Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor.

Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV.     

Structure-based design of novel human Pin1 inhibitors (I)

Pin1 is a member of the cis–trans peptidyl-prolyl isomerase family with potential anti-cancer therapeutic value. Here we report structure-based de novo design and optimization of novel Pin1 inhibitors. Without a viable lead from internal screenings, we designed a series of novel Pin1 inhibitors by interrogating and exploring a protein crystal structure of Pin1. The ligand efficiency of the initial concept molecule was optimized with integrated SBDD and parallel chemistry approaches, resulting in a more attractive lead series.     

TRAF family proteins interact with the common neurotrophin receptor and modulate apoptosis induction.

The common neurotrophin receptor, p75(NTR), has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-kappaB. However, the mechanisms by which p75(NTR) initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75(NTR). Furthermore, TRAF4 interacted with dimeric p75(NTR), whereas TRAF2 interacted preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulated p75(NTR)-induced cell death and NF-kappaB activation with contrasting effects. Coexpression of TRAF2 with p75(NTR) enhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75(NTR). TRAF4 also inhibited the NF-kappaB response, whereas TRAF2 and TRAF6 enhanced p75(NTR)-induced NF-kappaB activation. These results demonstrate that TRAF family proteins interact with p75(NTR) and differentially modulate its NF-kappaB activation and cell death induction.     

Apoptosis mediated by the TNF-related cytokine and receptor families

T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)-related family of membrane-anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin β-receptor (LTβR), among others. The MHC-encoded cytokines, TNF and LTα, form homomeric trimers, whereas LTβ assembles into heterotrimers with LTα, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LTβR are involved in induction NFκB and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the “death domain” homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways. © 1996 Wiley-Liss, Inc.     

Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.     

Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis

To probe signal propagation and genetic actuation in microbial consortia, we have coopted the components of both redox and quorum sensing (QS) signaling into a communication network for guiding composition by “programming” cell lysis. Here, we use an electrode to generate hydrogen peroxide as a redox cue that determines consortia composition. The oxidative stress regulon of Escherichia coli, OxyR, is employed to receive and transform this signal into a QS signal that coordinates the lysis of a subpopulation of cells. We examine a suite of information transfer modalities including “monoculture” and “transmitter-receiver” models, as well as a series of genetic circuits that introduce time-delays for altering information relay, thereby expanding design space. A simple mathematical model aids in developing communication schemes that accommodate the transient nature of redox signals and the “collective” attributes of QS signals. We suggest this platform methodology will be useful in understanding and controlling synthetic microbial consortia for a variety of applications, including biomanufacturing and biocontainment.