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 concentrated on questions related to the functions of the lipid kinase enzyme, phosphatidylinositol 4-kinase alpha (PI4K2A). This enzyme was found recently important for HIV-1 virus assembly and exit from infected cells. In these studies, we found that PI4K2A associates with one of the soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). SNARE proteins are required for the fusion of various membrane compartments with one another and hence are critical for the vesicular trafficking of all membrane components. Using affinity purification coupled to mass spectrometry (AP-MS) we identified PI4K2A as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport of numerous molecules. We found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A depletion by RNAi-mediated gene silencing strongly inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE, Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of its lipid product, PI4P on endosomes significantly delayed VAMP3 trafficking. Phospholipid modulation of SNARE function has been previously proposed based on in vitro studies, our study now provides mechanistic evidence in support of these claims by identifying PI4K2A and PI4P as regulators of an R-SNARE using intact cells. Together with the presence of PI4P in Rab7 positive late endosomes, these results suggest that PI4K2A and PI4P are key players in decision-making as to which molecules should be recycled and which molecules should be degraded via lysosomal degradation. PI4K2A has been show earlier to control EGF receptor degradation, a process that is often impaired in various forms of cancer. Our results provide further details of this important regulatory mechanism. An additional role of PI4K2A in late endosomal function was revealed in a collaborative study with Dr. Albert Haas in Germany, in which PI4K2A was found to be critical in the late phase of phagosome maturation and fusion with lysosomes. A major break-through in our efforts to characterize PI4K2A was solving the high resolution X-ray structure of the protein. The catalytic properties and sequence comparisons already predicted that type-II PI4Ks, such as PI4K2A belong to a unique family of lipid kinases distinct form other PI kinases. Determining the structure of these proteins has been an important goal of our laboratory on a collaborative basis. This led us to form a strong alliance with one of the trainees of Jim Hurleys laboratory, Dr. Evzen Boura, who set up his group in Prague, Czech Republic. In close collaboration with us, Dr. Bouras group has solved the X-ray structure of PI4K2A and confirmed that it represents a unique fold not seen previously in type III PI4Ks or PI3Ks. Some functional predictions based on the structural information have been tested in situ in live cells in our laboratory. These included mutagenesis studies on the role of a hydrophobic pocket facing the membrane surface in catalysis. The structural information obtained by these studies will greatly facilitate the development of new specific inhibitors against this group of enzymes. We have initiated a high throughput screen to identify PI4K2-specific inhibitors with the NCATS Center. For this we set up a miniature PI4K assay platform that is compatible with high-throughput screening. Using recombinant human PI4K2A, produced in bacteria, NCATs has run a screen of 500,000 compounds. This screen has identified several activator and inhibitor compounds that were counter-screened with PI4KB (a type-III PI4K) and narrowed down to 12 inhibitory and 10 activator compounds for further analysis in intact cells. These promising studies are still in progress, but an important phase of the study has been completed. Another important achievement of this years research was the completion of a study that addressed the question of how structurally important lipids are exchanged between the endoplasmic reticulum (ER) and the plasma membrane (PM) during the actions of hormones and neurotransmitters. Our studies focused on the question of how phosphatidylinositol (PI) the precursor lipid of the regulatory phosphoinositides is transported from the ER to other membranes. We tested all known PI transfer proteins (PITPs) by RNA-mediated knock-down and found that only Nir2 had a profound effect on phosphoinositide levels. Nir2 is a large PITP, first described in Drosophila as the RdgB mutant, which develops light-induced retinal degeneration. We found that Nir2- depleted cells not only had a problem in supplying the PM with PI, but also had a problem with PI synthesis, due to a defect in phosphatidic acid (PA) transport from the PM to the ER. We also found that Nir2 was enriched in ER-PM contact sites after stimulation of phospholipase C (PLC) enzymes and that the Nir2 protein was anchored to the ER via interaction with VAP-A and B proteins. These studies have solved a long-standing question of how the lipid products of PLC activation get recycled to maintain the signaling competence of cells under chronic stimulation. Since some familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrigs disease) are caused by mutations within the VAP-B protein, we also tested these mutants for their ability to interact with the Nir2 protein. We found that some disease-causing mutations largely interrupted the interaction between Nir2 and VAB-B, while others had more moderate effects. These studies may give us further clues regarding the molecular details of the processes that are critical for the pathology of ALS.

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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
Balla, Tamas (2018) Ca2+ and lipid signals hold hands at endoplasmic reticulum-plasma membrane contact sites. J Physiol 596:2709-2716
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