The adaptor protein CRK is a pro-apoptotic transducer of endoplasmic reticulum stress

Excessive demands on the protein-folding capacity of the endoplasmic reticulum (ER) cause irremediable ER stress and contribute to cell loss in a number of cell degenerative diseases, including type 2 diabetes and neurodegeneration. The signals communicating catastrophic ER damage to the mitochondrial apoptotic machinery remain poorly understood. We used a biochemical approach to purify a cytosolic activity induced by ER stress that causes release of cytochrome c from isolated mitochondria. We discovered that the principal component of the purified pro-apoptotic activity is the proto-oncoprotein CRK (CT10-regulated kinase), an adaptor protein with no known catalytic activity. Crk(-/-) cells are strongly resistant to ER-stress-induced apoptosis. Moreover, CRK is cleaved in response to ER stress to generate an amino-terminal M(r)~14K fragment with greatly enhanced cytotoxic potential. We identified a putative BH3 (BCL2 homology 3) domain within this N-terminal CRK fragment, which sensitizes isolated mitochondria to cytochrome c release and when mutated significantly reduces the apoptotic activity of CRK in vivo. Together these results identify CRK as a pro-apoptotic protein that signals irremediable ER stress to the mitochondrial execution machinery.     

Hu-K4 is a ubiquitously expressed type 2 transmembrane protein associated with the endoplasmic reticulum

Hu-K4 is a human protein homologous to the K4L protein of vaccinia virus. Due to the presence of two HKD motifs, Hu-K4 was assigned to the family of Phospholipase D proteins although so far no catalytic activity has been shown. The Hu-K4 mRNA is found in many human organs with highest expression levels in the central nervous system. We extended the ORF of Hu-K4 to the 5' direction. As a consequence the protein is 53 amino acids larger than originally predicted, now harbouring a putative transmembrane domain. The exon/intron structure of the Hu-K4 gene reveals extensive alternative splicing in the 5' untranslated region. Due to the absence of G/C-rich regions and upstream ATG codons, the mRNA isoform in brain may be translated with higher efficacy leading to a high Hu-K4 protein concentration in this tissue. Using a specific antiserum produced against Hu-K4 we found that Hu-K4 is a membrane-bound protein colocalizing with protein disulfide isomerase, a marker of the endoplasmic reticulum. Glycosylation of Hu-K4 as shown by treatment with peptide N-glycosidase F or tunicamycin indicates that Hu-K4 has a type 2 transmembrane topology.     

Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum

We have identified a novel endoplasmic reticulum (ER)-resident protein, named "calumin", which is expressed in various tissues. This protein has a molecular mass of approximately 60 kDa and is composed of an ER-luminal domain rich in acidic residues, a single transmembrane segment, and a large cytoplasmic domain. Biochemical experiments demonstrated that the amino-terminal luminal domain is capable of binding Ca2+ with a high capacity and moderate affinity. In embryonic fibroblasts derived from calumin-knockout mice exhibiting embryonic and neonatal lethality, fluorometric Ca2+ imaging detected insufficient Ca2+ contents in intracellular stores and attenuated store-operated Ca2+ entry. Moreover, the mutant fibroblasts were highly sensitive to cell death induced by ER stress. These observations suggest that calumin plays an essential role in ER Ca2+ handling and is also implicated in signaling from the ER, which is closely associated with cell-fate decision.     

CRELD2 is a novel endoplasmic reticulum stress-inducible gene

Recently, endoplasmic reticulum (ER) stress responses have been suggested to play important roles in maintaining various cellular functions and to underlie many tissue dysfunctions. In this study, we first identified cysteine-rich with EGF-like domains 2 (CRELD2) as an ER stress-inducible gene by analyzing a microarray analysis of thapsigargin (Tg)-inducible genes in Neuro2a cells. CRELD2 mRNA is also shown to be immediately induced by treatment with the ER stress-inducing reagents tunicamycin and brefeldin A. In the genomic sequence of the mouse CRELD2 promoter, we found a typical ER stress responsible element (ERSE), which is well conserved among various species. Using a luciferase reporter analyses, we demonstrated that the ERSE in mouse CRELD2 is functional and responds to Tg and ATF6-overexpression. Each mutation of ATF6- or NF-Y-binding sites in the ERSE of the mouse CRELD2 promoter dramatically decreased both the basal activity and responsiveness toward the ER stress stimuli. Our study suggests that CRELD2 could be a novel mediator in regulating the onset and progression of various ER stress-associated diseases.     

The Nrf1 CNC/bZIP protein is a nuclear envelope-bound transcription factor that is activated by t-butyl hydroquinone but not by endoplasmic reticulum stressors

In rat liver RL-34 cells, endogenous Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is localized in the ER (endoplasmic reticulum) where it exists as a glycosylated protein. Electron microscopy has demonstrated that ectopic Nrf1 in COS-1 cells is located in the ER and the NE (nuclear envelope). Subcellular fractionation, together with a membrane proteinase protection assay, revealed that Nrf1 is an integral membrane protein with both luminal and cytoplasmic domains. The N-terminal 65 residues of Nrf1 direct its integration into the ER and NE membranes and tether it to a Triton X-100-resistant membrane microdomain that is associated with lipid rafts. The activity of Nrf1 was increased by the electrophile tBHQ (t-butyl hydroquinone) probably through an N-terminal domain-dependent process. We found that the NST (Asn/Ser/Thr-rich) domain, along with AD1 (acidic domain 1), contributes positively to the transactivation activity of full-length Nrf1. Furthermore, the NST domain contains seven putative -Asn-Xaa-Ser/Thr- glycosylation sites and, when glycosylation was prevented by replacing all of the seven asparagine residues with either glutamine (Nrf1(1-7xN/Q)) or aspartic acid (Nrf1(1-7xN/D)), the former multiple point mutant possessed less activity than the wild-type factor, whereas the latter mutant exhibited substantially greater activity. Lastly, the ER stressors tunicamycin, thapsigargin and Brefeldin A were found to inhibit basal Nrf1 activity by approximately 25%, and almost completely prevented induction of Nrf1-mediated transactivation by tBHQ. Collectively, these results suggest that the activity of Nrf1 critically depends on its topology within the ER, and that this is modulated by redox stressors, as well as by its glycosylation status.     

Movement protein of a closterovirus is a type III integral transmembrane protein localized to the endoplasmic reticulum

Cell-to-cell movement of beet yellows closterovirus requires four structural proteins and a 6-kDa protein (p6) that is a conventional, nonstructural movement protein. Here we demonstrate that either virus infection or p6 overexpression results in association of p6 with the rough endoplasmic reticulum. The p6 protein possesses a single-span, transmembrane, N-terminal domain and a hydrophilic, C-terminal domain that is localized on the cytoplasmic face of the endoplasmic reticulum. In the infected cells, p6 forms a disulfide bridge via a cysteine residue located near the protein's N terminus. Mutagenic analyses indicated that each of the p6 domains, as well as protein dimerization, is essential for p6 function in virus movement.     

An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin.

A putative G protein-coupled transmembrane polypeptide, named Pael receptor, was identified as an interacting protein with Parkin, a gene product responsible for autosomal recessive juvenile Parkinsonism (AR-JP). When overexpressed in cells, this receptor tends to become unfolded, insoluble, and ubiquitinated in vivo. The insoluble Pael receptor leads to unfolded protein-induced cell death. Parkin specifically ubiquitinates this receptor in the presence of ubiquitin-conjugating enzymes resident in the endoplasmic reticulum and promotes the degradation of insoluble Pael receptor, resulting in suppression of the cell death induced by Pael receptor overexpression. Moreover, the insoluble form of Pael receptor accumulates in the brains of AR-JP patients. Here, we show that the unfolded Pael receptor is a substrate of Parkin, the accumulation of which may cause selective neuronal death in AR-JP.     

H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.     

Isolation and characterization of a putative transducer of endoplasmic reticulum stress in Oryza sativa

Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress.