Joanna Jung

ERp57 Modulates STAT3 Signaling from the Lumen of the Endoplasmic Reticulum

ERp57 is an endoplasmic reticulum (ER) resident thiol disulfide oxidoreductase. Using the gene trap technique, we created a ERp57-deficient mouse model. Targeted deletion of the Pdia3 gene, which encodes ERp57, in mice is embryonic lethal at embryonic day (E) 13.5. β-Galactosidase reporter gene analysis revealed that ERp57 is expressed early on during blastocyst formation with the highest expression in the inner cell mass. In early stages of mouse embryonic development (E11.5) there is a relatively low level of expression of ERp57. As the embryos developed, ERp57 became highly expressed in both the brain and the lungs (E15.5 and E18.5). The absence of ERp57 has no impact on ER morphology; expression of ER-associated chaperones and folding enzymes, ER stress, or apoptosis. ERp57 has been reported to interact with STAT3 (signal transducer and activator of transcription)-DNA complexes. We show here that STAT3-dependent signaling is increased in the absence of ERp57 and this can be rescued by expression of ER-targeted ERp57 but not by cytoplasmic-targeted protein, indicating that ERp57 affects STAT3 signaling from the lumen of the ER. ERp57 effects on STAT3 signaling are enhanced by ER luminal complex formation between ERp57 and calreticulin. In conclusion, we show that ERp57 deficiency in mouse is embryonic lethal at E13.5 and ERp57-dependent modulation of STAT3 signaling may contribute to this phenotype.

Functional characterization of Arabidopsis Calreticulin1a: a key alleviator of endoplasmic reticulum stress

The chaperone calreticulin plays important roles in a variety of processes in the endoplasmic reticulum (ER) of animal cells, such as Ca2+ signaling and protein folding. Although the functions of calreticulin are well characterized in animals, only indirect evidence is available for plants. To increase our understanding of plant calreticulins we introduced one of the Arabidopsis isoforms, AtCRT1a, into calreticulin-deficient (crt-/-) mouse embryonic fibroblasts. As a result of calreticulin deficiency, the mouse crt-/- fibroblasts have decreased levels of Ca2+ in the ER and impaired protein folding abilities. Expression of the AtCRT1a in mouse crt-/- fibroblasts rescued these phenotypes, i.e. AtCRT1a restored the Ca2+-holding capacity and chaperone functions in the ER of the mouse crt-/- fibroblasts, demonstrating that the animal sorting machinery was also functional for a plant protein, and that basic calreticulin functions are conserved across the Kingdoms. Expression analyses using a beta-glucuronidase (GUS)-AtCRT1a promoter construct revealed high expression of CRT1a in root tips, floral tissues and in association with vascular bundles. To assess the impact of AtCRT1a in planta, we generated Atcrt1a mutant plants. The Atcrt1a mutants exhibited increased sensitivity to the drug tunicamycin, an inducer of the unfolded protein response. We therefore conclude that AtCRT1a is an alleviator of the tunicamycin-induced unfolded protein response, and propose that the use of the mouse crt-/- fibroblasts as a calreticulin expression system may prove useful to assess functionalities of calreticulins from different species.

Endoplasmic reticulum stress in the absence of calnexin

Calnexin is a type I integral endoplasmic reticulum (ER) membrane chaperone involved in folding of newly synthesized (glycol)proteins. In this study, we used β-galactosidase reporter gene knock-in and reverse transcriptase polymerase chain reaction (RT-PCR) to investigate activation of the calnexin gene during embryonic development. We showed that the calnexin gene was activated in neuronal tissue at the early stages of embryonic development but remained low in the heart, intestine, and smooth muscle. At early stages of embryonic development, large quantities of calnexin messenger RNA (mRNA) were also found in neuronal tissue and liver. There was no detectable calnexin mRNA in the heart, lung, and intestine. The absence of calnexin had no significant effect on ER stress response (unfolded protein response, UPR) at the tissue level as tested by IRE1-dependent splicing of Xbp1 mRNA. In contrast, non-stimulated calnexin-deficient cells showed increased activation of IRE1, as measured by RT-PCR and luciferase reporter gene analysis of splicing of Xbp1 mRNA and activation of the BiP promoter. This indicates that cnx−/− cells have increased constitutively active UPR. Importantly, cnx−/− cells have significantly increased proteasomal activity, which may play a role in the adaptive mechanisms addressing the acute ER stress observed in the absence of calnexin.

The many functions of the endoplasmic reticulum chaperones and folding enzymes.

Endoplasmic reticulum (ER) is an essential sub‐cellular compartment of the eukaryotic cell performing many diverse functions essential for the cell and the whole organism. ER molecular chaperones and folding enzymes are multidomain proteins that are designed to support nascent proteins entering ER lumen to achieve their native conformation, mediate post‐translational modification, prevent misfolded protein aggregation, and facilitate exit from the ER. Typically the role of ER chaperones expands beyond protein folding. Here, we illustrate the multifunctional nature of many ER associated molecular chaperones and folding enzymes and unique functional overlap of these proteins all designed to support the many functions of the ER membrane.

