Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex

Copyright: Copyright 2008 Elsevier B.V., All rights reserved.Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated colocalization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.publishersversionPeer reviewe

ERp29 triggers a conformational change in polyomavirus to stimulate membrane binding

Funding Information: We thank Tom Rapoport and Kristen Verhey for critically reading the manuscript. B.T. is a Biological Scholar at the University of Michigan Medical School. E.K.R. is supported by a training grant from the National Science Foundation. S.M. was supported by the Swedish Medical Research Council and the Swedish Society of Medicine. M.B. received a scholarship from the Royal Swedish Academy. The work was supported in part by a grant to T.B. from the National Cancer Institute (CA 082395). Copyright: Copyright 2008 Elsevier B.V., All rights reserved.Membrane penetration of nonenveloped viruses is a poorly understood process. We have investigated early stages of this process by studying the conformational change experienced by polyomavirus (Py) in the lumen of the endoplasmic reticulum (ER), a step that precedes its transport into the cytosol. We show that a PDI-like protein, ERp29, exposes the C-terminal arm of Py's VP1 protein, leading to formation of a hydrophobic particle that binds to a lipid bilayer; this reaction likely mimics initiation of Py penetration across the ER membrane. Expression of a dominant-negative ERp29 decreases Py infection, indicating ERp29 facilitates viral infection. Interestingly, cholera toxin, another toxic agent that crosses the ER membrane into the cytosol, is unfolded by PDI in the ER. Our data thus identify an ER factor that mediates membrane penetration of a nonenveloped virus and suggest that PDI family members are generally involved in ER remodeling reactions.publishersversionPeer reviewe

Computational Methods for the Pharmacogenetic Interpretation of Next Generation Sequencing Data

Up to half of all patients do not respond to pharmacological treatment as intended. A substantial fraction of these inter-individual differences is due to heritable factors and a growing number of associations between genetic variations and drug response phenotypes have been identified. Importantly, the rapid progress in Next Generation Sequencing technologies in recent years unveiled the true complexity of the genetic landscape in pharmacogenes with tens of thousands of rare genetic variants. As each individual was found to harbor numerous such rare variants they are anticipated to be important contributors to the genetically encoded inter-individual variability in drug effects. The fundamental challenge however is their functional interpretation due to the sheer scale of the problem that renders systematic experimental characterization of these variants currently unfeasible. Here, we review concepts and important progress in the development of computational prediction methods that allow to evaluate the effect of amino acid sequence alterations in drug metabolizing enzymes and transporters. In addition, we discuss recent advances in the interpretation of functional effects of non-coding variants, such as variations in splice sites, regulatory regions and miRNA binding sites. We anticipate that these methodologies will provide a useful toolkit to facilitate the integration of the vast extent of rare genetic variability into drug response predictions in a precision medicine framework

Unfolded protein response is involved in the pathology of human congenital hypothyroid goiter and rat non-goitrous congenital hypothyroidism

Copyright: Copyright 2008 Elsevier B.V., All rights reserved.The unfolded protein response (UPR) is an intracellular signaling pathway that regulates the protein folding and processing capacity of the endoplasmic reticulum (ER). The UPR is induced by the pharmacological agents that perturb ER functions but is also activated upon excessive accumulation of the mutant secretory proteins that are unable to attain correct three-dimensional structure and are thus retained in the ER. Such defects in intracellular protein transport underlie the development of a number of phenotypically diverse inherited pathologies, termed endoplasmic reticulum storage diseases (ERSD). We have studied UPR development in two similar ERSDs, human congenital goiter caused by the C1264R and C1996S mutations in the thyroglobulin (Tg) gene and non-goitrous congenital hypothyroidism in rdw dwarf rats determined by the G2320R Tg mutation. In both cases, these mutations rendered Tg incapable of leaving the ER. A major ER chaperone immunoglobulin-binding protein (BiP), and a novel putative escort chaperone endoplasmic reticulum protein 29 KDa (ERp29) were found to be associated with Tg, which might be interpreted as the contribution of the quality control machinery to the previously shown retention of Tg in the ER. We have extended our earlier observations of ER chaperone induction with the identification of the additional ER (ERp29, ERp72, calreticulin, protein disulfide isomerase (PDI)), cytoplasmic (heat shock protein (HSP)70, HSP90) and mitochondrial (mtHSP70) upregulated chaperones and folding enzymes. Activation of the transcriptional arm of UPR, as judged by the appearance of the spliced (active) form of X-box binding protein (XBP1) and processed activating transcription factor 6 (ATF6) transcription factors was suggested to contribute to the overexpression of the ER chaperones. The processing of ATF6 was observed in both human and rat tissues with Tg mutations. Whereas, in human tissues, weak splicing of XBP1 mRNA was detected only in the C1264R mutant, all rat thyroids including wild-type contained significant amounts of the spliced form of XBP1 as opposed to human liver and rat brain tissues, implying the existence of a previously unknown tissue-specific regulation of XBP1 processing.publishersversionPeer reviewe

