TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2

Melo, Eduardo P.; Lopes, Carlos; Gollwitzer, Peter; Lortz, Stephan; Lenzen, Sigurd; Mehmeti, Ilir; Kaminski, Clemens F.; Ron, David; Avezov, Edward

doi:10.1186/s12915-017-0367-5

Cited by 40 publications

(42 citation statements)

References 46 publications

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“…The current genetically encoded proteins are unlikely to work well in more oxidizing compartments, such as the apoplast and endoplasmic reticulum (ER) lumen, where the probe is likely to become fully oxidized. A recent modification of HyPer (TriPer) is able to operate in the ER lumen of mammalian cells (Melo et al ., ).…”

Section: Measurement and Imaging Of H2o2mentioning

confidence: 97%

Hydrogen peroxide metabolism and functions in plants

2018

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1 I. Introduction 2 II. Measurement and imaging of H O 2 III. H O and O toxicity 3 IV. Production of H O : enzymes and subcellular locations 4 V. H O transport 9 VI. Control of H O concentration: how and where? 9 VII. Metabolic functions of H O 11 VIII. H O signalling 11 IX. Where next? 13 Acknowledgements 13 References 13 SUMMARY: Hydrogen peroxide (H O ) is produced, via superoxide and superoxide dismutase, by electron transport in chloroplasts and mitochondria, plasma membrane NADPH oxidases, peroxisomal oxidases, type III peroxidases and other apoplastic oxidases. Intracellular transport is facilitated by aquaporins and H O is removed by catalase, peroxiredoxin, glutathione peroxidase-like enzymes and ascorbate peroxidase, all of which have cell compartment-specific isoforms. Apoplastic H O influences cell expansion, development and defence by its involvement in type III peroxidase-mediated polymer cross-linking, lignification and, possibly, cell expansion via H O -derived hydroxyl radicals. Excess H O triggers chloroplast and peroxisome autophagy and programmed cell death. The role of H O in signalling, for example during acclimation to stress and pathogen defence, has received much attention, but the signal transduction mechanisms are poorly defined. H O oxidizes specific cysteine residues of target proteins to the sulfenic acid form and, similar to other organisms, this modification could initiate thiol-based redox relays and modify target enzymes, receptor kinases and transcription factors. Quantification of the sources and sinks of H O is being improved by the spatial and temporal resolution of genetically encoded H O sensors, such as HyPer and roGFP2-Orp1. These H O sensors, combined with the detection of specific proteins modified by H O , will allow a deeper understanding of its signalling roles.

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Section: Measurement and Imaging Of H2o2mentioning

confidence: 97%

Hydrogen peroxide metabolism and functions in plants

2018

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“…15 The introduction of this domain turns HyPer into a fluorescent ratiometric sensor that responds in a concentration-dependent manner to H 2 O 2 in vitro and in cells. 16 The response of HyPer to H 2 O 2 relies on the oxidation and concomitant disulfide bond formation between cysteines C199 and C208 from the OxyR domain. The fluorescence intensity ratio of HyPer excited at 488 and 405 nm (emission at 530 nm) reflects directly the degree of thiol oxidation and disulfide bond formation.…”

Section: Refolding Of Hyper Catalyzed By Protein Disulfide Isomerasementioning

confidence: 99%

Stability of Protein Formulations at Subzero Temperatures by Isochoric Cooling

Correia

Tavares

Lopes

et al. 2020

Journal of Pharmaceutical Sciences

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Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and L-arginine, significantly increased the stability of protein disulfide isomerase A1 at-20 C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP b-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at À20 C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at À20 C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.

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“…Fusing roGFP probes to peroxide sensors instead of GRX1 shifts the probe specificity toward H 2 O 2 detection . Targeted to the ER, these probes, however, also behave as PDI substrate and thus do not strictly report on ER H 2 O 2 production . To overcome this caveat, several modifications of disulfide‐based reporters and protocols were suggested .…”

Section: Approaches To Assess Er Stressmentioning

confidence: 99%

“…Targeted to the ER, these probes, however, also behave as PDI substrate and thus do not strictly report on ER H 2 O 2 production . To overcome this caveat, several modifications of disulfide‐based reporters and protocols were suggested . A better way to solve this issue would be the development of disulfide independent ER probes that are actively being searched for .…”

Section: Approaches To Assess Er Stressmentioning

confidence: 99%

A guide to assessing endoplasmic reticulum homeostasis and stress in mammalian systems

et al. 2019

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The endoplasmic reticulum (ER) is a multifunctional organelle that constitutes the entry into the secretory pathway. The ER contributes to the maintenance of cellular calcium homeostasis, lipid synthesis and productive secretory, and transmembrane protein folding. Physiological, chemical, and pathological factors that compromise ER homeostasis lead to endoplasmic reticulum stress (ER stress). To cope with this situation, cells activate an adaptive signaling pathway termed the unfolded protein response (UPR) that aims at restoring ER homeostasis. The UPR is transduced through post‐translational, translational, post‐transcriptional, and transcriptional mechanisms initiated by three ER‐resident sensors, inositol‐requiring protein 1α, activating transcription factor 6α, and PRKR‐like endoplasmic reticulum kinase. Determining the in and out of ER homeostasis control and UPR activation still represents a challenge for the community. Hence, standardized criteria and methodologies need to be proposed for monitoring ER homeostasis and ER stress in different model systems. Here, we summarize the pathways that are activated during ER stress and provide approaches aimed at assess ER homeostasis and stress in vitro and in vivo mammalian systems that can be used by researchers to plan and interpret experiments. We recommend the use of multiple assays to verify ER stress because no individual assay is guaranteed to be the most appropriate one.

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TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2

Cited by 40 publications

References 46 publications

Hydrogen peroxide metabolism and functions in plants

Hydrogen peroxide metabolism and functions in plants

Stability of Protein Formulations at Subzero Temperatures by Isochoric Cooling

A guide to assessing endoplasmic reticulum homeostasis and stress in mammalian systems

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