SummaryMethods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.
Store-operated Ca2+ entry (SOCE) occurs when loss of Ca2+ from the endoplasmic reticulum (ER) stimulates the Ca2+ sensor, STIM, to cluster and activate the plasma membrane Ca2+ channel Orai (encoded by Olf186-F in flies). Inositol 1,4,5-trisphosphate receptors (IP3Rs, which are encoded by a single gene in flies) are assumed to regulate SOCE solely by mediating ER Ca2+ release. We show that in Drosophila neurons, mutant IP3R attenuates SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and the IP3R to accumulate near the plasma membrane, association of STIM with Orai, clustering of STIM and Orai at ER–plasma-membrane junctions and activation of SOCE. These responses were attenuated in neurons with mutant IP3Rs and were rescued by overexpression of STIM with Orai. We conclude that, after depletion of Ca2+ stores in Drosophila, translocation of the IP3R to ER–plasma-membrane junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE.
The ability of Ca(2+), the simplest of all intracellular messengers, selectively to regulate so many cellular behaviours is due largely to the complex spatiotemporal organization of intracellular Ca(2+) signals. Most signalling pathways, including those that culminate in Ca(2+) signals, comprise sequences of protein-protein interactions linked by diffusible messengers. Using specific examples to illustrate key principles, we consider the roles of both components in defining the spatial organization of Ca(2+) signals. We discuss evidence that regulation of most Ca(2+) channels by Ca(2+) contributes to controlling the duration of Ca(2+) signals, to signal integration and, via Ca(2+)-induced Ca(2+) release, to defining the spatial spread of Ca(2+) signals. We distinguish two types of protein-protein interaction: scaffolds that allow rapid local transfer of diffusible messengers between signalling proteins, and interactions that directly transfer information between signalling proteins. Store-operated Ca(2+) entry provides a ubiquitous example of the latter, and it serves also to illustrate how Ca(2+) signals can be organized at different levels of spatial organization - from interactions between proteins to interactions between organelles.
Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors are the channels responsible for Ca(2+)release from the endoplasmic and sarcoplasmic reticulum. Research inScience Signalingby Alzayadyet al show that all four IP3-binding sites within the tetrameric IP3R must bind IP3before the channel can open, which has important consequences for the distribution of both IP3and IP3R activity within cells.
Analogues of the Ca 2+ -releasing intracellular messenger D-myoinositol 1,4,5-trisphosphate [1, Ins(1,4,5)P 3 ] are important synthetic targets. Replacement of the α-glucopyranosyl motif in the natural product mimic adenophostin 2 by D-chiro-inositol in D-chiro-inositol adenophostin 4 increased the potency. Similar modification of the non-nucleotide Ins(1,4,5)P 3 mimic ribophostin 6 may increase the activity. D-chiro-Inositol ribophostin 10 was synthesized by coupling as building blocks suitably protected ribose 12 with L-(+)-3-O-trifluoromethylsulfonyl-6-O-p-methoxybenzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 11. Separable diastereoisomeric 3-O-camphanate esters of (±)-6-O-p-methoxy-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol allowed the preparation of 11. Selective trans-isopropylidene deprotection in coupled 13, then monobenzylation gave separable regioisomers 15 and 16. p-Methoxybenzyl group deprotection of 16, phosphitylation/oxidation, then deprotection afforded 10, which was a full agonist in Ca 2+ -release assays; its potency and binding affinity for Ins(1,4,5)P 3 R were similar to those of adenophostin. Both 4 and 10 elicited a store-operated Ca 2+ current I CRAC in patch-clamped cells, unlike Ins(1,4,5)P 3 consistent with resistance to metabolism. D-chiro-Inositol ribophostin is the most potent small-molecule Ins(1,4,5)P 3 receptor agonist without a nucleobase yet synthesized.
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