Calcium Signaling in Liver Injury and Regeneration

The liver fulfills central roles in metabolic control and detoxification and, as such, is continuously exposed to a plethora of insults. Importantly, the liver has a unique ability to regenerate and can completely recoup from most acute, non-iterative insults. However, multiple conditions, including viral hepatitis, non-alcoholic fatty liver disease (NAFLD), long-term alcohol abuse and chronic use of certain medications, can cause persistent injury in which the regenerative capacity eventually becomes dysfunctional, resulting in hepatic scaring and cirrhosis. Calcium is a versatile secondary messenger that regulates multiple hepatic functions, including lipid and carbohydrate metabolism, as well as bile secretion and choleresis. Accordingly, dysregulation of calcium signaling is a hallmark of both acute and chronic liver diseases. In addition, recent research implicates calcium transients as essential components of liver regeneration. In this review, we provide a comprehensive overview of the role of calcium signaling in liver health and disease and discuss the importance of calcium in the orchestration of the ensuing regenerative response. Furthermore, we highlight similarities and differences in spatiotemporal calcium regulation between liver insults of different etiologies. Finally, we discuss intracellular calcium control as an emerging therapeutic target for liver injury and summarize recent clinical findings of calcium modulation for the treatment of ischemic-reperfusion injury, cholestasis and NAFLD

Neuroinflammatory markers associate with cognitive decline after major surgery:Findings of an explorative study

OBJECTIVE Long‐term cognitive decline is an adverse outcome after major surgery associated with increased risk for mortality and morbidity. We studied the cerebrospinal fluid (CSF) and serum biochemical inflammatory response to a standardized orthopedic surgical procedure and the possible association with long‐term changes in cognitive function. We hypothesized that the CSF inflammatory response pattern after surgery would differ in patients having long‐term cognitive decline defined as a composite cognitive z score of ≥1.0 compared to patients without long‐term cognitive decline at 3 months postsurgery. METHODS Serum and CSF biomarkers of inflammation and blood–brain barrier (BBB) integrity were measured preoperatively and up to 48 hours postoperatively, and cognitive function was assessed preoperatively and at 2 to 5 days and 3 months postoperatively. RESULTS Surgery was associated with a pronounced increase in inflammatory biomarkers in both CSF and blood throughout the 48‐hour study period. A principal component (PC) analysis was performed on 52 inflammatory biomarkers. The 2 first PC (PC1 and PC2) construct outcome variables on CSF biomarkers were significantly associated with long‐term cognitive decline at 3 months, but none of the PC construct serum variables showed a significant association with long‐term cognitive decline at 3 months. Patients both with and patients without long‐term cognitive decline showed early transient increases of the astroglial biomarkers S‐100B and glial fibrillary acidic protein in CSF, and in BBB permeability (CSF/serum albumin ratio). INTERPRETATION Surgery rapidly triggers a temporal neuroinflammatory response closely associated with long‐term cognitive outcome postsurgery. The findings of this explorative study require validation in a larger surgical patient cohort. ANN NEUROL 202

