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

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

Endoplasmic stress sensor Ire1 is involved in cytosolic/nuclear protein quality control in Pichia pastoris cells independent of HAC1

In eukaryotic species, dysfunction of the endoplasmic reticulum (ER), namely, ER stress, provokes a cytoprotective transcription program called the unfolded protein response (UPR). The UPR is triggered by transmembrane ER-stress sensors, including Ire1, which acts as an endoribonuclease to splice and mature the mRNA encoding the transcription factor Hac1 in many fungal species. Through analyses of the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii), we revealed a previously unknown function of Ire1. In P. pastoris cells, the IRE1 knockout mutation (ire1Δ) and HAC1 knockout mutation (hac1Δ) caused only partially overlapping gene expression changes. Protein aggregation and the heat shock response (HSR) were induced in ire1Δ cells but not in hac1Δ cells even under non-stress conditions. Moreover, Ire1 was further activated upon high-temperature culturing and conferred heat stress resistance to P. pastoris cells. Our findings cumulatively demonstrate an intriguing case in which the UPR machinery controls cytosolic protein folding status and the HSR, which is known to be activated upon the accumulation of unfolded proteins in the cytosol and/or nuclei

Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases

Self-association status-dependent inactivation of the endoplasmic reticulum stress sensor Ire1 by C-terminal tagging with artificial peptides

Upon endoplasmic reticulum (ER) stress, eukaryotic cells commonly induce unfolded protein response (UPR), which is triggered, at least partly, by the ER stress sensor Ire1. Upon ER stress, Ire1 is dimerized or forms oligomeric clusters, resulting in the activation of Ire1 as an endoribonuclease. In ER-stressed Saccharomyces cerevisiae cells, HAC1 mRNA is spliced by Ire1 and then translated into a transcription factor that promotes the UPR. Herein, we report that Ire1 tagged artificially with irrelevant peptides at the C terminus is almost completely inactive when only dimerized, while it induced the UPR as well as untagged Ire1 when clustered. This finding suggests a fundamental difference between the dimeric and clustered forms of Ire1. By comparing UPR levels in S. cerevisiae cells carrying artificially peptide-tagged Ire1 to that in cells carrying untagged Ire1, we estimated the self-association status of Ire1 under various ER stress conditions

Induction of the Unfolded Protein Response at High Temperature in Saccharomyces cerevisiae

Ire1 is an endoplasmic reticulum (ER)-located endoribonuclease that is activated in response to ER stress. In yeast Saccharomyces cerevisiae cells, Ire1 promotes HAC1-mRNA splicing to remove the intron sequence from the HAC1u mRNA (“u” stands for “uninduced”). The resulting mRNA, which is named HAC1i mRNA (“i” stands for “induced”), is then translated into a transcription factor that is involved in the unfolded protein response (UPR). In this study, we designed an oligonucleotide primer that specifically hybridizes to the exon-joint site of the HAC1i cDNA. This primer allowed us to perform real-time reverse transcription-PCR to quantify HAC1i mRNA abundance with high sensitivity. Using this method, we detected a minor induction of HAC1-mRNA splicing in yeast cells cultured at their maximum growth temperature of 39 °C. Based on our analyses of IRE1-gene mutant strains, we propose that when yeast cells are cultured at or near their maximum growth temperature, protein folding in the ER is disturbed, leading to a minor UPR induction that supports cellular growth

Induction and Aggravation of the Endoplasmic-Reticulum Stress by Membrane-Lipid Metabolic Intermediate Phosphatidyl-N-Monomethylethanolamine

Phosphatidylcholine (PC) is produced via two distinct pathways in both hepatocytes and yeast, Saccharomyces cerevisiae. One of these pathways involves the sequential methylation of phosphatidylethanolamine (PE). In yeast cells, the methyltransferase, Cho2, converts PE to phosphatidylmonomethylethanolamine (PMME), which is further modified to PC by another methyltransferase, Opi3. On the other hand, free choline is utilized for PC production via the Kennedy pathway. The blockage of PC production is well known to cause endoplasmic reticulum (ER) stress and activate the ER-stress sensor, Ire1, to induce unfolded protein response (UPR). Here, we demonstrate that even when free choline is sufficiently supplied, the opi3Δ mutation, but not the cho2 Δ mutation, induces the UPR. The UPR was also found to be induced by CHO2 overexpression. Further, monomethylethanolamine, which is converted to PMME probably through the Kennedy pathway, caused or potentiated ER stress in both mammalian and yeast cells. We thus deduce that PMME per se is an ER-stressing molecule. Interestingly, spontaneously accumulated PMME seemed to aggravate ER stress in yeast cells. Collectively, our findings demonstrate the multiple detrimental effects of the low-abundance phospholipid species, PMME

Stress-sensing and regulatory mechanism of the endoplasmic-stress sensors Ire1 and PERK

Ire1 and its family protein PERK are endoplasmic reticulum (ER)-stress sensors that initiate cellular responses against ER accumulation of unfolded proteins. As reviewed in this article, many publications describe molecular mechanisms by which yeast Ire1 senses ER conditions and gets regulated. We also cover recent studies which reveal that mammalian Ire1 (IRE1α) and PERK are controlled in a similar but not exactly the same manner. ER-located molecular chaperone BiP captures these ER-stress sensors and suppresses their activity. Intriguingly, Ire1 is associated with BiP not as a chaperone substrate, but as a unique ligand. Unfolded proteins accumulated in the ER promote dissociation of the Ire1-BiP complex. Moreover, Ire1 is directly bound with unfolded proteins, leading to its cluster formation and potent activation. PERK also captures unfolded proteins and then forms self-oligomers. Meanwhile, membrane-lipid aberrancy is likely to activate these ER-stress sensors independently of ER accumulation of unfolded proteins. In addition, there exist a number of reports that touch on other factors that control activity of these ER-stress sensors. Such a multiplicity of regulatory mechanisms for these ER-stress sensors is likely to contribute to fine tuning of their activity