Martin Schröder

Endoplasmic reticulum stress responses.

Abstract.In homeostasis, cellular processes are in a dynamic equilibrium. Perturbation of homeostasis causes stress. In this review I summarize how perturbation of three major functions of the endoplasmic reticulum (ER) in eukaryotic cells–protein folding, lipid and sterol biosynthesis, and storing intracellular Ca2+ – causes ER stress and activates signaling pathways collectively termed the unfolded protein response (UPR). I discuss how the UPR reestablishes homeostasis, and summarize our current understanding of how the transition from protective to apoptotic UPR signaling is controlled, and how the UPR induces inflammatory signaling.

Ligand-independent Dimerization Activates the Stress Response Kinases IRE1 and PERK in the Lumen of the Endoplasmic Reticulum

IRE1 and PERK are type I transmembrane serine/threonine protein kinases that are activated by unfolded proteins in the endoplasmic reticulum (ER) to signal adaptive responses. IRE1 is present in all eukaryotic cells and signals the unfolded protein response through its kinase and endoribonuclease activities. PERK signals phosphorylation of a translation initiation factor to inhibit protein synthesis in higher eukaryotic cells but is absent in the Saccharomyces cerevisiae genome. The amino acid sequences of the amino-terminal ER luminal domains (NLDs) from IRE1 and PERK display limited homology and have diverged among species. In this study, we have demonstrated that the NLD of yeast Ire1p is required for signaling. However, the NLDs from human IRE1α and murine IRE1β and the Caenorhabditis elegans IRE1 and PERK function as replacements for the S. cerevisiae Ire1p-NLD to signal the unfolded protein response. Replacement of the Ire1p-NLD with a functional leucine zipper dimerization motif yielded a constitutively active kinase that surprisingly was further activated by ER stress. These results demonstrate that ER stress-induced dimerization of the NLD is sufficient for IRE1 and PERK activation and is conserved through evolution. We propose that ligand-independent activation of IRE1 and PERK permits homodimerization upon accumulation of unfolded proteins in the lumen of the ER.

Divergent Roles of IRE1α and PERK in the Unfolded Protein Response

The endoplasmic reticulum (ER) provides unique machinery for the folding and posttranslational modification of many secretory and transmembrane proteins in eukaryotic cells. The unfolded protein response (UPR) is a signal transduction network from the ER to the nucleus activated when the folding demand imposed by nascent, unfolded polypeptide chains exceeds the capacity of the ER protein folding machinery. In all eukaryotes the UPR maintains the physiological balance between folding demand and capacity of the ER by regulating adaptive responses to this stress situation. These include an increase in the folding capacity of the ER through induction of ER resident molecular chaperones and protein foldases, and a decrease in the folding demand on the ER by upregulation of ER associated degradation (ERAD), attenuation of general translation in metazoans, and stimulation of ER synthesis to dilute the unfolded protein load. In higher eukaryotes the UPR gained control over inflammatory and immune responses by controlling the activity of the transcription factor NF-kappaB to combat viral infections associated with an increased synthesis of viral glycoproteins. Similarly, in multicellular organisms apoptotic programs are controlled by the UPR to eliminate cells whose folding problems in the ER cannot be resolved by coordinated regulation of adaptive, inflammatory, and immune responses. In this review we will summarize our current understanding of signal transduction mechanisms involved in the mammalian UPR, and discuss examples to highlight the regulation of adaptive, inflammatory, immune, and apoptotic responses by the UPR.

The Unfolded Protein Response

The unfolded protein response (UPR) is a signal transduction network activated by inhibition of protein folding in the endoplasmic reticulum (ER). The UPR coordinates adaptive responses to this stress situation, including induction of ER resident molecular chaperone and protein foldase expression to increase the protein folding capacity of the ER, induction of phospholipid synthesis, attenuation of general translation, and upregulation of ER-associated degradation to decrease the unfolded protein load of the ER, and an antioxidant response. Upon severe or prolonged ER stress the UPR induces apoptosis to eliminate unhealthy cells from an organism or a population. In this review, I will summarize our current knowledge about signal transduction pathways involved in transducing the unfolded protein signal from the ER to the nucleus or the cytosol.

Sensing Endoplasmic Reticulum Stress

This chapter provides an overview of our present understanding of mechanisms of sensing protein folding status and endoplasmic reticulum (ER) stress in eukaryotic cells. The ER folds and matures most secretory and transmembrane proteins. Mis- or unfolded proteins are sensed by specialized ER stress sensors, such as IRE1, PERK and ATF6, which initiate several cellular responses and signaling pathways to restore ER homeostasis. These intracellular signaling events are called the unfolded protein response (UPR). Here we focus on how ER stress and protein folding status in the ER are sensed by the ER stress sensors by summarizing results from recent structural, biochemical and genetic approaches.

Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Overexpression of recombinant proteins in animal cells is commonly achieved by using gene amplification techniques. Gene amplified cells possess up to several thousand genes coding for the target protein. Constitutive expression of these genes leads to high levels of the corresponding mRNA species and the immature protein in the cell. Inefficient processing of these precursors may result from their great abundance in the cell. To study the influence of elevated intracellular levels of a recombinant protein on its maturation and secretion, we examined the maturation and secretion of human antithrombin III (hATIII) in Chinese hamster ovary (CHO) cells at different levels of gene amplification. No loss of vitality was caused by elevated secretion of hATIII. As the intracellular hATIII content increased, the efficiency of hATIII secretion decreased steadily. The state of intracellular hATIII from the different cell lines was studied by determining the specific heparin cofactor activity of hATIII. Intracellular hATIII from the highest amplified cell line displayed a lowered specific heparin cofactor activity indicating the presence of malfolded, only partially folded, or incompletely or incorrectly posttranslationally modified hATIII in this cell line. Thus, the ability of CHO cells to fold and/or introduce posttranslational modifications and subsequently to secrete the recombinant protein becomes saturated, and therefore these processes may become limiting for protein secretion at highly elevated expression levels. This limitation was not due to a general exhaustion of the secretory capacity of the cells because hATIII constituted only a minor fraction of the secreted proteins, even at high expression levels.

The unfolded protein response represses differentiation through the RPD3-SIN3 histone deacetylase

In Saccharomyces cerevisiae, splicing of HAC1 mRNA is initiated in response to the accumulation of unfolded proteins in the endoplasmic reticulum by the transmembrane kinase‐endoribonuclease Ire1p. Spliced Hac1p (Hac1ip) is a negative regulator of differentiation responses to nitrogen starvation, pseudohyphal growth, and meiosis. Here we show that the RPD3‐SIN3 histone deacetylase complex (HDAC), its catalytic activity, recruitment of the HDAC to the promoters of early meiotic genes (EMGs) by Ume6p, and the Ume6p DNA‐binding site URS1 in the promoters of EMGs are required for nitrogen‐mediated negative regulation of EMGs and meiosis by Hac1ip. Co‐immunoprecipitation experiments demonstrated that Hac1ip can interact with the HDAC in vivo. Systematic analysis of double deletion strains revealed that HAC1 is a peripheral component of the HDAC. In summary, nitrogen‐induced synthesis of Hac1ip and association of Hac1ip with the HDAC are physiological events in the regulation of EMGs by nutrients. These data also define for the first time a gene class that is under negative control by the UPR, and provide the framework for a novel mechanism through which bZIP proteins repress transcription.

IRE1- and HAC1 -independent transcriptional regulation in the unfolded protein response of yeast

The unfolded protein response (UPR) is a signalling pathway leading to transcriptional activation of genes that protect cells from accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). In yeast, the only known ER stress signalling pathway originates at the type I transmembrane protein kinase/endoribonuclease Ire1p. Ire1p regulates synthesis of the basic leucine‐zipper (bZIP)‐containing transcription factor Hac1p by controlling splicing of HAC1 mRNA. Only spliced HAC1 mRNA (HAC1i) is translated, and Hac1ip activates transcription of genes that contain a conserved UPR element (UPRE) in their promoters. Here, we demonstrate that in addition to this well‐understood ER stress signalling pathway, a second, IRE1, HAC1 and UPRE‐independent mechanism for transcriptional activation upon ER stress, exists in yeast. A genetic screen identified recessive SIN4 alleles as suppressors of a defective UPR in ire1Δ strains. Elevation of basal transcription in sin4 strains or by tethering the RNA polymerase II holoenzyme with LexAp‐holoenzyme component fusion proteins to a promoter allowed for activation of the promoter by ER stress in an IRE1, HAC1 and UPRE‐independent manner. We propose that this novel second ER‐to‐nucleus signal transduction pathway culminates in core promoter activation (CPA) through stimulation of RNA polymerase II holoenzyme activity. Core promoter activation was observed upon diverse cellular stresses, suggesting it represents a primordial stress‐induced gene activation mechanism.

Ime1 and Ime2 Are Required for Pseudohyphal Growth of Saccharomyces cerevisiae on Nonfermentable Carbon Sources

ABSTRACT Pseudohyphal growth and meiosis are two differentiation responses to nitrogen starvation of diploid Saccharomyces cerevisiae. Nitrogen starvation in the presence of fermentable carbon sources is thought to induce pseudohyphal growth, whereas nitrogen and sugar starvation induces meiosis. In contrast to the genetic background routinely used to study pseudohyphal growth (Σ1278b), nonfermentable carbon sources stimulate pseudohyphal growth in the efficiently sporulating strain SK1. Pseudohyphal SK1 cells can exit pseudohyphal growth to complete meiosis. Two stimulators of meiosis, Ime1 and Ime2, are required for pseudohyphal growth of SK1 cells in the presence of nonfermentable carbon sources. Epistasis analysis suggests that Ime1 and Ime2 act in the same order in pseudohyphal growth as in meiosis. The different behaviors of strains SK1 and Σ1278b are in part attributable to differences in cyclic AMP (cAMP) signaling. In contrast to Σ1278b cells, hyperactivation of cAMP signaling using constitutively active Ras2G19V inhibited pseudohyphal growth in SK1 cells. Our data identify the SK1 genetic background as an alternative genetic background for the study of pseudohyphal growth and suggest an overlap between signaling pathways controlling pseudohyphal growth and meiosis. Based on these findings, we propose to include exit from pseudohyphal growth and entry into meiosis in the life cycle of S. cerevisiae.

Engineering of chaperone systems and of the unfolded protein response

Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR.