Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1α-JNK and mitochondrial apoptotic pathway

An increased intracellular methylglyoxal (MGO) under hyperglycemia led to pancreatic beta cell death. However, its mechanism in which way with MGO induced beta cell death remains unknown. We investigated both high glucose and MGO treatment significantly inclined intracellular MGO concentration and inhibited cell viability in vitro. MGO treatment also triggered intracellular advanced glycation end products (AGEs) formation, declined mitochondrial membrane potential (MMP), increased oxidative stress and the expression of ER stress mediators Grp78/Bip and p-PERK; activated mitochondrial apoptotic pathway, which could mimic by Glo1 knockdown. Aminoguanidine (AG), a MGO scavenger, however, prevented AGEs formation and MGO-induced cell death by inhibiting oxidative stress and ER stress. Furthermore, both antioxidant N-acetylcysteine (NAC) and ER stress inhibitor 4-phenylbutyrate (4-PBA) could attenuate MGO-induced cell death through ameliorating ER stress. MGO treatment down-regulated Ire1α, a key ER stress mediator, increased JNK phosphorylation and activated mitochondrial apoptosis; down-regulated Bcl-2 expression which could be attenuated by the JNK inhibitor SP600125 and further inhibited cytochrome c leakage from mitochondria and blocked the conversion of pro caspase 3 into cleaved caspase 3, all these might contribute to the inhibition of INS-1 cell apoptosis. Ire1α down-regulation by Ire1α siRNAs mimicked MGO-induced cytotoxicity by activating the JNK phosphorylation and mitochondrial apoptotic pathway. In summary, we demonstrated that increased intracellular MGO induced cytotoxicity in INS-1 cells primarily by activating oxidative stress and further triggering mitochondrial apoptotic pathway, and ER stress-mediated Ire1α-JNK pathway. These findings may have implication on new mechanism of glucotoxicity-mediated pancreatic beta-cell dysfunction.     

Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins

Chaperone protein BiP binds to Ire1 and dissociates in response to endoplasmic reticulum (ER) stress. However, it remains unclear how the signal transducer Ire1 senses ER stress and is subsequently activated. The crystal structure of the core stress-sensing region (CSSR) of yeast Ire1 luminal domain led to the controversial suggestion that the molecule can bind to unfolded proteins. We demonstrate that, upon ER stress, Ire1 clusters and actually interacts with unfolded proteins. Ire1 mutations that affect these phenomena reveal that Ire1 is activated via two steps, both of which are ER stress regulated, albeit in different ways. In the first step, BiP dissociation from Ire1 leads to its cluster formation. In the second step, direct interaction of unfolded proteins with the CSSR orients the cytosolic effector domains of clustered Ire1 molecules.     

BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response

The unfolded protein response (UPR) is an intracellular signaling pathway that counteracts variable stresses that impair protein folding in the endoplasmic reticulum (ER). As such, the UPR is thought to be a homeostat that finely tunes ER protein folding capacity and ER abundance according to need. The mechanism by which the ER stress sensor Ire1 is activated by unfolded proteins and the role that the ER chaperone protein BiP plays in Ire1 regulation have remained unclear. Here we show that the UPR matches its output to the magnitude of the stress by regulating the duration of Ire1 signaling. BiP binding to Ire1 serves to desensitize Ire1 to low levels of stress and promotes its deactivation when favorable folding conditions are restored to the ER. We propose that, mechanistically, BiP achieves these functions by sequestering inactive Ire1 molecules, thereby providing a barrier to oligomerization and activation, and a stabilizing interaction that facilitates de-oligomerization and deactivation. Thus BiP binding to or release from Ire1 is not instrumental for switching the UPR on and off as previously posed. By contrast, BiP provides a buffer for inactive Ire1 molecules that ensures an appropriate response to restore protein folding homeostasis to the ER by modulating the sensitivity and dynamics of Ire1 activity.     

A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1

In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.     

Membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor Ire1 in different ways

Eukaryotic cells activate the unfolded-protein response (UPR) upon endoplasmic reticulum (ER) stress, where the stress is assumed to be the accumulation of unfolded proteins in the ER. Consistent with previous in vitro studies of the ER-luminal domain of the mutant UPR initiator Ire1, our study show its association with a model unfolded protein in yeast cells. An Ire1 luminal domain mutation that compromises Ire1's unfolded-protein-associating ability weakens its ability to respond to stress stimuli, likely resulting in the accumulation of unfolded proteins in the ER. In contrast, this mutant was activated like wild-type Ire1 by depletion of the membrane lipid component inositol or by deletion of genes involved in lipid homeostasis. Another Ire1 mutant lacking the authentic luminal domain was up-regulated by inositol depletion as strongly as wild-type Ire1. We therefore conclude that the cytosolic (or transmembrane) domain of Ire1 senses membrane aberrancy, while, as proposed previously, unfolded proteins accumulating in the ER interact with and activate Ire1.     

Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase

The unfolded protein response (UPR) activates Ire1, an endoplasmic reticulum (ER) resident transmembrane kinase and ribonuclease (RNase), in response to ER stress. We used an in vivo assay, in which disappearance of the UPR-induced spliced HAC1 messenger ribonucleic acid (mRNA) correlates with the recovery of the ER protein-folding capacity, to investigate the attenuation of the UPR in yeast. We find that, once activated, spliced HAC1 mRNA is sustained in cells expressing Ire1 carrying phosphomimetic mutations within the kinase activation loop, suggesting that dephosphorylation of Ire1 is an important step in RNase deactivation. Additionally, spliced HAC1 mRNA is also sustained after UPR induction in cells expressing Ire1 with mutations in the conserved DFG kinase motif (D828A) or a conserved residue (F842) within the activation loop. The importance of proper Ire1 RNase attenuation is demonstrated by the inability of cells expressing Ire1-D828A to grow under ER stress. We propose that the activity of the Ire1 kinase domain plays a role in attenuating its RNase activity when ER function is recovered.     

Categorization of endoplasmic reticulum stress as accumulation of unfolded proteins or membrane lipid aberrancy using yeast Ire1 mutants

Endoplasmic reticulum (ER)-located protein Ire1 triggers the unfolded protein response against ER-stressing stimuli, which are categorized as ER accumulation of unfolded proteins or membrane lipid-related aberrancy. Here we demonstrate that by using yeast Ire1 mutants, we can distinguish the category to which a stress-inducing stimulus belongs. For instance, ethanol was found to activate Ire1 through both types of cellular damage.     

A chimeric mutant analysis in yeast cells suggests BiP independent regulation of the mammalian endoplasmic reticulum-stress sensor IRE1α

An endoplasmic reticulum (ER)-located transmembrane protein, Ire1, triggers cytoprotective events upon ER stress. Chimeric yeast Ire1 carrying the luminal domain of the mammalian major Ire1 paralogue IRE1α is upregulated in ER-stressed yeast cells, but is poorly associated with the ER-located chaperone BiP even under non-stressed conditions. This observation contradicts the theory that BiP is the master regulator of IRE1α.     

Enlargement of the endoplasmic reticulum membrane in Saccharomyces cerevisiae is not necessarily linked to the unfolded protein response via Ire1p.

Conditions that stress the endoplasmic reticulum (ER) in Saccharomyces cerevisiae can elicit a combination of an unfolded protein response (UPR) and an inositol response (IR). This results in increased synthesis of ER protein-folding factors and of enzymes participating in phospholipid biosynthesis. It was suggested that in cells grown on glucose or galactose medium, the UPR and the IR are linked and controlled by the ER stress sensor Ire1p. However, our studies suggest that during growth on oleate the IR is controlled both by an Ire1p-dependent pathway and by an Ire1p-independent pathway.     

Null mutation in IRE1 gene inhibits overproduction of microsomal cytochrome P450Alk1 (CYP 52A3) and proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae

Overproduction of microsomal cytochrome P450Alk1 (P450Alk1) of Candida maltosa in Saccharomyces cerevisiae resulted in an extensive proliferation of endoplasmic reticulum (ER) and induction of Kar2p and Pdi1p. The ire1 null mutation severely suppressed ER proliferation, reduced the level of functional P450Alk1, and showed no induction of these ER chaperones, suggesting that the function of Ire1p is required for ER proliferation upon the overproduction of P450Alk1. Cerulenin, a potent inhibitor of lipid biosynthesis, also induced these chaperones in an Ire1p-dependent manner and limited the production of functional P450Alk1. These results imply that Ire1p may function to restore the balance between membrane proteins and lipids of the ER when the ER is relatively overcrowded by membrane proteins.     

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.