Specialization of endoplasmic reticulum chaperones for the folding and function of myelin glycoproteins P0 and PMP22

Peripheral myelin protein 22 (PMP22) and protein 0 (P0) are major peripheral myelin glycoproteins, and mutations in these two proteins are associated with hereditary demyelinating peripheral neuropathies. Calnexin, calreticulin, and ERp57 are critical components of protein quality control responsible for proper folding of newly synthesized glycoproteins. Here, using confocal microscopy, we show that cell surface targeting of P0 and PMP22 is not affected in the absence of the endoplasmic reticulum chaperones. However, the folding and function (adhesiveness) of PMP22 and P0, measured using the adhesion assay, are affected significantly in the absence of calnexin but not in the absence of calreticulin. Deficiency in oxidoreductase ERp57 results in impaired folding and function of P0, a disulfide bond‐containing protein, but does not have any effect on folding or function of PMP22 (a protein that does not contain a disulfide bond). We concluded that calnexin and ERp57, but not calreticulin, play an important role in the biology of peripheral myelin proteins PMP22 and P0, and, consequently, these chaperones may contribute to the pathogenesis of peripheral neuropathies and the diversity of these neurological disorders.—Jung, J., Coe, H., Michalak, M. Specialization of endoplasmic reticulum chaperones for the folding and function of myelin glycoproteins P0 and PMP22. FASEB J. 25, 3929–3937 (2011). www.fasebj.org

The role of N-glycan in folding, trafficking and pathogenicity of myelin oligodendrocyte glycoprotein (MOG).

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane protein that is expressed in the central nervous system. MOG has a single N-glycosylation site within its extracellular domain. MOG has been linked with pathogenesis of multiple sclerosis; anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. N-glycosylation is an important post-translational modification of protein that might impact their folding, localization and function. However, the role of sugar in the biology of MOG is not well understood. In this study, we created a mutant MOG lacking N-linked glycan and tested its properties. We concluded that the lack of sugar did not impact on MOG abundance in the absence of endoplasmic reticulum molecular chaperone calnexin. We also show that the absence of N-glycan did not interfere with MOGs subcellular localization and it did not result in activation of endoplasmic reticulum stress. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Cell surface targeting of myelin oligodendrocyte glycoprotein (MOG) in the absence of endoplasmic reticulum molecular chaperones.

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane glycoprotein that localizes to myelin sheaths in the central nervous system. MOG has important implications in multiple sclerosis, as pathogenic anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. As a membrane protein, MOG achieves its native structure in the endoplasmic reticulum where its folding is expected to be controlled by endoplasmic reticulum chaperones. Calnexin, calreticulin, and ERp57 are essential components of the endoplasmic reticulum quality control where they assist in the proper folding of newly synthesized glycoproteins. In this study, we show that expression of MOG is not affected by the absence of the endoplasmic reticulum quality control proteins calnexin, calreticulin, or ERp57. We also show that calnexin forms complexes with MOG and these interactions might be glycan-independent. Importantly, we show that cell surface targeting of MOG is not disrupted in the absence of the endoplasmic reticulum chaperones. This article is part of a special issue entitled: 11th European Symposium on Calcium.

Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis.

Background Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR) pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function. Methodology/Principal Findings We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER) homeostasis, transient activation of the unfolded protein response (UPR) pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA) is sufficient to prevent cardiac fibrosis, and improved exercise tolerance. Conclusions We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function.

Endoplasmic Reticulum Malfunction in the Nervous System

Neurodegenerative diseases often have multifactorial causes and are progressive diseases. Some are inherited while others are acquired, and both vary greatly in onset and severity. Impaired endoplasmic reticulum (ER) proteostasis, involving Ca2+ signaling, protein synthesis, processing, trafficking, and degradation, is now recognized as a key risk factor in the pathogenesis of neurological disorders. Lipidostasis involves lipid synthesis, quality control, membrane assembly as well as sequestration of excess lipids or degradation of damaged lipids. Proteostasis and lipidostasis are maintained by interconnected pathways within the cellular reticular network, which includes the ER and Ca2+ signaling. Importantly, lipidostasis is important in the maintenance of membranes and luminal environment that enable optimal protein processing. Accumulating evidence suggest that the loss of coordinate regulation of proteostasis and lipidostasis has a direct and negative impact on the health of the nervous system.

Role of cysteine amino acid residues in calnexin.

Calnexin is an endoplasmic reticulum protein that has a role in folding newly synthesized glycoproteins. In this study, we used site-specific mutagenesis to disrupt cysteine and histidine amino acid residues in the N- and P-domains of calnexin and determined whether these mutations impact the structure and function of calnexin. We identified that disruption of the N-domain cysteines resulted in significant loss of the chaperone activity of calnexin toward the glycosylated substrate, IgY, while disruption of the P-domain cysteines only had a small impact toward IgY. We observed that wild-type calnexin as well as the P-domain double cysteine mutant contained an intramolecular disulfide bond which is lost when the N-domain cysteines are mutated. Mutation to the N-domain histidine and N-domain cysteines resulted in increased binding of ERp57. Mutations to the P-domain cysteines further enhanced ERp57 binding to calnexin. Taken together, these observations indicated that the cysteine residues within calnexin were important for the structure and function of calnexin.