Heather Harding, Ph.D.

Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides
Gao, Ningguo; Shang, Jie; Huynh, Dang; Manthati, Vijaya L; Arias, Carolina; Harding, Heather P; Kaufman, Randal J; Mohr, Ian; Ron, David; Falck, John R; Lehrman, Mark A
2011 Sep;22(17):2994-3009, Molecular biology of the cell
Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-alpha. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses
— id: 136999, year: 2011, vol: 22, page: 2994, stat: Journal Article,

Inhibition of nonsense-mediated RNA decay by the tumor microenvironment promotes tumorigenesis
Wang, Ding; Zavadil, Jiri; Martin, Leenus; Parisi, Fabio; Friedman, Eugene; Levy, David; Harding, Heather; Ron, David; Gardner, Lawrence B
2011 Sep;31(17):3670-3680, Molecular & cellular biology
While nonsense-mediated RNA decay (NMD) is an established mechanism to rapidly degrade select transcripts, the physiological regulation and biological significance of NMD are not well characterized. We previously demonstrated that NMD is inhibited in hypoxic cells. Here we show that the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) translation initiation factor by a variety of cellular stresses leads to the inhibition of NMD and that eIF2alpha phosphorylation and NMD inhibition occur in tumors. To explore the significance of this NMD regulation, we used an unbiased approach to identify approximately 750 NMD-targeted mRNAs and found that these mRNAs are overrepresented in stress response and tumor-promoting pathways. Consistent with these findings, the inhibition of NMD promotes cellular resistance to endoplasmic reticulum stress and encourages tumor formation. The transcriptional and translational regulations of gene expression by the microenvironment are established mechanisms by which tumor cells adapt to stress. These data indicate that NMD inhibition by the tumor microenvironment is also an important mechanism to dynamically regulate genes critical for the response to cellular stress and tumorigenesis
— id: 136513, year: 2011, vol: 31, page: 3670, stat: Journal Article,

A small molecule inhibitor of endoplasmic reticulum oxidation 1 (ERO1) with selectively reversible thiol reactivity
Blais, Jaime D; Chin, King-Tung; Zito, Ester; Zhang, Yuhong; Heldman, Nimrod; Harding, Heather P; Fass, Deborah; Thorpe, Colin; Ron, David
2010 Jul 2;285(27):20993-21003, Journal of biological chemistry
Endoplasmic reticulum oxidation 1 (ERO1) is a conserved eukaryotic flavin adenine nucleotide-containing enzyme that promotes disulfide bond formation by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen. Although disulfide bond formation is an essential process, recent experiments suggest a surprisingly broad tolerance to genetic manipulations that attenuate the rate of disulfide bond formation and that a hyperoxidizing ER may place stressed cells at a disadvantage. In this study, we report on the development of a high throughput in vitro assay for mammalian ERO1alpha activity and its application to identify small molecule inhibitors. The inhibitor EN460 (IC(50), 1.9 mum) interacts selectively with the reduced, active form of ERO1alpha and prevents its reoxidation. Despite rapid and promiscuous reactivity with thiolates, EN460 exhibits selectivity for ERO1. This selectivity is explained by the rapid reversibility of the reaction of EN460 with unstructured thiols, in contrast to the formation of a stable bond with ERO1alpha followed by displacement of bound flavin adenine dinucleotide from the active site of the enzyme. Modest concentrations of EN460 and a functionally related inhibitor, QM295, promote signaling in the unfolded protein response and precondition cells against severe ER stress. Together, these observations point to the feasibility of targeting the enzymatic activity of ERO1alpha with small molecule inhibitors
— id: 110660, year: 2010, vol: 285, page: 20993, stat: Journal Article,

Flavonol activation defines an unanticipated ligand-binding site in the kinase-RNase domain of IRE1
Wiseman, R Luke; Zhang, Yuhong; Lee, Kenneth P K; Harding, Heather P; Haynes, Cole M; Price, Joshua; Sicheri, Frank; Ron, David
2010 Apr 23;38(2):291-304, Molecular cell
Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1's RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide-binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1's kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence of enhanced dimer formation in quercetin's mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site
— id: 109521, year: 2010, vol: 38, page: 291, stat: Journal Article,

ERO1-{beta}, a pancreas-specific disulfide oxidase, promotes insulin biogenesis and glucose homeostasis
Zito, Ester; Chin, King-Tung; Blais, Jaime; Harding, Heather P; Ron, David
2010 Mar 22;188(6):821-832, Journal of cell biology
Mammals have two genes encoding homologues of the endoplasmic reticulum (ER) disulfide oxidase ERO1 (ER oxidoreductin 1). ERO1-beta is greatly enriched in the endocrine pancreas. We report in this study that homozygosity for a disrupting allele of Ero1lb selectively compromises oxidative folding of proinsulin and promotes glucose intolerance in mutant mice. Surprisingly, concomitant disruption of Ero1l, encoding the other ERO1 isoform, ERO1-alpha, does not exacerbate the ERO1-beta deficiency phenotype. Although immunoglobulin-producing cells normally express both isoforms of ERO1, disulfide bond formation and immunoglobulin secretion proceed at nearly normal pace in the double mutant. Moreover, although the more reducing environment of their ER protects cultured ERO1-beta knockdown Min6 cells from the toxicity of a misfolding-prone mutant Ins2(Akita), the diabetic phenotype and islet destruction promoted by Ins2(Akita) are enhanced in ERO1-beta compound mutant mice. These findings point to an unexpectedly selective function for ERO1-beta in oxidative protein folding in insulin-producing cells that is required for glucose homeostasis in vivo
— id: 108799, year: 2010, vol: 188, page: 821, stat: Journal Article,

Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development
Harding, Heather P; Zhang, Yuhong; Scheuner, Donalyn; Chen, Jane-Jane; Kaufman, Randal J; Ron, David
2009 Feb 10;106(6):1832-1837, Proceedings of the National Academy of Sciences of the United States of America
Diverse cellular stress responses are linked to phosphorylation of serine 51 on the alpha subunit of translation initiation factor 2. The resultant attenuation of protein synthesis and activation of gene expression figure heavily in the adaptive response to stress, but dephosphorylation of eIF2(alphaP), which terminates signaling in this pathway, is less well understood. GADD34 and CReP, the products of the related mammalian genes Ppp1r15a and Ppp1r15b, can recruit phosphatase catalytic subunits of the PPP1 class to eIF2(alphaP), but the significance of their contribution to its dephosphorylation has not been explored systematically. Here we report that unlike Ppp1r15a mutant mice, which are superficially indistinguishable from wild type, Ppp1r15b(-/-) mouse embryos survive gestation but exhibit severe growth retardation and impaired erythropoiesis, and loss of both Ppp1r15 genes leads to early embryonic lethality. These loss-of-function phenotypes are rescued by a mutation, Eif2a(S51A), that prevents regulated phosphorylation of eIF2alpha. These findings reveal that the essential process of eIF2(alphaP) dephosphorylation is the predominant role of PPP1R15 proteins in mammalian development
— id: 94497, year: 2009, vol: 106, page: 1832, stat: Journal Article,

Role of ERO1-alpha-mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress-induced apoptosis
Li, G; Mongillo, M; Chin, KT; Harding, H; Ron, D; Marks, AR; Tabas, I
2009 SEP 21 ;186(6):783-792, Journal of cell biology
Endoplasmic reticulum (ER) stress-induced apoptosis is involved in many diseases, but the mechanisms linking ER stress to apoptosis are incompletely understood. Based on roles for C/EPB homologous protein (CHOP) and ER calcium release in apoptosis, we hypothesized that apoptosis involves the activation of inositol 1,4,5-triphosphate (IP3) receptor (IP3R) via CHOP-induced ERO1-alpha (ER oxidase 1 alpha). In ER-stressed cells, ERO1-alpha is induced by CHOP, and small interfering RNA (siRNA) knockdown of ERO1-alpha suppresses apoptosis. IP3-induced calcium release (IICR) is increased during ER stress, and this response is blocked by siRNA-mediated silencing of ERO1-alpha or IP3R1 and by loss-of-function mutations in Ero1a or Chop. Reconstitution of ERO1-alpha in Chop(-/-) macrophages restores ER stress-induced IICR and apoptosis. In vivo, macrophages from wild-type mice but not Chop(-/-) mice have elevated IICR when the animals are challenged with the ER stressor tunicamycin. Macrophages from insulin-resistant ob/ob mice, another model of ER stress, also have elevated IICR. These data shed new light on how the CHOP pathway of apoptosis triggers calcium-dependent apoptosis through an ERO1-alpha-IP3R pathway
— id: 102950, year: 2009, vol: 186, page: 783, stat: Journal Article,

Adaptive suppression of the ATF4-CHOP branch of the unfolded protein response by toll-like receptor signalling
Woo, CW; Cui, DY; Arellano, J; Dorweiler, B; Harding, H; Fitzgerald, KA; Ron, D; Tabas, I
2009 DEC ;11(12):1473-U203, Nature cell biology
The endoplasmic reticulum ( ER) unfolded protein response (UPR) restores equilibrium to the ER, but prolonged expression of the UPR effector CHOP (GADD153) is cytotoxic. We found that CHOP expression induced by ER stress was suppressed by prior engagement of toll-like receptor (TLR) 3 or 4 through a TRIF-dependent pathway. TLR engagement did not suppress phosphorylation of PERK or eIF-2 alpha, which are upstream of CHOP, but phospho-eIF-2 alpha failed to promote translation of the CHOP activator ATF4. In mice subjected to systemic ER stress, pretreatment with low dose lipopolysaccharide (LPS), a TLR4 ligand, suppressed CHOP expression and apoptosis in splenic macrophages, renal tubule cells and hepatocytes, and prevented renal dysfunction and hepatosteatosis. This protective effect of LPS did not occur in Trif(-/-) mice or in wildtype mice in which CHOP expression was genetically restored. Thus, TRIF-mediated signals from TLRs selectively attenuate translational activation of ATF4 and its downstream target gene CHOP. We speculate that this mechanism evolved to promote survival of TLR-expressing cells that experience prolonged levels of physiological ER stress in the course of the host response to invading pathogens
— id: 105630, year: 2009, vol: 11, page: 1473, stat: Journal Article,

An intact unfolded protein response in Trpt1 knockout mice reveals phylogenic divergence in pathways for RNA ligation
Harding, Heather P; Lackey, Jeremy G; Hsu, Hao-Chi; Zhang, Yuhong; Deng, Jing; Xu, Rui-Ming; Damha, Masad J; Ron, David
2008 Feb;14(2):225-232, RNA
Unconventional mRNA splicing by an endoplasmic reticulum stress-inducible endoribonuclease, IRE1, is conserved in all known eukaryotes. It controls the expression of a transcription factor, Hac1p/XBP-1, that regulates gene expression in the unfolded protein response. In yeast, the RNA fragments generated by Ire1p are ligated by tRNA ligase (Trl1p) in a process that leaves a 2'-PO4(2-) at the splice junction, which is subsequently removed by an essential 2'-phosphotransferase, Tpt1p. However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin the cleaved Xbp-1 mRNA fragments. We report that inactivation of the Trpt1 gene, encoding the only known mammalian homolog of Tpt1p, eliminates all detectable 2'-phosphotransferase activity from cultured mouse cells but has no measurable effect on spliced Xbp-1 translation. Furthermore, the relative translation rates of tyrosine-rich proteins is unaffected by the Trpt1 genotype, suggesting that the pool of (normally spliced) tRNA(Tyr) is fully functional in the Trpt1-/- mouse cells. These observations argue against the presence of a 2'-PO4(2-) at the splice junction of ligated RNA molecules in Trpt1-/- cells, and suggest that Xbp-1 and tRNA ligation proceed by distinct pathways in yeast and mammals
— id: 76337, year: 2008, vol: 14, page: 225, stat: Journal Article,

Toward the discovery of new antifungal agents: the design and validation of a novel 2'P-RNA probe and high throughput screening assay against 2'-phosphotransferase Tpt1p
Lackey, Jeremy G; Ron, David; Damha, Masad J; Harding, Heather P
2008 ;(52):475-476, Nucleic acids symposium series (2004)
We report the solid-phase synthesis of novel 2'P-RNA probes for use in fluorescence polarization (FP) ligand binding assays that screens for inhibitors of the yeast 2'- phosphotransferase Tpt1p. The probe was synthesized by utilizing silyl phosphoramidite chemistry and a phosphoramidite synthon containing an orthogonal (DMT) protecting group at its 2'-position. Regioselective removal of the 2'-DMT group and phosphitylation of the unmasked 2'-hydroxyl group afforded the desired 2'P-RNA sequence
— id: 94502, year: 2008, vol: , page: 475, stat: Journal Article,

Enhanced integrated stress response promotes myelinating oligodendrocyte survival in response to interferon-gamma
Lin, Wensheng; Kunkler, Phillip E; Harding, Heather P; Ron, David; Kraig, Richard P; Popko, Brian
2008 Nov;173(5):1508-1517, American journal of pathology
The T-cell-derived, pleiotropic cytokine interferon (IFN)-gamma is believed to play a key regulatory role in immune-mediated demyelinating disorders of the central nervous system, including multiple sclerosis and experimental autoimmune encephalomyelitis. Our previous work has demonstrated that the endoplasmic reticulum (ER) stress response modulates the response of oligodendrocytes to this cytokine. The ER stress response activates the pancreatic ER kinase, which coordinates an adaptive program known as the integrated stress response by phosphorylating translation initiation factor 2alpha (eIF2alpha). In this study, we found that growth arrest and DNA damage 34 (GADD34), a stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2alpha, was selectively up-regulated in myelinating oligodendrocytes in mice that ectopically expressed IFN-gamma in the central nervous system. We also found that a GADD34 mutant strain of mice displayed increased levels of phosphorylated eIF2alpha (p-eIF2alpha) in myelinating oligodendrocytes when exposure to IFN-gamma, as well as diminished oligodendrocyte loss and hypomyelination. Furthermore, treatment with salubrinal, a small chemical compound that specifically inhibits protein phosphatase 1(PP1)-GADD34 phosphatase activity, increased the levels of p-eIF2alpha and ameliorated hypomyelination and oligodendrocyte loss in cultured hippocampal slices exposed to IFN-gamma. Thus, our data provide evidence that an enhanced integrated stress response could promote oligodendrocyte survival in immune-mediated demyelination diseases
— id: 94500, year: 2008, vol: 173, page: 1508, stat: Journal Article,

Novel function of PERK as a mediator of force-induced apoptosis
Mak, Baldwin C; Wang, Qin; Laschinger, Carol; Lee, Wilson; Ron, David; Harding, Heather P; Kaufman, Randal J; Scheuner, Donalyn; Austin, Richard C; McCulloch, Christopher A
2008 Aug 22;283(34):23462-23472, Journal of biological chemistry
Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response
— id: 94503, year: 2008, vol: 283, page: 23462, stat: Journal Article,

Dephosphorylation of translation initiation factor 2alpha enhances glucose tolerance and attenuates hepatosteatosis in mice
Oyadomari, Seiichi; Harding, Heather P; Zhang, Yuhong; Oyadomari, Miho; Ron, David
2008 Jun;7(6):520-532, Cell metabolism
The molecular mechanisms linking the stress of unfolded proteins in the endoplasmic reticulum (ER stress) to glucose intolerance in obese animals are poorly understood. In this study, enforced expression of a translation initiation factor 2alpha (eIF2alpha)-specific phosphatase, GADD34, was used to selectively compromise signaling in the eIF2(alphaP)-dependent arm of the ER unfolded protein response in liver of transgenic mice. The transgene resulted in lower liver glycogen levels and susceptibility to fasting hypoglycemia in lean mice and glucose tolerance and diminished hepatosteatosis in animals fed a high-fat diet. Attenuated eIF2(alphaP) correlated with lower expression of the adipogenic nuclear receptor PPARgamma and its upstream regulators, the transcription factors C/EBPalpha and C/EBPbeta, in transgenic mouse liver, whereas eIF2alpha phosphorylation promoted C/EBP translation in cultured cells and primary hepatocytes. These observations suggest that eIF2(alphaP)-mediated translation of key hepatic transcriptional regulators of intermediary metabolism contributes to the detrimental consequences of nutrient excess
— id: 79415, year: 2008, vol: 7, page: 520, stat: Journal Article,

Modulation of the eukaryotic initiation factor 2 alpha-subunit kinase PERK by tyrosine phosphorylation
Su, Qiaozhu; Wang, Shuo; Gao, Hong Qing; Kazemi, Shirin; Harding, Heather P; Ron, David; Koromilas, Antonis E
2008 Jan 4;283(1):469-475, Journal of biological chemistry
The endoplasmic reticulum (ER)-resident protein kinase PERK attenuates protein synthesis in response to ER stress through the phosphorylation of translation initiation factor eIF2alpha at serine 51. ER stress induces PERK autophosphorylation at several serine/threonine residues, a process that is required for kinase activation and phosphorylation of eIF2alpha. Herein, we demonstrate that PERK also possesses tyrosine kinase activity. Specifically, we show that PERK is capable of autophosphorylating on tyrosine residues in vitro and in vivo. We further show that tyrosine 615, which is embedded in a highly conserved region of the kinase domain of PERK, is essential for autocatalytic activity. That is, mutation of Tyr-615 to phenylalanine compromises the autophosphorylation capacity of PERK and the phosphorylation of eIF2alpha in vitro and in vivo. The Y615F mutation also impairs the ability of PERK to induce translation of ATF4. Immunoblot analyses with a phosphospecific antibody confirm the phosphorylation of PERK at Tyr-615 both in vitro and in vivo. Thus, our data classify PERK as a dual specificity kinase whose regulation by tyrosine phosphorylation contributes to its optimal activation in response to ER stress
— id: 94508, year: 2008, vol: 283, page: 469, stat: Journal Article,

The integrated stress response prevents demyelination by protecting oligodendrocytes against immune-mediated damage
Lin, Wensheng; Bailey, Samantha L; Ho, Hanson; Harding, Heather P; Ron, David; Miller, Stephen D; Popko, Brian
2007 Feb;117(2):448-456, Journal of clinical investigation
In response to ER stress, the pancreatic endoplasmic reticulum kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha). IFN-gamma, which activates the ER stress response in oligodendrocytes, is believed to play a critical role in the immune-mediated CNS disorder multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Here we report that CNS delivery of IFN-gamma before EAE onset ameliorated the disease course and prevented demyelination, axonal damage, and oligodendrocyte loss. The beneficial effects of IFN-gamma were accompanied by PERK activation in oligodendrocytes and were abrogated in PERK-deficient animals. Our results indicate that IFN-gamma activation of PERK in mature oligodendrocytes attenuates EAE severity and suggest that therapeutic approaches to activate the ISR could prove beneficial in MS
— id: 71592, year: 2007, vol: 117, page: 448, stat: Journal Article,

