a PI4P gradient generated between the PM and the ER. This PI4P gradient is maintained by PI4KA in the PM and the Sac1 phosphatase in the ER. While ours studies were in progress, it was published that oxysterol binding protein-related protein 5 and -8 (ORP5 and -8) is able to transport PS from the ER to the PM using the PI4P gradient set up between the PM and the ER. Indeed, in our studies ORP8 was shown to bridge the ER and the PM in contact sites and its PM-binding required PM PI4P. These discoveries have important implications as PI4KA is an essential host protein that supports hepatitis C virus (HCV) replication in the liver. It is likely that the important role of PI4KA in PS metabolism is critical for the HCV lifecycle. In additional studies in collaboration with the group of Dr. Boura in Prague, Czech Republic, the structural features of the interaction between PI4KB (another enzyme that can phosphorylate PI) and the ACBD3 protein was investigated. PI4KB and ACBD3 proteins were both found to be important host factors for several enteroviral virus replication in mammalian cells. It appears that the interaction of the two proteins is essential for the viral life cycle because the virus uses ACBD3 to recruit the lipid kinase to the replication organelle. Dr. Boura's group has identified the minimum interaction domains between the two proteins and solved the structure of this complex. These studies have identified key residues on both proteins essential for the interaction. Our group has used mutant ACBD3 proteins in intact cells to demonstrate that indeed these residues are essential for the ability of the ACDB3 protein to recruit the kinase to specific membrane compartments. These structural studies may help to identify pharmacological means to interrupt this association as a way of blocking enteroviral replication in mammalian cells. In a separate line of studies we pursued the goal of improving our abilities to quantitate and image inositol lipid and inositol phosphate changes in intact cells. We have performed a thorough study to investigate the claim that a tandem N-terminal piece of the MLN1 (Mucolipin 1 channel) protein (MLN1N2x) is capable of visualizing PI(3,5)P2 pools inside the cell. PI(3,5)P2 is a minor phosphoinositide species that has a critical role in a variety of cellular functions that rely upon vesicular transport between certain type of endosomes and lysosomes. Tools to show the location and dynamics of PI(3,5)P2 have long been sought after without convincing data. Hence, the report by Haoxing Xus group from the University of Michigan showing that GFP-tagged MLN1N2x is a faithful PI(3,5)P2 sensor has been a welcome addition to the phosphoinositide imaging toolbox. In this study, we have evaluated this recently reported PI(3,5)P2 biosensor, GFP-ML1Nx2, for its veracity as such a probe. Unfortunately, we found that, in live cells, the localization of this biosensor to sub-cellular compartments is largely independent of PI(3,5)P2, as assessed after pharmacological, chemical genetic or genomic interventions that block the lipid's synthesis. We therefore concluded that it is unwise to interpret the localization of ML1Nx2 as a true and unbiased biosensor for PI(3,5)P2. This study was done in collaboration with Dr. Sasakis group in Japan. Lastly, this year we also collaborated with our long-standing collaborator, Dr. Peter Varnai in Budapest to develop a method by which the level of inositol 1,4,5-trisphosphate (InsP3) can be followed in cell populations or in single living cells. For this we used the InsP3 binding domain of the type-I InsP3 receptor and introduced mutations to fine-tune its affinity for optimal on-off kinetics of InsP3 binding. These optimal sensors were then engineered as molecular sensors based on fluorescent resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) principles for single or cell population measurements, respectively. These new tools will be of great value in studies where InsP3 changes have to be assessed in living cells and they can be easily adapted for high throughput screening applications.
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