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. During this period we concentrated on several questions related to the functions of the lipid kinase enzyme, phosphatidylinositol 4-kinase alpha (PI4KA). This enzyme was found recently as an essential host protein for the replication of the hepatitis C virus (HCV). Inhibition of this enzyme seemed as a very promising strategy to combat HCV infections. HCV is a major health problem for which new therapies are constantly sought. Several pharmaceutical companies have been active in developing PI4KA specific inhibitors to test if they can be used as anti HCV agents. We have collaborated with researchers in Glaxo-Smith-Kline (GSK) to investigate the effects of several of their PI4KA specific inhibitors on cellular signal transduction processes. We showed that these compounds inhibit the synthesis of the important regulatory lipid, phosphatidylinositol 4-phosphate (PI4P) specifically in the plasma membrane (PM) but not in the Golgi. The inhibitors also impaired the maintenance of the critically important phosphoinositide, PI(4,5)P2 (which is made from PI4P in the plasma membrane) when the cells were stimulated by hormones that cause PLC activation. Importantly we found that the potency of these compounds to inhibit purified PI4KA in vitro and to inhibit PI4P synthesis in the PM in cells showed significant variations raising questions about the ability of the compounds to reach the relevant cellular compartments despite similar chemistries. However, the inhibitory effects of the compounds on PI4P in the PM and on PI(4,5)P2 levels in PLC-stimulated cells were closely correlated. Toxicity studies performed in animals by GSK showed that the most potent small molecule inhibitors of PI4P synthesis have interfered with PI(4,5)P2 maintenance and caused sudden death when applied at high doses with symptoms reminiscent of cardiovascular collapse. These deleterious outcomes may reflect the compounds ability to inhibit PI(4,5)P2 maintenance during Gq-coupled receptor signaling that is essential for maintaining vascular tone. Finally, genetic inactivation of the PI4KA enzyme in adult animals with a tamoxifen-induced conditional knockout mouse model caused a lethal gastrointestinal phenotype that was different from the acute drug-induced toxicity. These differences will require further studies to be fully understood but highlight the need for both genetic and pharmacological approaches in order to anticipate the results of pharmacological interventions on the biology of whole animals. Unfortunately, these studies raised significant doubts concerning the suitability of PI4KA inhibitors as anti HCV agents. In a separate but highly related study, we pursued the aim of developing a fluorescent reporter molecule that would be capable of detecting PI4P formation and dynamics in live cell microscopy applications. The importance of such a tool is obvious when considering the importance of PI4P in a variety of cellular organelles such as the PM (see above), the Golgi, and the various endosomes. Detection of PI4P in live cells has been previously attempted using fluorescent protein fusions of PI4P-binding pleckstrin homology (PH) domains from Golgi effector proteins, which invariably localize to the Golgi in a PI4P-dependent manner, apparently supporting a Golgi-selective enrichment of the lipid. A major caveat of this approach was that PH domains often exhibit tertiary inter-molecular interactions that bias the probes localization. In other words, PI4P alone is not sufficient for membrane targeting of these probes. Another PI4P-binding module, the Osh2p PH domain binds to the PM and Golgi in yeast, but localizes solely to the PM in mammalian cells, which could be interpreted as reflecting a relatively low concentration of available PI4P at the Golgi. However, this probe also binds PM PI(4,5)P2. Immunocytochemical methods have detected PI4P in both Golgi and PM, but this technique was limited to fixed specimens and could not answer the crucial question as to which pools of the lipid are available to recruit PI4P-regulated effector proteins. Many bacterial pathogens have evolved secreted effectors proteins with features that allow selective targeting of host cell membranes, which are then hijacked to support replication of the bacteria. The ability to bind or modify the phosphoinositide head group is a common mechanism employed by such proteins. We therefore sought to utilize the power of bacterial effector proteins as a tool to investigate PI4P distribution in living cells. We focused on the SidM protein, secreted by the intracellular pathogen, Legionella pneumophila. SidM is recruited to L. pneomophila-containing vacuoles where its guanine nucleotide exchange factor and AMPylation activities stimulate Rab1 signaling, causing the recruitment of ER-derived materials. Vacuole recruitment was shown to be facilitated through binding to PI4P by a unique P4M (PI4P-binding of SidM) domain). The P4M domain exhibits exquisite specificity and affinity for PI4P in vitro, and is structurally distinct from the catalytic and Rab1-interacting activities. The P4M domain from SidM seemed therefore to be an ideal candidate for an unbiased and sensitive biosensor for PI4P in living cells. In these studies we showed that PI4P is necessary and sufficient for membrane targeting of P4M in living cells, and that the probe therefore detects readily accessible pools of the lipid in the Golgi, PM and, unprecedentedly, in Rab7-positive endo/lysosomes. Each of the mammalian PI4Ks corresponded to at least one of these pools, and the PM and Golgi pools were found accessible to the ER/Golgi localized phosphatase Sac1. This distribution pointed to a more functionally diverse role for PI4P than had previously been appreciated. With the aid of this new PI4P probe we, and others will be able to study the dynamics of this lipids in various setting, including during infections with viruses, or during malignant transformation.
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