Translation attenuation by PERK balances ER glycoprotein synthesis with lipid-linked oligosaccharide flux
Shang, Jie; Gao, Ningguo; Kaufman, Randal J; Ron, David; Harding, Heather P; Lehrman, Mark A
2007 Feb 26;176(5):605-616, Journal of cell biology
Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc(3)Man(9)GlcNAc(2) (G(3)M(9)Gn(2)) from the lipid-linked oligosaccharide (LLO) glucose(3)mannose(9)N-acetylglucosamine(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) on Ser(51) by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G(3)M(9)Gn(2)-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2alpha Ser(51) phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser(51) kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway
— id: 71591, year: 2007, vol: 176, page: 605, stat: Journal Article,

Ubiquitin-like protein 5 positively regulates chaperone gene expression in the mitochondrial unfolded protein response
Benedetti, Cristina; Haynes, Cole M; Yang, Yun; Harding, Heather P; Ron, David
2006 Sep;174(1):229-239, Genetics
Perturbation of the protein-folding environment in the mitochondrial matrix selectively upregulates the expression of nuclear genes encoding mitochondrial chaperones. To identify components of the signal transduction pathway(s) mediating this mitochondrial unfolded protein response (UPR(mt)), we first isolated a temperature-sensitive mutation (zc32) that conditionally activates the UPR(mt) in C. elegans and subsequently searched for suppressors by systematic inactivation of genes. RNAi of ubl-5, a gene encoding a ubiquitin-like protein, suppresses activation of the UPR(mt) markers hsp-60::gfp and hsp-6::gfp by the zc32 mutation and by other manipulations that promote mitochondrial protein misfolding. ubl-5 (RNAi) inhibits the induction of endogenous mitochondrial chaperone encoding genes hsp-60 and hsp-6 and compromises the ability of animals to cope with mitochondrial stress. Mitochondrial morphology and assembly of multi-subunit mitochondrial complexes of biotinylated proteins are also perturbed in ubl-5(RNAi) worms, indicating that UBL-5 also counteracts physiological levels of mitochondrial stress. Induction of mitochondrial stress promotes accumulation of GFP-tagged UBL-5 in nuclei of transgenic worms, suggesting that UBL-5 effects a nuclear step required for mounting a response to the threat of mitochondrial protein misfolding
— id: 69577, year: 2006, vol: 174, page: 229, stat: Journal Article,

Antiviral effect of the mammalian translation initiation factor 2alpha kinase GCN2 against RNA viruses
Berlanga, Juan J; Ventoso, Ivan; Harding, Heather P; Deng, Jing; Ron, David; Sonenberg, Nahum; Carrasco, Luis; de Haro, Cesar
2006 Apr 19;25(8):1730-1740, EMBO journal
In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses
— id: 71597, year: 2006, vol: 25, page: 1730, stat: Journal Article,

Perk-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress
Blais, Jaime D; Addison, Christina L; Edge, Robert; Falls, Theresa; Zhao, Huijun; Wary, Kishore; Koumenis, Costas; Harding, Heather P; Ron, David; Holcik, Martin; Bell, John C
2006 Dec;26(24):9517-9532, Molecular & cellular biology
It has been well established that the tumor microenvironment can promote tumor cell adaptation and survival. However, the mechanisms that influence malignant progression have not been clearly elucidated. We have previously demonstrated that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR). Here, we show that tumors derived from K-Ras-transformed Perk(-/-) mouse embryonic fibroblasts (MEFs) are smaller and exhibit less angiogenesis than tumors with an intact ISR. Furthermore, Perk promotes a tumor microenvironment that favors the formation of functional microvessels. These observations were corroborated by a microarray analysis of polysome-bound RNA in aerobic and hypoxic Perk(+/+) and Perk(-/-) MEFs. This analysis revealed that a subset of proangiogenic transcripts is preferentially translated in a Perk-dependent manner; these transcripts include VCIP, an adhesion molecule that promotes cellular adhesion, integrin binding, and capillary morphogenesis. Taken with the concomitant Perk-dependent translational induction of additional proangiogenic genes identified by our microarray analysis, this study suggests that Perk plays a role in tumor cell adaptation to hypoxic stress by regulating the translation of angiogenic factors necessary for the development of functional microvessels and further supports the contention that the Perk pathway could be an attractive target for novel antitumor modalities
— id: 71594, year: 2006, vol: 26, page: 9517, stat: Journal Article,

ER stress disrupts Ca2+-signaling complexes and Ca2+ regulation in secretory and muscle cells from PERK-knockout mice
Huang, Guojin; Yao, Jian; Zeng, Weizhong; Mizuno, Yusuke; Kamm, Kristine E; Stull, James T; Harding, Heather P; Ron, David; Muallem, Shmuel
2006 Jan 1;119(Pt 1):153-161, Journal of cell science
Disruption of protein synthesis and folding results in ER stress, which is associated with the pathophysiology of diverse diseases affecting secretory and muscle cells. Cells are protected against ER stress by activation of the unfolded protein response (UPR) that is regulated by the protein kinase PERK, which phosphorylates the translation initiation factor 2 eIF2alpha to attenuate protein synthesis. PERK-/- cells are unable to modulate ER protein load and experience high levels of ER stress. In addition to its role in protein synthesis, the ER also orchestrates many signaling events essential for cell survival, prominent among which is Ca2+ signaling. It is not known, however, whether there is a relationship between ER stress and the function of the Ca2+-signaling pathway in muscle and non-muscle cells. To directly address this question we characterized Ca2+ signaling in the secretory pancreatic and parotid acinar cells and in urinary bladder smooth muscle (UBSM) cells obtained from PERK-/- and wild-type mice. Deletion of PERK that results in high levels of ER stress, and distention and fragmentation of the ER slowed the rate of agonist-mediated Ca2+ release from the ER and reduced Ca2+-induced Ca2+ release, although IP3 production, localization of the IP3 receptors, IP3-mediated Ca2+ release, Ca(v)1.2 current and RyRs activity remained unaltered. On the other hand, ER stress disrupted the integrity of the Ca2+-signaling complexes in both secretory and UBSM cells, as revealed by markedly reduced co-immunoprecipitation of plasma membrane- and ER-resident Ca2+-signaling proteins. These findings establish a relationship between the unfolding protein response, ER stress and Ca2+ signaling and highlight the importance of communication within the terminal ER-plasma membrane microdomain for propagation of the Ca2+ signal from the plasma membrane into the cell
— id: 71600, year: 2006, vol: 119, page: 153, stat: Journal Article,

Interferon-gamma inhibits central nervous system remyelination through a process modulated by ER stress
Lin, W; Kemper, A; Dupree, JL; Harding, HP; Ron, D; Popko, B
2006 MAR ;96(1):57-57, Journal of neurochemistry
— id: 62903, year: 2006, vol: 96, page: 57, stat: Journal Article,

Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress
Lin, Wensheng; Kemper, April; Dupree, Jeffrey L; Harding, Heather P; Ron, David; Popko, Brian
2006 May;129(Pt 5):1306-1318, Brain
Interferon-gamma (IFN-gamma) is believed to play a deleterious role in the immune-mediated demyelinating disorder multiple sclerosis. Here we have exploited transgenic mice that ectopically express IFN-gamma in a temporally controlled manner in the CNS to specifically study its effects on remyelination in the cuprizone-induced demyelination model and in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. CNS delivery of IFN-gamma severely suppressed remyelination in both models and impaired the clinical recovery of the mice experiencing EAE. These observations correlated with a dramatic reduction of oligodendroglial repopulation in the demyelinated lesions. Moreover, we found that in cuprizone-treated mice the detrimental actions of IFN-gamma were associated with endoplasmic reticulum (ER) stress in remyelinating oligodendrocytes. Compared with a wild-type genetic background, the presence of IFN-gamma in mice heterozygous for a loss of function mutation in the pancreatic ER kinase (PERK), a kinase that responds specifically to ER stress, further reduced the percentage of remyelinated axons and oligodendrocyte numbers in cuprizone-induced demyelinated lesions. Thus, these data suggest that IFN-gamma is capable of inhibiting remyelination in demyelinated lesions and that ER stress modulates the response of remyelinating oligodendrocytes to this cytokine
— id: 71598, year: 2006, vol: 129, page: 1306, stat: Journal Article,

Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK
Marciniak, Stefan J; Garcia-Bonilla, Lidia; Hu, Junjie; Harding, Heather P; Ron, David
2006 Jan 16;172(2):201-209, Journal of cell biology
Regulated phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) by the endoplasmic reticulum (ER) stress-activated protein kinase PERK modulates protein synthesis and couples the production of ER client proteins with the organelle's capacity to fold and process them. PERK activation by ER stress is known to involve transautophosphorylation, which decorates its unusually long kinase insert loop with multiple phosphoserine and phosphothreonine residues. We report that PERK activation and phosphorylation selectively enhance its affinity for the nonphosphorylated eIF2 complex. This switch correlates with a marked change to the protease sensitivity pattern, which is indicative of a major conformational change in the PERK kinase domain upon activation. Although it is dispensable for catalytic activity, PERK's kinase insert loop is required for substrate binding and for eIF2alpha phosphorylation in vivo. Our findings suggest a novel mechanism for eIF2 recruitment by activated PERK and for unidirectional substrate flow in the phosphorylation reaction
— id: 71599, year: 2006, vol: 172, page: 201, stat: Journal Article,

Cotranslocational degradation protects the stressed endoplasmic reticulum from protein overload
Oyadomari, Seiichi; Yun, Chi; Fisher, Edward A; Kreglinger, Nicola; Kreibich, Gert; Oyadomari, Miho; Harding, Heather P; Goodman, Alan G; Harant, Hanna; Garrison, Jennifer L; Taunton, Jack; Katze, Michael G; Ron, David
2006 Aug 25;126(4):727-739, Cell
The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58(IPK)/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58(IPK) recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58(IPK-/-) mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58(IPK) is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells
— id: 69025, year: 2006, vol: 126, page: 727, stat: Journal Article,

Translation attenuation by PERK in response to ER stress rectifies impaired Glc(3)Man(9)GlcNAc(2)-P-P-dolichol synthesis and N-linked glycosylation
Shang, J; Gao, NG; Kaufman, RJ; Ron, D; Harding, HP; Lehrman, MA
2006 NOV ;16(11):1120-1121, Glycobiology
— id: 69303, year: 2006, vol: 16, page: 1120, stat: Journal Article,

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth
Bi, Meixia; Naczki, Christine; Koritzinsky, Marianne; Fels, Diane; Blais, Jaime; Hu, Nianping; Harding, Heather; Novoa, Isabelle; Varia, Mahesh; Raleigh, James; Scheuner, Donalyn; Kaufman, Randal J; Bell, John; Ron, David; Wouters, Bradly G; Koumenis, Constantinos
2005 Oct 5;24(19):3470-3481, EMBO journal
Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2alpha or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities
— id: 71605, year: 2005, vol: 24, page: 3470, stat: Journal Article,

Bioactive small molecules reveal antagonism between the integrated stress response and sterol-regulated gene expression
Harding, Heather P; Zhang, Yuhong; Khersonsky, Sonya; Marciniak, Stefan; Scheuner, Donalyn; Kaufman, Randal J; Javitt, Norman; Chang, Young-Tae; Ron, David
2005 Dec;2(6):361-371, Cell metabolism
Phosphorylation of translation initiation factor 2alpha (eIF2alpha) coordinates a translational and transcriptional program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. A screen for small molecule activators of the ISR identified two related compounds that also activated sterol-regulated genes by blocking cholesterol biosynthesis at the level of CYP51. Ketoconazole, a known CYP51 inhibitor, had similar effects, establishing that perturbed flux of precursors to cholesterol activates the ISR. Surprisingly, compound-mediated activation of sterol-regulated genes was enhanced in cells with an ISR-blocking mutation in the regulatory phosphorylation site of eIF2alpha. Furthermore, induction of the ISR by an artificial drug-activated eIF2alpha kinase reduced the level of active sterol regulatory element binding protein (SREBP) and sterol-regulated mRNAs. These findings suggest a mechanism by which interactions between sterol metabolism, the ISR, and the SREBP pathway affect lipid metabolism during ER stress
— id: 71601, year: 2005, vol: 2, page: 361, stat: Journal Article,

ER stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma
Lin, W; Harding, HP; Ron, D; Popko, B
2005 JUN ;94(3):127-127, Journal of neurochemistry
— id: 57663, year: 2005, vol: 94, page: 127, stat: Journal Article,

[GCN2 regulates feeding behavior to maintain amino acid homeostasis in omnivores]
Maurin, Anne-Catherine; Jousse, Celine; Balage, Michelle; Averous, Julien; Parry, Laurent; Bruhat, Alain; Cherasse, Yoan; Zeng, Huiqing; Zhang, Yuhong; Harding, Heather; Ron, David; Fafournoux, Pierre
2005 Oct;21(10):799-801, M/S: medecine sciences
— id: 71603, year: 2005, vol: 21, page: 799, stat: Journal Article,

Rapid B cell receptor-induced unfolded protein response in nonsecretory B cells correlates with pro- versus antiapoptotic cell fate
Skalet, Alison H; Isler, Jennifer A; King, Leslie B; Harding, Heather P; Ron, David; Monroe, John G
2005 Dec 2;280(48):39762-39771, Journal of biological chemistry
The adaptive unfolded protein response (UPR) is essential for the development of antibody-secreting plasma cells. B cells induced by lipopolysaccharide (LPS) to differentiate into plasma cells exhibit a nonclassical UPR reported to anticipate endoplasmic reticulum stress prior to immunoglobulin production. Here we demonstrate that activation of a physiologic UPR is not limited to cells undergoing secretory cell differentiation. We identify B cell receptor (BCR) signaling as an unexpected physiologic UPR trigger and demonstrate that in mature B cells, BCR stimulation induces a short lived UPR similar to the LPS-triggered nonclassical UPR. However, unlike LPS, BCR stimulation does not induce plasma cell differentiation. Furthermore, the BCR-induced UPR is not limited to cells in which BCR induces activation, since a UPR is also induced in transitional immature B cells that respond to BCR stimulation with a rapid apoptotic fate. This response involves sustained up-regulation of Chop mRNA indicative of a terminal UPR. Whereas sustained Chop expression correlates with the ultimate fate of the BCR-triggered B cell and not its developmental stage, Chop-/- B cells undergo apoptosis, indicating that CHOP is not required for this process. These studies establish a system whereby a terminal or adaptive UPR can be alternatively triggered by physiologic stimuli
— id: 71604, year: 2005, vol: 280, page: 39762, stat: Journal Article,

Activating transcription factor 4 is translationally regulated by hypoxic stress
Blais, Jaime D; Filipenko, Vasilisa; Bi, Meixia; Harding, Heather P; Ron, David; Koumenis, Costas; Wouters, Bradly G; Bell, John C
2004 Sep;24(17):7469-7482, Molecular & cellular biology
Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2alpha at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2alpha was able to attenuate the phosphorylation of eIF2alpha and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2alpha-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress
— id: 45318, year: 2004, vol: 24, page: 7469, stat: Journal Article,

Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2
Deng, Jing; Lu, Phoebe D; Zhang, Yuhong; Scheuner, Donalyn; Kaufman, Randal J; Sonenberg, Nahum; Harding, Heather P; Ron, David
2004 Dec;24(23):10161-10168, Molecular & cellular biology
Numerous stressful conditions activate kinases that phosphorylate the alpha subunit of translation initiation factor 2 (eIF2alpha), thus attenuating mRNA translation and activating a gene expression program known as the integrated stress response. It has been noted that conditions associated with eIF2alpha phosphorylation, notably accumulation of unfolded proteins in the endoplasmic reticulum (ER), or ER stress, are also associated with activation of nuclear factor kappa B (NF-kappaB) and that eIF2alpha phosphorylation is required for NF-kappaB activation by ER stress. We have used a pharmacologically activable version of pancreatic ER kinase (PERK, an ER stress-responsive eIF2alpha kinase) to uncouple eIF2alpha phosphorylation from stress and found that phosphorylation of eIF2alpha is both necessary and sufficient to activate both NF-kappaB DNA binding and an NF-kappaB reporter gene. eIF2alpha phosphorylation-dependent NF-kappaB activation correlated with decreased levels of the inhibitor IkappaBalpha protein. Unlike canonical signaling pathways that promote IkappaBalpha phosphorylation and degradation, eIF2alpha phosphorylation did not increase phosphorylated IkappaBalpha levels or affect the stability of the protein. Pulse-chase labeling experiments indicate instead that repression of IkappaBalpha translation plays an important role in NF-kappaB activation in cells experiencing high levels of eIF2alpha phosphorylation. These studies suggest a direct role for eIF2alpha phosphorylation-dependent translational control in activating NF-kappaB during ER stress
— id: 47770, year: 2004, vol: 24, page: 10161, stat: Journal Article,

Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response
Jiang, Hao-Yuan; Wek, Sheree A; McGrath, Barbara C; Lu, Dan; Hai, Tsonwin; Harding, Heather P; Wang, Xiaozhong; Ron, David; Cavener, Douglas R; Wek, Ronald C
2004 Feb;24(3):1365-1377, Molecular & cellular biology
In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed
— id: 42130, year: 2004, vol: 24, page: 1365, stat: Journal Article,

Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response
Lu, Phoebe D; Harding, Heather P; Ron, David
2004 Oct 11;167(1):27-33, Journal of cell biology
Stress-induced eukaryotic translation initiation factor 2 (eIF2) alpha phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program. Previous studies implicated the 5' end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation. Here, we report on mutation analysis of the ATF4 mRNA which revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs. In unstressed cells, low levels of eIF2alpha phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR. In stressed cells high levels of eIF2alpha phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2. These features are common to regulated translation of GCN4 in yeast. The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details
— id: 45315, year: 2004, vol: 167, page: 27, stat: Journal Article,

Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2
Lu, Phoebe D; Jousse, Celine; Marciniak, Stefan J; Zhang, Yuhong; Novoa, Isabel; Scheuner, Donalyn; Kaufman, Randal J; Ron, David; Harding, Heather P
2004 Jan 14;23(1):169-179, EMBO journal
Transient phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2alpha phosphorylation-dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2alpha phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress-inducible eIF2alpha kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2alpha kinase, Fv2E-PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E-PERK activation enhanced the expression of numerous stress-induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2alpha phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress-induced signals and that activation of this pathway can single-handedly promote a stress-resistant preconditioned state
— id: 42131, year: 2004, vol: 23, page: 169, stat: Journal Article,

CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum
Marciniak, Stefan J; Yun, Chi Y; Oyadomari, Seiichi; Novoa, Isabel; Zhang, Yuhong; Jungreis, Rivka; Nagata, Kazuhiro; Harding, Heather P; Ron, David
2004 Dec 15;18(24):3066-3077, Genes & development
Unfolded and malfolded client proteins impose a stress on the endoplasmic reticulum (ER), which contributes to cell death in pathophysiological conditions. The transcription factor C/EBP homologous protein (CHOP) is activated by ER stress, and CHOP deletion protects against its lethal consequences. We find that CHOP directly activates GADD34, which promotes ER client protein biosynthesis by dephosphorylating phospho-Ser 51 of the alpha-subunit of translation initiation factor 2 (eIF2alpha) in stressed cells. Thus, impaired GADD34 expression reduces client protein load and ER stress in CHOP(-/-) cells exposed to perturbations that impair ER function. CHOP(-/-) and GADD34 mutant cells accumulate less high molecular weight protein complexes in their stressed ER than wild-type cells. Furthermore, mice lacking GADD34-directed eIF2alpha dephosphorylation, like CHOP(-/-) mice, are resistant to renal toxicity of the ER stress-inducing drug tunicamycin. CHOP also activates ERO1alpha, which encodes an ER oxidase. Consequently, the ER of stressed CHOP(-/-) cells is relatively hypo-oxidizing. Pharmacological and genetic manipulations that promote a hypo-oxidizing ER reduce abnormal high molecular weight protein complexes in the stressed ER and protect from the lethal consequences of ER stress. CHOP deletion thus protects cells from ER stress by decreasing ER client protein load and changing redox conditions within the organelle
— id: 48881, year: 2004, vol: 18, page: 3066, stat: Journal Article,

Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones
Yoneda, Takunari; Benedetti, Cristina; Urano, Fumihiko; Clark, Scott G; Harding, Heather P; Ron, David
2004 Aug 15;117(Pt 18):4055-4066, Journal of cell science
Protein folding in the mitochondria is assisted by nuclear-encoded compartment-specific chaperones but regulation of the expression of their encoding genes is poorly understood. We found that the mitochondrial matrix HSP70 and HSP60 chaperones, encoded by the Caenorhabditis elegans hsp-6 and hsp-60 genes, were selectively activated by perturbations that impair assembly of multi-subunit mitochondrial complexes or by RNAi of genes encoding mitochondrial chaperones or proteases, which lead to defective protein folding and processing in the organelle. hsp-6 and hsp-60 induction was specific to perturbed mitochondrial protein handling, as neither heat-shock nor endoplasmic reticulum stress nor manipulations that impair mitochondrial steps in intermediary metabolism or ATP synthesis activated the mitochondrial chaperone genes. These observations support the existence of a mitochondrial unfolded protein response that couples mitochondrial chaperone gene expression to changes in the protein handling environment in the organelle
— id: 45319, year: 2004, vol: 117, page: 4055, stat: Journal Article,

The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages
Feng, Bo; Yao, Pin Mei; Li, Yankun; Devlin, Cecilia M; Zhang, Dajun; Harding, Heather P; Sweeney, Michele; Rong, James X; Kuriakose, George; Fisher, Edward A; Marks, Andrew R; Ron, David; Tabas, Ira
2003 Sep;5(9):781-792, Nature cell biology
Excess cellular cholesterol induces apoptosis in macrophages, an event likely to promote progression of atherosclerosis. The cellular mechanism of cholesterol-induced apoptosis is unknown but had previously been thought to involve the plasma membrane. Here we report that the unfolded protein response (UPR) in the endoplasmic reticulum is activated in cholesterol-loaded macrophages, resulting in expression of the cell death effector CHOP. Cholesterol loading depletes endoplasmic reticulum calcium stores, an event known to induce the UPR. Furthermore, endoplasmic reticulum calcium depletion, the UPR, caspase-3 activation and apoptosis are markedly inhibited by selective inhibition of cholesterol trafficking to the endoplasmic reticulum, and Chop(-/-) macrophages are protected from cholesterol-induced apoptosis. We propose that cholesterol trafficking to endoplasmic reticulum membranes, resulting in activation of the CHOP arm of the UPR, is the key signalling step in cholesterol-induced apoptosis in macrophages
— id: 37270, year: 2003, vol: 5, page: 781, stat: Journal Article,

An integrated stress response regulates amino acid metabolism and resistance to oxidative stress
Harding, Heather P; Zhang, Yuhong; Zeng, Huiquing; Novoa, Isabel; Lu, Phoebe D; Calfon, Marcella; Sadri, Navid; Yun, Chi; Popko, Brian; Paules, Richard; Stojdl, David F; Bell, John C; Hettmann, Thore; Leiden, Jeffrey M; Ron, David
2003 Mar;11(3):619-633, Molecular cell
Eukaryotic cells respond to unfolded proteins in their endoplasmic reticulum (ER stress), amino acid starvation, or oxidants by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). This adaptation inhibits general protein synthesis while promoting translation and expression of the transcription factor ATF4. Atf4(-/-) cells are impaired in expressing genes involved in amino acid import, glutathione biosynthesis, and resistance to oxidative stress. Perk(-/-) cells, lacking an upstream ER stress-activated eIF2alpha kinase that activates Atf4, accumulate endogenous peroxides during ER stress, whereas interference with the ER oxidase ERO1 abrogates such accumulation. A signaling pathway initiated by eIF2alpha phosphorylation protects cells against metabolic consequences of ER oxidation by promoting the linked processes of amino acid sufficiency and resistance to oxidative stress
— id: 39258, year: 2003, vol: 11, page: 619, stat: Journal Article,

Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells
Jousse, Celine; Oyadomari, Seiichi; Novoa, Isabel; Lu, Phoebe; Zhang, Yuhong; Harding, Heather P; Ron, David
2003 Nov 24;163(4):767-775, Journal of cell biology
Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) on serine 51 is effected by specific stress-activated protein kinases. eIF2alpha phosphorylation inhibits translation initiation promoting a cytoprotective gene expression program known as the integrated stress response (ISR). Stress-induced activation of GADD34 feeds back negatively on this pathway by promoting eIF2alpha dephosphorylation, however, GADD34 mutant cells retain significant eIF2alpha-directed phosphatase activity. We used a somatic cell genetic approach to identify a gene encoding a novel regulatory subunit of a constitutively active holophosphatase complex that dephosphorylates eIF2alpha. RNAi of this gene, which we named constitutive repressor of eIF2alpha phosphorylation (CReP, or PPP1R15B), repressed the constitutive eIF2alpha-directed phosphatase activity and activated the ISR. CReP RNAi strongly protected mammalian cells against oxidative stress, peroxynitrite stress, and more modestly against accumulation of malfolded proteins in the endoplasmic reticulum. These findings suggest that therapeutic inhibition of eIF2alpha dephosphorylation by targeting the CReP-protein-phosphatase-1 complex may be used to access the salubrious qualities of the ISR
— id: 42133, year: 2003, vol: 163, page: 767, stat: Journal Article,

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes
Kimball, Scot R; Horetsky, Rick L; Ron, David; Jefferson, Leonard S; Harding, Heather P
2003 Feb;284(2):C273-C284, American journal of physiology. Cell physiology
In eukaryotic cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA(i) (Met-tRNA(i)) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2. GTP. Met-tRNA(i) complex is thought to bind to the 40S ribosomal subunit before the eIF4G. eIF4E. mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the alpha-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes
— id: 38134, year: 2003, vol: 284, page: C273, stat: Journal Article,

Stress-induced gene expression requires programmed recovery from translational repression
Novoa, Isabel; Zhang, Yuhong; Zeng, Huiqing; Jungreis, Rivka; Harding, Heather P; Ron, David
2003 Mar 3;22(5):1180-1187, EMBO journal
Active repression of protein synthesis protects cells against protein malfolding during endoplasmic reticulum stress, nutrient deprivation and oxidative stress. However, long-term adaptation to these conditions requires synthesis of new stress-induced proteins. Phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation in diverse stressful conditions. GADD34 is a stress-inducible regulatory subunit of a holophosphatase complex that dephosphorylates eIF2alpha, and has been hypothesized to play a role in translational recovery. Here, we report that GADD34 expression correlated temporally with eIF2alpha dephosphorylation late in the stress response. Inactivation of both alleles of GADD34 prevented eIF2alpha dephosphorylation and blocked the recovery of protein synthesis, normally observed late in the stress response. Furthermore, defective recovery of protein synthesis markedly impaired translation of stress-induced proteins and interfered with programmed activation of stress-induced genes in the GADD34 mutant cells. These observations indicate that GADD34 controls a programmed shift from translational repression to stress-induced gene expression, and reconciles the apparent contradiction between the translational and transcriptional arms of cellular stress responses
— id: 38135, year: 2003, vol: 22, page: 1180, stat: Journal Article,

IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA
Calfon, Marcella; Zeng, Huiqing; Urano, Fumihiko; Till, Jeffery H; Hubbard, Stevan R; Harding, Heather P; Clark, Scott G; Ron, David
2002 Jan 3;415(6867):92-96, Nature
The unfolded protein response (UPR), caused by stress, matches the folding capacity of endoplasmic reticulum (ER) to the load of client proteins in the organelle. In yeast, processing of HAC1 mRNA by activated Ire1 leads to synthesis of the transcription factor Hac1 and activation of the UPR. The responses to activated IRE1 in metazoans are less well understood. Here we demonstrate that mutations in either ire-1 or the transcription-factor-encoding xbp-1 gene abolished the UPR in Caenorhabditis elegans. Mammalian XBP-1 is essential for immunoglobulin secretion and development of plasma cells, and high levels of XBP-1 messenger RNA are found in specialized secretory cells. Activation of the UPR causes IRE1-dependent splicing of a small intron from the XBP-1 mRNA both in C. elegans and mice. The protein encoded by the processed murine XBP-1 mRNA accumulated during the UPR, whereas the protein encoded by unprocessed mRNA did not. Purified mouse IRE1 accurately cleaved XBP-1 mRNA in vitro, indicating that XBP-1 mRNA is a direct target of IRE1 endonucleolytic activity. Our findings suggest that physiological ER load regulates a developmental decision in higher eukaryotes
— id: 27253, year: 2002, vol: 415, page: 92, stat: Journal Article,

