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Role and regulation of luminal disulfide switches in calcium signaling from the endoplasmic reticulum

English title Role and regulation of luminal disulfide switches in calcium signaling from the endoplasmic reticulum
Applicant Appenzeller-Herzog Christian
Number 126625
Funding scheme Ambizione
Research institution Institut für Molekulare und Systemische Toxikologie Departement Pharmazeutische Wissenschaften
Institution of higher education University of Basel - BS
Main discipline Cellular Biology, Cytology
Start/End 01.10.2009 - 30.09.2012
Approved amount 609'154.00
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All Disciplines (3)

Discipline
Cellular Biology, Cytology
Biochemistry
Molecular Biology

Keywords (10)

Endoplasmic Reticulum; Calcium; Disulfide Bond; Oxidative Stress; Apoptosis; mitochondrium; cysteine; endoplasmic reticulum stress; SERCA; IP3 receptor

Lay Summary (English)

Lead
Lay summary
Intracellular synthesis of secreted and cell surface proteins takes place in a membraneous network called the endoplasmic reticulum (ER). The physiological condition of deregulated homeostasis in the lumen of the ER is commonly referred to as ER stress. It is now increasingly recognized that ER stress-evoked calcium transmission from the ER to another membrane-bound cell organelle, the mitochondrium, can impart and amplify a cell death signal. This process involves a complicated network of signaling cascades. In most non-muscle tissues, calcium signals emanating from the ER depend on calcium pumping into the ER by sarco(endo)plasmic reticulum calcium ATPase isoform 2b (SERCA2b) and calcium release through inositol 1,4,5-trosphosphate receptor (IP3R) channel proteins. Here, we hypothesize a regulatory mechanism that integrates oxidative stress in the ER into the propagation of death signaling pathways via redox-dependent modulation of SERCA2b and/or IP3R activity. Both of these ER-resident multispanning membrane proteins comprise a conserved pair of luminal cysteines. To investigate if ER environment-dependent thiol-disulfide conversion of these cysteines may represent a regulatory redox switch, we will establish a quantitative procedure to determine their in vivo redox state. Because both SERCA2b and IP3Rs are very large proteins comprising many cysteines, different strategies to trim the polypeptides and isolate the fragments harboring the luminal peptides will be applied. In a next step, we will search for conditions that impact the in situ thiol-disulfide distribution of the presumable switch cysteines. To achieve this, we will study their redox modulation induced both by over-expression or knockdown of selected ER proteins and by treatment of the cells with different concentrations of reductants and oxidants as well as with general inducers of ER stress. Having established conditions of most specific SERCA2b and IP3R thiol-disulfide manipulation, we will test the functionality of the redox switches by analyzing the activity of SERCA2b and IP3Rs in treated versus control cells. In parallel experiments, the mechanistic aspect of redox switch catalysis will be investigated by hunting mixed-disulfide interaction partners of the SERCA2b and IP3Rs luminal cysteines. Overall, the anticipated results are expected to enable a more profound comprehension of the processes that determine cell viability during physiological situations of stress. Such comprehension is of crucial importance and will aid the targeted treatment of devastating diseases such as neurodegenerative disorders and cancer.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Suppression of the Nrf2-dependent antioxidant response by glucocorticoids and 11β-HSD1-mediated glucocorticoid activation in hepatic cells.
Kratschmar Denise V, Calabrese Diego, Walsh Jo, Lister Adam, Birk Julia, Appenzeller-Herzog Christian, Moulin Pierre, Goldring Chris E, Odermatt Alex (2012), Suppression of the Nrf2-dependent antioxidant response by glucocorticoids and 11β-HSD1-mediated glucocorticoid activation in hepatic cells., in PloS one, 7(5), 36774-36774.
Hyperactivity of the Ero1α oxidase elicits endoplasmic reticulum stress but no broad antioxidant response.
Hansen Henning Gram, Schmidt Jonas Damgaard, Soeltoft Cecilie Lutzen, Ramming Thomas, Geertz-Hansen Henrik Marcus, Christensen Brian, Sorensen Esben Skipper, Juncker Agnieszka Sierakowska, Appenzeller-Herzog Christian, Ellgaard Lars (2012), Hyperactivity of the Ero1α oxidase elicits endoplasmic reticulum stress but no broad antioxidant response., in The Journal of biological chemistry, in press(in press), in press-in press.
Bidirectional crosstalk between endoplasmic reticulum stress and mTOR signaling.
Appenzeller-Herzog Christian, Hall Michael N (2012), Bidirectional crosstalk between endoplasmic reticulum stress and mTOR signaling., in Trends in cell biology, 22(5), 274-82.
The physiological functions of mammalian endoplasmic oxidoreductin 1: on disulfides and more.
Ramming Thomas, Appenzeller-Herzog Christian (2012), The physiological functions of mammalian endoplasmic oxidoreductin 1: on disulfides and more., in Antioxidants & redox signaling, 16(10), 1109-18.
Updates on "endoplasmic reticulum redox".
Appenzeller-Herzog Christian (2012), Updates on "endoplasmic reticulum redox"., in Antioxidants & redox signaling, 16(8), 760-2.
Glutathione- and non-glutathione-based oxidant control in the endoplasmic reticulum.
Appenzeller-Herzog Christian (2011), Glutathione- and non-glutathione-based oxidant control in the endoplasmic reticulum., in Journal of cell science, 124(Pt 6), 847-55.
Identification of the PDI-Family Member ERp90 as an Interaction Partner of ERFAD
Riemer J, Hansen HG, Appenzeller-Herzog C, Johansson L, Ellgaard L (2011), Identification of the PDI-Family Member ERp90 as an Interaction Partner of ERFAD, in PLOS ONE, 6(2), e17037-e17037.
Disulphide production by Ero1α-PDI relay is rapid and effectively regulated.
Appenzeller-Herzog Christian, Riemer Jan, Zito Ester, Chin King-Tung, Ron David, Spiess Martin, Ellgaard Lars (2010), Disulphide production by Ero1α-PDI relay is rapid and effectively regulated., in The EMBO journal, 29(19), 3318-29.

