IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.8-formyl-umbelliferone | unfolded protein response | high-throughput screening | reversible covalent inhibitor | aldehydes P erturbation of the protein folding environment in the endoplasmic reticulum (ER) leads to rectifying changes in gene expression and protein synthesis. These are mediated by an unfolded protein response (UPR), whose most conserved arm is initiated by an ER localized transmembrane protein, IRE1 (1, 2). The lumenal domain of IRE1 senses the perturbation in the ER and transmits the ER stress signal across the ER membrane to the effector cytosolic domain of the protein. This effector domain is endowed with two linked enzymatic activities: a protein kinase and an RNase, and both are activated by ER stress.The most conserved output of IRE1 signaling is the sitespecific cleavage of an mRNA, the product of the yeast HAC1 gene (3) and its metazoan orthologue Xbp1 (4, 5). Cleavage occurs at two distinct sites and is followed by ligation of the 5′ and 3′ fragments to generate an ER stress-dependent spliced mRNA encoding a potent transcription factor. The target genes of spliced XBP1 (and Hac1p) enhance the ability of the ER to cope with unfolded proteins (6) and also act more broadly to upregulate secretory capacity (7). In addition, mammalian IRE1 contributes to the promiscuous degradation of membrane-associated mRNAs in a process known as regulated IRE1-dependent degradation (or RIDD), but whose mechanistic basis and functional consequences are incompletely understood (8, 9).Fluctuating levels of ER stress accompany diverse physiological conditions. In metazoans, IRE1 signaling constitutes one arm of a three-pronged UPR. The other two arms are mediated by the translation initiation factor 2α (eIF2α) kinase PERK, which attenuates ER load by inhibiting protein synthesis in stressed cells, and by a parallel transcriptional pathway mediated by ATF6. Redundancy between the long-term transcriptional programs mediated by the three arms of the UPR has obscured the interp...

Interactions of lipids are central to the folding and stability of membrane proteins. Coarse-grained molecular simulations have been used to reveal the mechanisms of self-assembly of protein/ membrane and protein/detergent complexes for representatives of two classes of membrane protein, namely glycophorin (a simple α-helical bundle) and OmpA (a β-barrel). The accuracy of the coarse-grained simulations is established via comparison with the equivalent atomistic simulations of self-assembly of protein/detergent micelles. The simulation of OmpA/bilayer selfassembly reveals how a folded outer membrane protein can be inserted in a bilayer. The glycophorin/bilayer simulation supports the two-state model of membrane folding, in which transmembrane helix insertion precedes dimer self-assembly within a bilayer. The simulations also suggest a dynamic equilibrium exists between the glycophorin helix monomer and dimer within a bilayer. The simulated glycophorin helix dimer is remarkably close in structure to that revealed by NMR. Thus, coarse-grained methods may help to define mechanisms of membrane protein (re)folding, and will prove suitable for simulation of larger scale dynamic rearrangements of biological membranes.

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Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation in vivo, via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters cellular metabolism, gene expression, lipid metabolic pathways, and membrane lipid composition, changes that are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.