Activation of GCN2 in UV-irradiated cells inhibits translation
Deng, J; Harding, HP; Raught, B; Gingras, AC; Berlanga, JJ; Scheuner, D; Kaufman, RJ; Ron, D; Sonenberg, N
2002 Aug 6;12(15):1279-1286, Current biology. CB
Background: Mammalian cells subjected to ultraviolet (UV) irradiation actively repress DNA replication, transcription, and mRNA translation. While the effects of UV irradiation on DNA replication and transcription have been extensively studied, the mechanism(s) responsible for translational repression are poorly understood. Results: Here, we demonstrate that UV irradiation elicits phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) by activating the kinase GCN2 in a manner that does not require SAPK/JNK or p38 MAP kinase. GCN2-/- cells, and cells expressing nonphosphorylatable eIF2alpha as their only source of eIF2alpha protein, fail to repress translation in response to UV irradiation. Conclusions: These results provide a mechanism for translation inhibition by UV irradiation and identify a hitherto unrecognized role for mammalian GCN2 as a mediator of the cellular response to UV stress
— id: 32380, year: 2002, vol: 12, page: 1279, stat: Journal Article,

Transcriptional and translational control in the Mammalian unfolded protein response
Harding, Heather P; Calfon, Marcella; Urano, Fumihiko; Novoa, Isabel; Ron, David
2002 ;18(3):575-599, Annual review of cell & developmental biology
Cells monitor the physiological load placed on their endoplasmic reticulum (ER) and respond to perturbations in ER function by a process known as the unfolded protein response (UPR). In metazoans the UPR has a transcriptional component that up-regulates expression of genes that enhance the capacity of the organelle to deal with the load of client proteins and a translational component that insures tight coupling between protein biosynthesis on the cytoplasmic side and folding in the ER lumen. Together, these two components adapt the secretory apparatus to physiological load and protect cells from the consequences of protein malfolding
— id: 39419, year: 2002, vol: 18, page: 575, stat: Journal Article,

Endoplasmic reticulum stress and the development of diabetes: a review
Harding, Heather P; Ron, David
2002 Dec;51 Suppl 3(12):S455-S461, Diabetes
The early steps of insulin biosynthesis occur in the endoplasmic reticulum (ER), and the beta-cell has a highly developed and active ER. All cells regulate the capacity of their ER to fold and process client proteins and they adapt to an imbalance between client protein load and folding capacity (so-called ER stress). Mutations affecting the ER stress-activated pancreatic ER kinase (PERK) and its downstream effector, the translation initiation complex eukaryotic initiation factor 2 (eIF2), have a profound impact on islet cell development, function, and survival. PERK mutations are associated with the Wolcott-Rallison syndrome of infantile diabetes and mutations that prevent the alpha-subunit of eIF2 from being phosphorylated by PERK, block beta-cell development, and impair gluconeogenesis. We will review this and other rare forms of clinical and experimental diabetes and consider the role of ER stress in the development of more common forms of the disease
— id: 39351, year: 2002, vol: 51 Suppl 3, page: S455, stat: Journal Article,

Transmission of cell stress from endoplasmic reticulum to mitochondria: enhanced expression of Lon protease
Hori, O; Ichinoda, F; Tamatani, T; Yamaguchi, A; Sato, N; Ozawa, K; Kitao, Y; Miyazaki, M; Harding, HP; Ron, D; Tohyama, M; Stern, DM; Ogawa, S
2002 Jun 24;157(7):1151-1160, Journal of cell biology
T he rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial- encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP- dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (-/-) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX 1-containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex
— id: 30699, year: 2002, vol: 157, page: 1151, stat: Journal Article,

Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease
Ryu, EJ; Harding, HP; Angelastro, JM; Vitolo, OV; Ron, D; Greene, LA
2002 DEC 15 ;22(24):10690-10698, Journal of neuroscience
6-Hydroxydopamine, 1-methyl-4-phenyl-pyridinium (MPP+), and rotenone cause the death of dopaminergic neurons in vitro and in vivo and are widely used to model Parkinson's disease. To identify regulated genes in such models, we performed serial analysis of gene expression on neuronal PC12 cells exposed to 6-hydroxydopamine. This revealed a striking increase in transcripts associated with the unfolded protein response. Immunoblotting confirmed phosphorylation of the key endoplasmic reticulum stress kinases IRE1alpha and PERK (PKR-like ER kinase) and induction of their downstream targets. There was a similar response to MPP+ and rotenone, but not to other apoptotic initiators. As evidence that endoplasmic reticulum stress contributes to neuronal death, sympathetic neurons from PERK null mice in which the capacity to respond to endoplasmic reticulum stress is compromised were more sensitive to 6- hydroxydopamine. Our findings, coupled with evidence from familial forms of Parkinson's disease, raise the possibility of widespread involvement of endoplasmic reticulum stress and the unfolded protein response in the pathophysiology of this disease
— id: 33272, year: 2002, vol: 22, page: 10690, stat: Journal Article,

Translational regulation in the cellular response to biosynthetic load on the endoplasmic reticulum
Harding HP; Novoa I; Bertolotti A; Zeng H; Zhang Y; Urano F; Jousse C; Ron D
2001 ;66(6):499-508, Cold Spring Harbor symposia on quantitative biology
— id: 39220, year: 2001, vol: 66, page: 499, stat: Journal Article,

Diabetes mellitus and exocrine pancreatic dysfunction in perk-/- mice reveals a role for translational control in secretory cell survival
Harding HP; Zeng H; Zhang Y; Jungries R; Chung P; Plesken H; Sabatini DD; Ron D
2001 Jun;7(6):1153-1163, Molecular cell
The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. PERK is highly expressed in mouse pancreas, an organ active in protein secretion. Under physiological conditions, PERK was partially activated, accounting for much of the phosphorylated eIF2alpha in the pancreas. The exocrine and endocrine pancreas developed normally in Perk-/- mice. Postnatally, ER distention and activation of the ER stress transducer IRE1alpha accompanied increased cell death and led to progressive diabetes mellitus and exocrine pancreatic insufficiency. These findings suggest a special role for translational control in protecting secretory cells from ER stress
— id: 21161, year: 2001, vol: 7, page: 1153, stat: Journal Article,

Brain ischemia and reperfusion activates the eukaryotic initiation factor 2alpha kinase, PERK
Kumar R; Azam S; Sullivan JM; Owen C; Cavener DR; Zhang P; Ron D; Harding HP; Chen JJ; Han A; White BC; Krause GS; DeGracia DJ
2001 Jun;77(5):1418-1421, Journal of neurochemistry
Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons, which persists in vulnerable neurons, that is caused by the inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha). To identify kinases responsible for eIF2alpha phosphorylation [eIF2alpha(P)] during brain reperfusion, we induced ischemia by bilateral carotid artery occlusion followed by post-ischemic assessment of brain eIF2alpha(P) in mice with homozygous functional knockouts in the genes encoding the heme-regulated eIF2alpha kinase (HRI), or the amino acid-regulated eIF2alpha kinase (GCN2). A 10-fold increase in eIF2alpha(P) was observed in reperfused wild-type mice and in the HRI-/- or GCN2-/- mice. However, in all reperfused groups, the RNA-dependent protein kinase (PKR)-like endoplasmic reticulum eIF2alpha kinase (PERK) exhibited an isoform mobility shift on SDS-PAGE, consistent with the activation of the kinase. These data indicate that neither HRI nor GCN2 are required for the large increase in post-ischemic brain eIF2alpha(P), and in conjunction with our previous report that eIF2alpha(P) is produced in the brain of reperfused PKR-/- mice, provides evidence that PERK is the kinase responsible for eIF2alpha phosphorylation in the early post-ischemic brain
— id: 27773, year: 2001, vol: 77, page: 1418, stat: Journal Article,

Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha
Novoa I; Zeng H; Harding HP; Ron D
2001 May 28;153(5):1011-1022, Journal of cell biology
Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2alpha-dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196-4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2alpha kinases PERK (also known as PEK) and GCN2, phospho-eIF2alpha levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2alpha in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2alpha and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK(-)/- and GCN2(-)/- cells. These findings implicate GADD34-mediated dephosphorylation of eIF2alpha in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response
— id: 21193, year: 2001, vol: 153, page: 1011, stat: Journal Article,

Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response
Bertolotti A; Zhang Y; Hendershot LM; Harding HP; Ron D
2000 Jun;2(6):326-332, Nature cell biology
PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains
— id: 11648, year: 2000, vol: 2, page: 326, stat: Journal Article,

Regulated translation initiation controls stress-induced gene expression in mammalian cells
Harding HP; Novoa I; Zhang Y; Zeng H; Wek R; Schapira M; Ron D
2000 Nov;6(5):1099-1108, Molecular cell
Protein kinases that phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) are activated in stressed cells and negatively regulate protein synthesis. Phenotypic analysis of targeted mutations in murine cells reveals a novel role for eIF2alpha kinases in regulating gene expression in the unfolded protein response (UPR) and in amino acid starved cells. When activated by their cognate upstream stress signals, the mammalian eIF2 kinases PERK and GCN2 repress translation of most mRNAs but selectively increase translation of Activating Transcription Factor 4 (ATF4), resulting in the induction of the downstream gene CHOP (GADD153). This is the first example of a mammalian signaling pathway homologous to the well studied yeast general control response in which eIF2alpha phosphorylation activates genes involved in amino acid biosynthesis. Mammalian cells thus utilize an ancient pathway to regulate gene expression in response to diverse stress signals
— id: 27774, year: 2000, vol: 6, page: 1099, stat: Journal Article,

