Browsing by Author "Kaminski, Clemens F."
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- Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulumPublication . Avezov, Edward; Konno, Tasuku; Zyryanova, Alisa; Chen, Weiyue; Laine, Romain; Crespillo-Casado, Ana; Melo, Eduardo; Ushioda, Ryo; Nagata, Kazuhiro; Kaminski, Clemens F.; Harding, Heather P.; Ron, DavidBackground: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. Results: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca2+ concentrations approached those normally found in the ER lumen ([Ca2+] K-0.5max = 190 mu M). Conclusions: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.
- TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2Publication . Melo, Eduardo; Rafael dos Reis Lopes, Carlos; Gollwitzer, Peter; Lortz, Stephan; Lenzen, Sigurd; Mehmeti, Ilir; Kaminski, Clemens F.; Ron, David; Avezov, EdwardBackground: The fate of hydrogen peroxide (H2O2) in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER-localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Over the years hints have suggested that glutathione, puzzlingly abundant in the ER lumen, might have a role in reducing the heavy burden of H2O2 produced by the luminal enzymatic machinery for disulfide bond formation. However, limitations in existing organelle-targeted H2O2 probes have rendered them inert in the thiol-oxidizing ER, precluding experimental follow-up of glutathione's role in ER H2O2 metabolism. Results: Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide-bonded protein of pancreatic beta-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in the lumen of the ER further enhanced the luminal H2O2 signal and eroded beta-cell viability. Conclusions: A tri-cysteine system with a single peroxidatic thiol enables H2O2 detection in oxidizing milieux such as that of the ER. Tracking ER H2O2 in live pancreatic beta-cells points to a role for glutathione in H2O2 turnover.