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. As detailed below, 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. The first set of experiments focused on the activation mechanism of a specific Ca2+ entry pathway, namely that formed by the STIM1/Orai1 complex. Calcium ions (Ca2+) are one of the most ubiquitously used signaling molecules in eukaryotic cell regulation. They regulate a range of cellular processes such as muscle contraction, hormone secretion or gene transcription. Ca2+ enters the cells via multiple Ca2+ entry routes formed by Ca2+ channels and transporters and is very efficiently eliminated by Ca2+ pumps and exchangers. The tight control of the cells Ca2+ homeostasis is essential for cellular signaling and for the maintenance of cellular integrity. Recent studies identified two protein families (STIM and Orai/CRACM) that mediate a specific form of Ca2+ entry, termed store-operated calcium entry (SOCE). STIM1, is an endoplasmatic reticulum (ER) resident protein that rapidly translocates to the plasma membrane (PM)-adjacent compartment of the ER upon depletion of the ER Ca2+ stores where it activates the calcium channel, Orai1. The importance of this calcium entry pathway is highlighted by the fact that mutations in Orai1 has been linked to severe inborn human immunodeficiencies and that this route of calcium entry is key to the calcium regulated activation of T-cells mediated by the NFAT transcription factors. In a series of studies we showed that the cytoplasmic domain of the ER resident protein, STIM1 (STIM1cyto) is capable of activating the Orai1 Ca2+ channels but only when the STIM1 molecules are clustered. This led us to conclude that the small Orai1 activating segment within STIM1cyto (previously identified by the Lewis and Muallem groups and called CAD and SOAR, respectively) is cryptic and only gets unmasked during the STIM1 clustering process. We then identified the membrane adjacent coiled-coil (CC) domain of STIM1cyto as a segment necessary for keeping the CAD/SOAR domain silent and showed that an acidic sequence within the CC1 domain plays a critical role in maintaining an intramolecular interaction with a basic stretch within the CAD/SOAR domains. This finding suggested that the activation cascade of the STIM1/Orai1 complex, which is known to be initiated by Ca2+ unbinding at the ER luminal segment of STIM1, involves the breaking of an electrostatic interaction that occurs parallel or in response to the clustering of the STIM1 molecules. These studies shed new lights to the molecular sequence of STIM1 activation and will help us identify critical points where this process can be antagonized by molecular or pharmacological interventions. In another set of experiments we investigated the genetic organization and expression patterns of one of the PI 4-kinases, PI4KIIIα. This enzyme is a key regulator of the production of more complex phosphoinositides and also has been identified as a critical host factor for hepatitis C (HCV) replication in liver. Two isoforms of the mammalian PI4KIIIαhave been described and annotated in GenBank: a larger, 230kDa (isoform 2) and a shorter splice variant containing only the 97 kDa C-terminus that includes the catalytic domain (isoform 1). However, Northern analysis of human tissues and cancer cells showed only a single transcript of 7.5kb with the exception of the proerythroleukemia line K562, which contained significantly higher level of the 7.5 kb transcript along with smaller ones of 2.4, 3.5 and 4.2 kb size. Bioinformatic analysis also confirmed the high copy number of PI4KIIIαtranscript in K562 cells along with several genes located in the same region in Chr22, including two pseudogenes that cover most exons coding for isoform 1, consistent with chromosome amplification. A panel of polyclonal antibodies raised against peptides within the C-terminal half of PI4KIIIαfailed to detect the shorter isoform 1 either in COS-7 cells or K562 cells even though they detected the larger isoform 2. Moreover, expression of a cDNA encoding isoform 1 yielded a protein of 97 kDa that showed no catalytic activity and failed to rescue hepatitis C virus replication. These data drew attention to PI4KIIIαas one of the genes found in Chr22q11, a region affected by chromosomal instability, but did not substantiate the existence of a functionally relevant short form of PI4KIIIα. Parallel to these studies, our group collaborated with Dr. Bartenschlagers group (Department for Molecular Virology, Heidelberg University, Germany) to investigate the role of PI4KIIIαin hepatitis C (HCV) replication in liver. In this study an elevated level of PtdIns4P was found in HCV-infected cultured hepatocytes and in liver tissue from chronic hepatitis C patients. The enzymatic activity of PI4KIIIαwas also found to be critical for HCV replication and the viral nonstructural protein 5A (NS5A) was shown to interact with PI4KIIIαand stimulate its kinase activity. The absence of PI4KIIIαactivity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. These findings suggest that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex. These findings revealed how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication. These results highlighted PI4KIIIαas an important potential drug target for the control of HCV infection and therefore, we also entered collaboration with Dr. Andrew Tai (Department of Internal Medicine, Division of Gastroenterology, Michigan University) and set up a non-isotopic PI4K fluorescent assay system that would facilitate the identification of PI4K inhibitors in high-throughput screening. This method will be used to identify novel inhibitory compounds specific for the various classes of PI 4-kinase enzymes. These latter studies also exemplified how basic research can yield unforeseeable benefits with potentially high impact in public health.

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