Perk is essential for translational regulation and cell survival during the unfolded protein response
Harding HP; Zhang Y; Bertolotti A; Zeng H; Ron D
2000 May;5(5):897-904, Molecular cell
Malfolded proteins in the endoplasmic reticulum (ER) inhibit translation initiation. This response is believed to be mediated by increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and is hypothesized to reduce the work load imposed on the folding machinery during stress. Here we report that mutating the gene encoding the ER stress-activated eIF2alpha kinase PERK abolishes the phosphorylation of eIF2alpha in response to accumulation of malfolded proteins in the ER resulting in abnormally elevated protein synthesis and higher levels of ER stress. Mutant cells are markedly impaired in their ability to survive ER stress and inhibition of protein synthesis by cycloheximide treatment during ER stress ameliorates this impairment. PERK thus plays a major role in the ability of cells to adapt to ER stress
— id: 11620, year: 2000, vol: 5, page: 897, stat: Journal Article,

PERK and translational control by stress in the endoplasmic reticulum
Ron, David; Harding, Heather P
Translation control of gene expression Plainview, NY : Cold Spring Harbor Laboratory Press ,
— id: 2555, year: 2000, vol: , page: 547, stat: Chapter,

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1
Urano F; Wang X; Bertolotti A; Zhang Y; Chung P; Harding HP; Ron D
2000 Jan 28;287(5453):664-666, Science
Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals
— id: 8577, year: 2000, vol: 287, page: 664, stat: Journal Article,

Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase [published erratum appears in Nature 1999 Mar 4;398(6722):90] [see comments]
Harding HP; Zhang Y; Ron D
1999 Jan 21;397(6716):271-274, Nature
Protein synthesis and the folding of the newly synthesized proteins into the correct three-dimensional structure are coupled in cellular compartments of the exocytosis pathway by a process that modulates the phosphorylation level of eukaryotic initiation factor-2alpha (eIF2alpha) in response to a stress signal from the endoplasmic reticulum (ER). Activation of this process leads to reduced rates of initiation of protein translation during ER stress. Here we describe the cloning of perk, a gene encoding a type I transmembrane ER-resident protein. PERK has a lumenal domain that is similar to the ER-stress-sensing lumenal domain of the ER-resident kinase Ire1, and a cytoplasmic portion that contains a protein-kinase domain most similar to that of the known eIF2alpha kinases, PKR and HRI. ER stress increases PERK's protein-kinase activity and PERK phosphorylates eIF2alpha on serine residue 51, inhibiting translation of messenger RNA into protein. These properties implicate PERK in a signalling pathway that attenuates protein translation in response to ER stress
— id: 7985, year: 1999, vol: 397, page: 271, stat: Journal Article,

Amino acid limitation regulates CHOP expression through a specific pathway independent of the unfolded protein response
Jousse C; Bruhat A; Harding HP; Ferrara M; Ron D; Fafournoux P
1999 Apr 9;448(2-3):211-216, FEBS letters
The gene encoding CHOP (C/EBP-homologous protein) is transcriptionally activated by many stimuli and by amino acid deprivation. CHOP induction was considered to be due to an accumulation of unfolded protein into the ER (unfolded protein response (UPR)). We investigate the role of the UPR in the induction of CHOP by amino acid deprivation and show that this induction is not correlated with BiP expression (an UPR marker). Moreover, amino acid deprivation and UPR inducers regulate the CHOP promoter activity using distinct cis elements. We conclude that amino acid deprivation does not activate the UPR and regulates CHOP expression through a pathway that is independent of the UPR
— id: 27775, year: 1999, vol: 448, page: 211, stat: Journal Article,

CHOP-Dependent stress-inducible expression of a novel form of carbonic anhydrase VI
Sok J; Wang XZ; Batchvarova N; Kuroda M; Harding H; Ron D
1999 Jan;19(1):495-504, Molecular & cellular biology
CHOP (also called GADD153) is a stress-inducible nuclear protein that dimerizes with members of the C/EBP family of transcription factors and was initially identified as an inhibitor of C/EBP binding to classic C/EBP target genes. Subsequent experiments suggested a role for CHOP-C/EBP heterodimers in positively regulating gene expression; however, direct evidence that this is the case has so far not been uncovered. Here we describe the identification of a positively regulated direct CHOP-C/EBP target gene, that encoding murine carbonic anhydrase VI (CA-VI). The stress-inducible form of the gene is expressed from an internal promoter and encodes a novel intracellular form of what is normally a secreted protein. Stress-induced expression of CA-VI is both CHOP and C/EBPbeta dependent in that it does not occur in cells deficient in either gene. A CHOP-responsive element was mapped to the inducible CA-VI promoter, and in vitro footprinting revealed binding of CHOP-C/EBP heterodimers to that site. Rescue of CA-VI expression in c/ebpbeta-/- cells by exogenous C/EBPbeta and a shorter, normally inhibitory isoform of the protein known as LIP suggests that the role of the C/EBP partner is limited to targeting the CHOP-containing heterodimer to the response element and points to a preeminent role for CHOP in CA-VI induction during stress
— id: 7451, year: 1999, vol: 19, page: 495, stat: Journal Article,

Cloning of mammalian Ire1 reveals diversity in the ER stress responses
Wang XZ; Harding HP; Zhang Y; Jolicoeur EM; Kuroda M; Ron D
1998 Oct 1;17(19):5708-5717, EMBO journal
Cells modify their gene expression pattern in response to stress signals emanating from the endoplasmic reticulum (ER). The well-characterized aspect of this response consists of the activation of genes that encode protein chaperones and other ER resident proteins, and is conserved between mammals and yeast. In mammalian cells, however, ER stress also activates other pathways, including the expression of the transcription factor CHOP/GADD153 and its downstream target genes. ER stress is also linked to the development of programmed cell death, a phenomenon in which CHOP plays an important role. Here we report on the cloning of a murine homolog of yeast IRE1, an essential upstream component of the ER stress-response in yeast. The mammalian Ire1 is located in the ER membrane and its over-expression in mammalian cells activates both the endogenous ER chaperone GRP78/BiP and CHOP-encoding genes. Over-expression of a dominant-negative form of Ire1 blocks the induction of GRP78/BiP and CHOP in response to the ER stress induced by tunicamycin treatment. Over-expression of murine Ire1 also leads to the development of programmed cell death in transfected cells. These results indicate that a single upstream component, Ire1, plays a role in multiple facets of the ER stress-response in mammalian cells
— id: 7841, year: 1998, vol: 17, page: 5708, stat: Journal Article,

Transcriptional activation and repression by RORalpha, an orphan nuclear receptor required for cerebellar development
Harding HP; Atkins GB; Jaffe AB; Seo WJ; Lazar MA
1997 Oct;11(11):1737-1746, Molecular endocrinology
Mutation of the orphan nuclear receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. We have found that RORalpha activates transcription from only a subset of sites to which it binds strongly as a monomer. RORalpha also selectively binds as a homodimer to a direct repeat of this monomer site with a 2-bp spacing between the AGGTCA sequences (Rev-DR2 site) and is a much more potent transcriptional activator on this site than on monomer sites or other direct repeats. To better understand the transcriptional regulatory functions of RORalpha, we fused its C terminus to a heterologous DNA-binding domain. Mutational analysis revealed that RORalpha contains both transcriptional activation and transcriptional repression domains, with the repression domain being more active in some cell types. The abilities of RORalpha polypeptides to repress transcription correlate with their abilities to interact with the nuclear receptor corepressors N-CoR and SMRT in vitro. However, the AF2 region of RORalpha inhibits corepressor interaction on DNA, consistent with the lack of repression by the full-length receptor. Thus, transcriptional regulation by RORalpha is complex and likely to be regulated in a cell type- and target gene-specific manner
— id: 27776, year: 1997, vol: 11, page: 1737, stat: Journal Article,

A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with distinct repression domains
Zamir I; Harding HP; Atkins GB; Horlein A; Glass CK; Rosenfeld MG; Lazar MA
1996 Oct;16(10):5458-5465, Molecular & cellular biology
Ligand-independent transcriptional repression is an important function of nuclear hormone receptors. An interaction screen with the repression domain of the orphan receptor RevErb identified N-CoR, the corepressor for thyroid hormone receptor (TR) and retinoic acid receptor (RAR). N-CoR is likely to be a bona fide transcriptional corepressor for RevErb because (i) RevErb interacts with endogenous N-CoR, (ii) ectopic N-CoR potentiates RevErb-mediated repression, and (iii) transcriptional repression by RevErb correlates with its ability to bind N-CoR. Remarkably, a region homologous to the CoR box which is necessary for TR and RAR to interact with N-CoR is not required for RevErb. Rather, two short regions of RevErb separated by approximately 200 amino acids are required for interaction with N-CoR. The primary amino acid sequence of the N-terminal region of RevErb essential for N-CoR interaction is not homologous to that of TR or RAR, whereas similarities exist among the C-terminal domains of the receptors. N-CoR contains two adjacent but distinct interaction domains, one of which binds tightly to both RevErb and TR whereas the other binds more weakly and differentially interacts with the nuclear receptors. These results indicate that multiple nuclear receptors, utilizing different primary amino acid sequences, repress transcription by interacting with N-CoR
— id: 27777, year: 1996, vol: 16, page: 5458, stat: Journal Article,