Collaboration

Group / person Country
Types of collaboration
University of Michigan United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
University of Utrecht Netherlands (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
EMBO conference: The physiology of the Endoplasmic Reticulum (ER): Function and Dysfunction 15.10.2012 Girona, Spain


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
DKFZ Redox Seminar 14.12.2011 Heidelberg, Germany


Associated projects

Number Title Start Funding scheme
133859 Purchase of a laser scanning microscope / LSM710 Carl Zeiss 01.12.2010 R'EQUIP
142964 Redox processes in the mammalian endoplasmic reticulum: Sensors, mechanisms, and consequences 01.10.2012 Ambizione
113072 Redox regulation in the endoplasmic reticulum of human cells 01.10.2006 Fellowships for advanced researchers

Abstract

The physiological condition of deregulated endoplasmic reticulum (ER) homeostasis is commonly referred to as ER stress. It is now increasingly recognized that ER stress-evoked calcium transmission from the ER to mitochondria can impart and amplify an apoptotic signal which plays a vital role in a host of human pathologies ranging from neurodegenerative diseases to obesity and diabetes. This process involves a complicated network of signaling cascades that is regulated at several stages. In most non-muscle tissues, calcium signals emanating from the ER depend on calcium pumping into the ER by sarco(endo)plasmic reticulum calcium ATPase isoform 2b (SERCA2b) and calcium release through inositol 1,4,5-trisphosphate receptors (IP3Rs). Here, we hypothesize a regulatory mechanism that integrates oxidative stress in the ER into the propagation of death signaling pathways via redox-dependent modulation of SERCA2b and/or IP3R activity. Both of these ER-resident multispanning membrane proteins comprise a conserved pair of cysteine residues in a luminal peptide loop. To investigate if ER environment-dependent thiol-disulfide conversion of these cysteines may represent a regulatory redox switch we will - based on the differential alkylation of thiols and disulfides - establish a quantitative procedure to determine their in vivo redox state. Because both SERCA2b and IP3Rs are very large proteins comprising many cysteines, different strategies to trim the polypeptides and isolate the fragments harboring the luminal loops in question will be applied. In a next step, we will search for conditions that impact the in situ thiol-disulfide distribution of the presumable switch cysteines as specifically as possible. To achieve this, we will study their redox modulation induced both by over-expression or knockdown of selected ER proteins and by treatment of the cells with different concentrations of reductants and oxidants as well as with general inducers of ER stress. These experiments will include the assessment of the known interaction partners ERp57 (of SERCA2b), chromogranin B (of IP3R), ERp44 (of IP3R1), and sigma-1 receptor (of IP3R3). Having established conditions of most specific SERCA2b and IP3R thiol-disulfide manipulation, we will test the functionality of the redox switches by analyzing the activity of SERCA2b and IP3Rs in treated versus control cells. In parallel experiments, the mechanistic aspect of redox switch catalysis will be investigated by hunting mixed-disulfide interaction partners of the luminal switch cysteines in SERCA2b and IP3Rs. Moreover, the potential involvement of reactive oxygen species (ROS) will be addressed by analyzing the effect of ROS quenchers on the in vivo redox state of the luminal loops. Overall, the anticipated results are expected to enable a more profound comprehension of the processes that determine cell viability during physiological situations of stress. Such comprehension is of crucial importance and will aid the targeted treatment of devastating diseases such as neurodegenerative disorders and cancer.
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