Oligomerization properties of ERp29, an endoplasmic reticulum stress protein

Funding Information: This work was supported by the Swedish Medical Research Council. We thank Prof. H.F. Gilbert for helpful discussions and critical reading of the manuscript. Copyright: Copyright 2007 Elsevier B.V., All rights reserved. Correction(s) for this article: Oligomerization properties of ERp29, an endoplasmic reticulum stress protein. FEBS Letters,Vol.433, N.3, p.335-335. - First Published online: June 28, 1999.ERp29, a novel and ubiquitously expressed endoplasmic reticulum (ER) stress-inducible protein, was recently isolated and cDNA cloned in our laboratory. Using size exclusion chromatography and chemical cross-linking we have assessed the oligomerization properties of ERp29. Purified ERp29 in solution as well as in rat hepatoma cells self-associates predominantly into homodimers. Labeling of the cells with [35S]methionine with subsequent cross-linking and immunprecipitation showed that ERp29 interacts with a number of ER proteins, one of which was previously identified as BiP/GRP78. Secondary structure prediction and fold recognition methods indicate that the native conformation of ERp29 resembles the thioredoxin fold, a structural motif characteristic of a number of enzymes with the redox function, including protein disulfide isomerase (with which ERp29 shares limited sequence similarity). Dimerization of the protein is suggested to be advantageous for the protein binding potential of ERp29.publishersversionPeer reviewe

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI

The human carotid body transcriptome with focus on oxygen sensing and inflammation-a comparative analysis

et al.The carotid body (CB) is the key oxygen sensing organ. While the expression of CB specific genes is relatively well studied in animals, corresponding data for the human CB are missing. In this study we used five surgically removed human CBs to characterize the CB transcriptome with microarray and PCR analyses, and compared the results with mice data. In silico approaches demonstrated a unique gene expression profile of the human and mouse CB transcriptomes and an unexpected upregulation of both human and mouse CB genes involved in the inflammatory response compared to brain and adrenal gland data. Human CBs express most of the genes previously proposed to be involved in oxygen sensing and signalling based on animal studies, including NOX2, AMPK, CSE and oxygen sensitive K+ channels. In the TASK subfamily of K+ channels, TASK-1 is expressed in human CBs, while TASK-3 and TASK-5 are absent, although we demonstrated both TASK-1 and TASK-3 in one of the mouse reference strains. Maxi-K was expressed exclusively as the spliced variant ZERO in the human CB. In summary, the human CB transcriptome shares important features with the mouse CB, but also differs significantly in the expression of a number of CB chemosensory genes. This study provides key information for future functional investigations on the human carotid body. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.This work was supported by grants from the Swedish Research Council (No. 521-2011-152 (L.I.E.)), the Stockholm County Council (ALF project 20090321 (L.I.E.)), Post-Doc project 108042 (M.J.F.), Karolinska Foundations (M.J.F., L.I.E.), The Swedish Society of Medicine (L.I.E.), LPS Medical Foundation (M.J.F.), and Fraenkel Foundation (M.J.F., L.I.E.). C.G. is supported by the Ministerio de Ciencia e Innovación of Spain, grant no. BFU2007-61848, and by the Instituto Carlos III, grant no. CIBER CB06/06/0050. D.S. is supported by the MICINN grant BFU2008-01170. E.W.K. and M.S. are supported by NHLBI HL81345 of the USA.Peer Reviewe

The C-Terminal Domain of ERp29 Mediates Polyomavirus Binding, Unfolding, and Infection▿

Penetration of the endoplasmic reticulum (ER) membrane by polyomavirus (PyV) is a decisive step in virus entry. We showed previously that the ER-resident factor ERp29 induces the local unfolding of PyV to initiate the ER membrane penetration process. ERp29 contains an N-terminal thioredoxin domain (NTD) that mediates its dimerization and a novel C-terminal all-helical domain (CTD) whose function is unclear. The NTD-mediated dimerization of ERp29 is critical for its unfolding activity; whether the CTD plays any role in PyV unfolding is unknown. We now show that three hydrophobic residues within the last helix of the ERp29 CTD that were individually mutated to either lysine or alanine abolished ERp29's ability to stimulate PyV unfolding and infection. This effect was not due to global misfolding of the mutant proteins, as they dimerize and do not form aggregates or display increased protease sensitivity. Moreover, the mutant proteins stimulated secretion of the secretory protein thyroglobulin with an efficiency similar to that of wild-type ERp29. Using a cross-linking coimmunoprecipitation assay, we found that the physical interaction of the ERp29 CTD mutants with PyV is inefficient. Our data thus demonstrate that the ERp29 CTD plays a crucial role in PyV unfolding and infection, likely by serving as part of a substrate-binding domain