The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat
Harding HP; Lazar MA
1995 Sep;15(9):4791-4802, Molecular & cellular biology
Rev-Erb is an orphan nuclear receptor which binds as a monomer to the thyroid/retinoic acid receptor half-site AGGTCA flanked 5' by an A/T-rich sequence, referred to here as a Rev monomer site. Fusion of Rev-Erb to the DNA binding domain of yeast GAL4 strongly repressed basal transcription of a GAL4-luciferase reporter gene as a result of the presence of a C-terminal domain containing both the hinge and heptad repeat regions. Nevertheless, wild-type Rev-Erb did not repress basal transcription from the Rev monomer binding site. Therefore, a DNA binding site selection strategy was devised to test the hypothesis that Rev-Erb may function on a different site as a dimer. This approach identified sequences containing two Rev monomer sites arranged as direct repeats with the AGGTCA motifs separated by 2 bp (Rev-DR2). Remarkably, Rev-Erb bound as a homodimer to Rev-DR2 but not to other direct repeats or to a standard DR2 sequence. The DNA binding domain contained all of the determinants for Rev-DR2-specific homodimerization. Rev-Erb bound cooperatively as a homodimer to Rev-DR2, and this interaction was 5 to 10 times more stable than Rev-Erb monomer binding to the Rev monomer site. Functionally, Rev-Erb markedly repressed the basal activity of a variety of promoters with a strong Rev-DR2 specificity. The C terminus was required for this repression, consistent with the GAL4 results. However, the Rev-DR2 specificity did not require the C terminus in vivo, since fusion of C-terminally truncated Rev-Erb to a heterologous transactivation domain created a transcriptional activator specific for Rev-DR2. In addition to idealized Rev-DR2 sites, Rev-Erb also repressed basal as well as retinoic acid-induced transcription from a naturally occurring Rev-DR2 in the CRBPI gene. Thus, although Rev-Erb is distinguished from other thyroid/steroid receptor superfamily members by its ability to bind DNA as a monomer, it functions as a homodimer to repress transcription of genes containing a novel DR2 element
— id: 27779, year: 1995, vol: 15, page: 4791, stat: Journal Article,

Differential activation of peroxisome proliferator-activated receptors by eicosanoids
Yu K; Bayona W; Kallen CB; Harding HP; Ravera CP; McMahon G; Brown M; Lazar MA
1995 Oct 13;270(41):23975-23983, Journal of biological chemistry
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate gene transcription in response to peroxisome proliferators and fatty acids. PPARs also play an important role in the regulation of adipocyte differentiation. It is unclear, however, what naturally occurring compounds activate each of the PPAR subtypes. To address this issue, a screening assay was established using heterologous fusions of the bacterial tetracycline repressor to several members of the peroxisome proliferator-activated receptor (PPAR) family. This assay was employed to compare the activation of PPAR family members by known PPAR activators including peroxisome proliferators and fatty acids. Interestingly, the activation of PPARs by fatty acids was partially inhibited by the cyclooxygenase inhibitor indomethacin, which prevents prostaglandin synthesis. Indeed, prostaglandins PGA1 and 2, PGD1 and 2, and PGJ2-activated PPARs, while a number of other prostaglandins had no effect. We also screened a variety of hydroxyeicosatetraenoic acids (HETEs) for the ability to activate PPARs. 8(S)-HETE, but not other (S)-HETEs, was a strong activator of PPAR alpha. Remarkably, PPAR activation by 8(S)-HETE was stereoselective. In addition, 8(S)-HETE was able to induce differentiation of 3T3-L1 preadipocytes. These results indicate that PPARs are differentially activated by naturally occurring eicosanoids and related molecules
— id: 27778, year: 1995, vol: 270, page: 23975, stat: Journal Article,

A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-Erb
Dumas B; Harding HP; Choi HS; Lehmann KA; Chung M; Lazar MA; Moore DD
1994 Aug;8(8):996-1005, Molecular endocrinology
We have isolated complementary DNA clones encoding a novel orphan member of the nuclear receptor superfamily, termed BD73. This protein shows strong amino acid sequence similarity to the previously described Rev-ErbA alpha. Unlike Rev-Erb, in which the opposite strand of the C-terminal coding region encodes the C-terminal portion of a variant thyroid hormone receptor isoform, the opposite strand of the C-terminal coding region of BD73 does not have any extensive open reading frames. BD73 messenger RNA is expressed in a wide variety of tissues and cell lines. In quiescent HepG2 cells, BD73 messenger RNA levels are strongly induced by planar aromatic antioxidants. Like Rev-Erb, BD73 binds as a monomer to a DNA sequence which consists of a specific A/T-rich sequence upstream of the consensus hexameric half-site specified by the P box of the DNA-binding domain. Amino acid sequence comparisons suggest that the A box sequence, which has been suggested to mediate monomer binding by other superfamily members, lies closer to the DNA-binding domain in BD73 and Rev-Erb than in other receptors. Under the conditions examined, neither BD73 nor Rev-Erb activated reporters containing multiple copies of their common binding site. Thus, these two orphans may require an as yet unidentified ligand or other signal for such activation. Together, BD73 and Rev-Erb define a subgroup of orphan receptors that bind as monomers to a half-site flanked by a specific and extended A/T-rich sequence
— id: 27780, year: 1994, vol: 8, page: 996, stat: Journal Article,

The orphan receptor Rev-ErbA alpha activates transcription via a novel response element
Harding HP; Lazar MA
1993 May;13(5):3113-3121, Molecular & cellular biology
Rev-ErbA alpha (Rev-Erb) is a nuclear hormone receptor-related protein encoded on the opposite strand of the alpha-thyroid hormone receptor (TR) gene. This unusual genomic arrangement may have a regulatory role, but the conservation of human and rodent Rev-Erb amino acid sequences suggests that the protein itself has an important function, potentially as a sequence-specific transcriptional regulator. However, despite its relationship to the TR, Rev-Erb bound poorly to TR binding sites. To determine its DNA-binding specificity in an unbiased manner, Rev-Erb was synthesized in Escherichia coli, purified, and used to select specific binding-sites from libraries of random double-stranded DNA sequences. We found that Rev-Erb binds to a unique site consisting of a specific 5-bp A/T-rich sequence adjacent to a TR half-site. Rev-Erb contacts this entire asymmetric 11-bp sequence, which is the longest nonrepetitive element specifically recognized by a member of the thyroid/steroid hormone receptor superfamily, and mutations in either the A/T-rich or TR half-site regions abolished specific binding. The binding specificity of wild-type Rev-Erb was nearly identical to that of C- and N-terminally truncated forms. This binding was not enhanced by retinoid X receptor, TR, or other nuclear proteins, none of which formed heterodimers with Rev-Erb. Rev-Erb also appeared to bind to the selected site as a monomer. Furthermore, Rev-Erb activates transcription through this binding site even in the absence of exogenous ligand.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 27781, year: 1993, vol: 13, page: 3113, stat: Journal Article,

Differential DNA binding by monomeric, homodimeric, and potentially heteromeric forms of the thyroid hormone receptor
Lazar MA; Berrodin TJ; Harding HP
1991 Oct;11(10):5005-5015, Molecular & cellular biology
Binding of the thyroid hormone receptor (TR) to thyroid hormone-responsive elements (TREs) is crucial for regulation of gene expression by thyroid hormone. The TR binds to each half-site of a palindromic TRE separately, as a monomer, or simultaneously, as a homodimer. In addition, the TR monomer interacts with a 42-kDa protein that may be responsible for an increase in the apparent size and stability of the TR-TRE complex after incubation with liver nuclear extract. The multiple DNA-binding forms of the TR contact the TRE differently but compete for binding in a dynamic equilibrium which is highly dependent on the relative concentrations of TR and nuclear protein. Thus, protein-protein interactions are likely to determine the context in which the TR binds to target genes and regulates the transcriptional response to thyroid hormone
— id: 27782, year: 1991, vol: 11, page: 5005, stat: Journal Article,

Postnatal stimulation of rat surfactant protein A synthesis by dexamethasone
Floros J; Phelps DS; Harding HP; Church S; Ware J
1989 Aug;257(2 Pt 1):L137-L143, American journal of physiology
The effects of postnatal dexamethasone treatment in vivo on the synthesis of surfactant protein A (SP-A) were examined at the protein and RNA levels. Rats ranging from 1 day old to adult were injected with 200 micrograms of dexamethasone/kg body wt or with vehicle alone and were killed 24 h after injection. One portion of the lung was metabolically labeled with [35S]methionine, the proteins immunoprecipitated using an antiserum to SP-A, and analyzed electrophoretically. Both newly synthesized intracellular and secreted SP-A levels were increased by dexamethasone, reaching averages of 2.3 and 4.5 times control values, respectively. Another portion of the lung tissue was used for RNA analysis. SP-A mRNA levels were also elevated an average of 1.4 times control values by hormone treatment. Dose-response experiments using 16-day-old pups showed that both total SP-A, as measured by enzyme-linked immunosorbent assay, and total SP-A mRNA levels were elevated with dexamethasone treatment, reaching maximal stimulation at 2 mg. We conclude that postnatal dexamethasone treatment in vivo results in increased levels of both newly synthesized SP-A and SP-A mRNA, suggesting that pretranslational events may in part contribute to this process
— id: 27783, year: 1989, vol: 257, page: L137, stat: Journal Article,

Immunohistochemical localization of a low molecular weight surfactant-associated protein in human lung
Phelps DS; Harding HP
1987 Nov;35(11):1339-1342, Journal of histochemistry & cytochemistry
We used an antiserum to a hydrophobic 6 KD surfactant-associated protein to localize this protein in human lung tissue. This antiserum does not crossreact with the 35 KD surfactant-associated protein. By light microscopy using the indirect immunoperoxidase technique, the protein appears to be localized within Type II alveolar epithelial cells. Staining is also detectable in alveolar macrophages and occasionally within the lumina of alveoli and bronchioles. No staining was detected within the alveolar septa or in association with blood vessels. An identical distribution is seen for the 35 KD surfactant-associated protein using an antiserum specific for that protein
— id: 27784, year: 1987, vol: 35, page: 1339, stat: Journal Article,