We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.cellular assays ͉ G protein-coupled receptor ͉ protein interaction A ll cells have evolved mechanisms to respond to rapid changes in the environment. Extracellular signals are detected by transmembrane receptors that translate binding into intracellular signaling events. Most signaling systems that respond to environmental cues exhibit adaptation mechanisms that afford the cell a facile response to rapid changes in their surroundings. Mechanisms to assure the rapid but transient response to environmental cues are of obvious advantage to the cell but seriously limit most assays for receptor function. We have genetically modified receptors such that transient responses to ligand result in the stable transcription of a reporter gene. The transformation of a transient intracellular response to a stable amplifiable readout provides a sensitive and quantitative assay for receptor function.We have developed an assay for receptor activation and more generally for protein-protein interaction that involves the fusion of a membrane receptor with a transcriptional activator. The membrane-bound receptor and transcription factor sequences are separated by a cleavage site for a highly specific viral protease. A second gene encodes a fusion of the viral protease with a cellular protein that interacts only with activated receptor. Ligand binding to the receptor will stimulate this proteinprotein interaction, recruiting the protease to its cleavage site. Site-specific cleavage will release the transcriptional regulator that can now enter the nucleus and activate reporter genes. Recently, a similar principle, based on the complementation of split tobacco etch virus (TEV) protease fragments, has been used to monitor protein interactions (1). Our experimental scheme derives conceptually from the mechanism of action of the Notch receptor in which ligand binding elicits proteolytic cleavage events in the receptor to release a Notch intracellular domain that translocates to the nucleus and modulates transcription of downstream target genes (2, 3) (Fig. 1A).The assay we have developed relies solely on exogenous genes in...
The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.
Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.
This article is available online at http://www.jlr.org Coronary atherosclerosis is the most prevalent disease in industrialized societies. Although numerous advances have been made in understanding the underlying causes of atherosclerosis and treatment thereof, this condition still remains the leading cause of death in the Western world. The most important risk factor for atherosclerosis is hyperlipidemia ( 1 ). Development of atherosclerosis correlates with high levels of low density lipoprotein cholesterol (LDL). As a result, several therapies have been developed for management of LDL levels. Among these, statins are most widely used ( 2 ). However, there is a range of statin response in humans, and a subset of familial hyperlipidemia patients is unresponsive to statins, prompting the development of additional therapies.
Members of the Frizzled family of serpentine transmembrane receptors are required to transduce Wingless͞Int (Wnt) signals and contain in their N-terminal regions a conserved Wnt-binding cysteine-rich domain (CRD). Each CRD has specific affinities for particular Wnts, and it is generally believed that signal transduction depends on the strength of this interaction. Here, we report in vivo evidence that the CRD is dispensable for Frizzled family receptors to transduce Wingless (Wg), the primary Wnt signal in Drosophila. Thus, we infer that signal transduction does not require binding of Wg to the CRD, but instead depends on interactions between Wg and other portions of the receptor, or other proteins of the receptor complex. W ingless͞Ints (Wnts) are secreted glycoproteins that play profound and pervasive roles in animal development (1). Most Wnts are transduced by the Frizzled (Fz) family of serpentine transmembrane receptors (2) in conjunction with the Arrow͞low-density lipoprotein receptor-related protein (LRP) family of coreceptors (3). Some, but not all, Fz family proteins transduce Wnts through the ''canonical'' Armadillo͞-catenin pathway (1). However, Fz proteins also can transduce Wnts, or other classes of ligands, through noncanonical pathways that are independent of Armadillo͞-catenin (4).Structure͞function analysis is essential to understand the mechanism by which Fz proteins are activated by Wnts. All Fz proteins contain a large extracellular N-terminal motif with 10 conserved cysteines, known as the cysteine-rich domain (CRD; Fig. 1A). In general, intact Fz proteins and their isolated CRDs can bind Wnts in cell culture assays, but different Fz CRDs have different binding affinities for specific Wnts (2, 5-8). The structures of two CRDs recently have been determined, and the surfaces that are likely to interact with Wnts were identified by mutational analysis of Wnt͞CRD-binding interactions in cell culture assays (9). These and other data (10) have led to the view that Wnt binding to the CRD is essential for activating signal transduction.Inferences about Fz protein structure and function from crystallographic analysis and cell culture experiments can be tested in vivo in Drosophila. The Drosophila genome encodes seven Wnts and four proteins of the Fz family (11). Of these, Wingless (Wg) is the predominant Wnt signal in Drosophila and is transduced by two of the four Fz proteins, Fz and Dfz2, through activation of the canonical Armadillo͞-catenin pathway (1). Fz and Dfz2 are fully redundant for Wg transduction, because loss of either has no detectable effect on Wg signaling at any stage of development, whereas loss of both abrogates virtually all known responses to Wg (12,13). A third protein, Dfz3, can bind Wg in cell culture assays but has little or no transducing activity and instead likely functions as an antagonist of Wg signaling (6,8). Dfz4 is a divergent Fz protein that lacks the capacity to bind Wg (8); furthermore, its overexpression or down-regulation by RNA interference appears to have n...
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