Coupling Of Endoplasmic Reticulum Research Articles

Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR.

Coupling of endoplasmic reticulum (ER) stress to dimerisation-dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex between the Hsp70 BiP and IRE1's stress-sensing luminal domain (IRE1LD) that favours the latter's monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1LD that mediated BiP-induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1LD induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1LD dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1LD to initiate active repression of UPR signalling.

Crosstalk between mitochondrial damage and NAD(P)H oxidase activity in vascular smooth muscle cells

Vascular aging and pathological processes involve mitochondrial (MT) DNA mutations, which lead to MT stress/dysfunction. Possible crosstalk between MT damage and oxidative stress sources such as NAD(P)H oxidase is unknown. Coupling of endoplasmic reticulum (ER) stress to disrupted MT Ca++ signaling was recently shown (Xu et al, 2004). We showed that interaction between ER-resident protein disulfide isomerase (PDI) and NAD(P)H oxidase is a pathway bridging ER and oxidative stress. Thus, we asked whether MT damage induces ER stress-linked NAD(P)H oxidase activation. We validated a model of acute mitochondrial stress/dysfunction with minimal DNA damage induced by 24 h-incubation of rabbit aortic smooth muscle (VSMC) or HEK293 cells with ethidium bromide (EB, 250ng/mL). In this model, VSMC do not undergo apoptosis nor change cell counts at baseline, with angiotensin II (AII) or with serum. Serum-stimulated proliferation decreased by 31% at 72 hs. VSMC membrane fraction NADPH oxidase activity (lucigenin or Amplex Red assays) was increased by 30–50% after EB, at levels similar to known VSMC oxidase agonists. However, oxidase activation due to AII (100nM, 4h) was markedly abrogated. GSH/GSSG ratio decreased ~40% after EB. After EB, there was increased expression of p22phox subunit and PDI and increased PDI migration to membrane fraction. However, we were unable to show increased expression of ER stress markers Grp78 and Grp94. Thus, MT stress/dysfunction leads to increased baseline NADPH oxidase activity in connection with ER stress-independent PDI overexpression, but decreased redox signals due to AII. (FAPESP)