The endoplasmic reticulum (ER) is an important site for protein folding and maturation in eukaryotes. The cellular requirement to synthesize proteins within the ER is matched by its folding capacity. However, the physiological demands or aberrations in folding may result in an imbalance which can lead to the accumulation of misfolded protein, also known as “ER stress.” The unfolded protein response (UPR) is a cell-signaling system that readjusts ER folding capacity to restore protein homeostasis. The key UPR signal activator, IRE1, responds to stress by propagating the UPR signal from the ER to the cytosol. Here, we discuss the structural and molecular basis of IRE1 stress signaling, with particular focus on novel mechanistic advances. We draw a comparison between the recently proposed allosteric model for UPR induction and the role of Hsp70 during polypeptide import to the mitochondrial matrix.
BiP is a major ER chaperone and suggested to act as primary sensor in the activation of the unfolded protein response (UPR). How BiP operates as a molecular chaperone and as an ER stress sensor is unknown. Here, by reconstituting components of human UPR, ER stress and BiP chaperone systems, we discover that the interaction of BiP with the luminal domains (LD) of UPR proteins, IRE1 and PERK, switch BiP from its chaperone cycle into an ER stress sensor cycle by preventing the binding of its cochaperones, with loss of ATPase stimulation. Furthermore, misfolded protein-dependent dissociation of BiP from IRE1 is primed by ATP but not ADP. Our data elucidate a previously unidentified mechanistic cycle of BiP function that explains its ability to act as a Hsp70 chaperone and ER stress sensor.
The unfolded protein response (UPR) is an essential cell signaling system that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER) and initiates a cellular response in order to maintain homeostasis. How cells detect the accumulation of misfolded proteins remains unclear. In this study, we identify a noncanonical interaction between the ATPase domain of the ER chaperone BiP and the luminal domains of the UPR sensors Ire1 and Perk that dissociates when authentic ER unfolded protein CH1 binds to the canonical substrate binding domain of BiP. Unlike the interaction between chaperone and substrates, we found that the interaction between BiP and UPR sensors was unaffected by nucleotides. Thus, we discover that BiP is dual functional UPR sensor, sensing unfolded proteins by canonical binding to substrates and transducing this event to noncanonical, signaling interaction to Ire1 and Perk. Our observations implicate BiP as the key component for detecting ER stress and suggest an allosteric mechanism for UPR induction.DOI: http://dx.doi.org/10.7554/eLife.03522.001
The unfolded protein response (UPR) is a key signaling system that regulates protein homeostasis within the endoplasmic reticulum (ER). The primary step in UPR activation is the detection of misfolded proteins, the mechanism of which is unclear. We have previously suggested an allosteric mechanism for UPR induction (Carrara et al., 2015) based on qualitative pull-down assays. Here, we develop an in vitro Förster resonance energy transfer (FRET) UPR induction assay that quantifies IRE1 luminal domain and BiP association and dissociation upon addition of misfolded proteins. Using this technique, we reassess our previous observations and extend mechanistic insight to cover other general ER misfolded protein substrates and their folded native state. Moreover, we evaluate the key BiP substrate-binding domain mutant V461F. The new experimental approach significantly enhances the evidence suggesting an allosteric model for UPR induction upon ER stress.
The genes coding for the enzymes of oxidative degradation of nicotinic acid have recently been identified in several species of aerobic bacteria, namely, Pseudomonas putida KT2440, Bordetella bronchiseptica RB50, and Bacillus niacini. One of the enzymes involved in an early step of this pathway is a flavin-dependent monooxygenase (6-hydroxynicotinic acid 3-monooxygenase; NicC) that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP), with concomitant oxidation of NADH to NAD+. The nicC genes from B. bronchiseptica RB50 and P. putida have been cloned, and the purified enzymes have been characterized functionally and structurally. Global fits of the steady-state kinetic data show that both enzymes are efficient catalysts, with an apparent k cat/K M 6‑HNA of 5.0 × 104 M–1 s–1 for B. bronchiseptica NicC. The pH dependence of V max/[E] t fits a double-bell model showing an optimum around pH 8 with apparent pK as of 7.24 ± 0.08 and 8.64 ± 0.08, whereas the apparent catalytic efficiency (k cat/K M 6‑HNA) is maximal around pH 7 and decreases at high pH with an apparent pK a of 7.60 ± 0.06. The enzyme’s relative affinity for 6-hydroxynicotinaldehyde, a neutral analogue that shows competitive inhibition with respect to 6-HNA, is weak (K i = 3000 ± 400 μM) in comparison to the apparent binding of 6-HNA (K M = 85 ± 13 μM). The crystal structure for P. putida NicC has been solved to 2.1 Å using SAD phasing, and the 6-HNA substrate has been modeled into the active site. Together these data provide insight into potential reaction mechanisms for this novel decarboxylative hydroxylation reaction.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.