The role of glutathione in disulphide bond formation and endoplasmic‐reticulum‐generated oxidative stress

ABSTRACT Spores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores’ metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.

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Native disulphide bond formation in protein biosynthesis: evidence for the role of protein disulphide isomerase

Trends in Biochemical Sciences ◽

10.1016/0968-0004(84)90152-x ◽

1984 ◽

Vol 9 (10) ◽

pp. 438-441 ◽

Cited By ~ 209

Author(s):

Robert B. Freedman

Keyword(s):

Protein Biosynthesis ◽

Bond Formation ◽

Disulphide Bond ◽

Protein Disulphide Isomerase ◽

Disulphide Bond Formation

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A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation

Nature Cell Biology ◽

10.1038/ncb1001-874 ◽

2001 ◽

Vol 3 (10) ◽

pp. 874-882 ◽

Cited By ~ 129

Author(s):

Carolyn S. Sevier ◽

John W. Cuozzo ◽

Andrea Vala ◽

Fredrik Åslund ◽

Chris A. Kaiser

Keyword(s):

Endoplasmic Reticulum ◽

Bond Formation ◽

Disulphide Bond ◽

Disulphide Bond Formation

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The synthesis and secretion of cartilage procollagen

Biochemical Journal ◽

10.1042/bj1480129 ◽

1975 ◽

Vol 148 (1) ◽

pp. 129-138 ◽

Cited By ~ 21

Author(s):

R Harwood ◽

A K Bhalla ◽

M E Grant ◽

D S Jackson

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Endoplasmic Reticulum ◽

Bond Formation ◽

Disulphide Bond ◽

Published Data ◽

Cellular Compartment ◽

Disulphide Bond Formation ◽

Tendon Cells ◽

Cartilage Cells ◽

Membrane Bound

1. Isolation of free and membrane-bound ribosomes from embryonic chick sternal-cartilage cells labelled for 4min with [14C]proline and their subsequent analysis for hydroxy[14C]proline indicated that cartilage procollagen biosynthesis occurs on bound ribosomes. 2. Nascent procollagen polypeptides on bound ribosomes isolated from cells labelled with [14C]lysine were found to contain hydroxy[14C]lysine indicating that hydroxylation of lysine commences while the growing chains are still attached to the ribosomes. 3. Analysis of bound ribosomes labelled with either [14C]proline or [14C]lysine on sucrose density gradients indicated that cartilage procollagen is synthesized on large polyribosomes in the range 250-400S. 4. Microsomal preparations isolated from cells pulse-labelled for 4 min with [14C]proline were used to determine the direction of release of nascent procollagen polypeptides. Puromycin induced the vectorial release of nascent procollagen polypeptides into the microsomal vesicles suggesting that the first step in the secretion of procollagen polypeptides is their transfer from the ribosomes through the membrane of the endoplasmic reticulum into the cisternal space. 5. The procollagen polypeptides secreted by cartilage cells were shown to be linked by inter-chain disulphide bonds. 6. Examination of the state of aggregation of pro-α chains in subcellular fractions isolated from cartilage cells labelled with [14C]proline for various periods of time have provided data on the timing and location of inter-chain disulphide-bond formation. This process commences in the rough endoplasmic reticulum after the release of completed pro-α chains from membrane-bound ribosomes. Pro-α chains isolated from fractions of smooth endoplasmic reticulum were virtually all present as disulphide-bonded aggregates, suggesting that either disulphide bonding is completed in this cellular compartment, or that procollagen needs to be in a disulphide-bonded form to be transferred to this region of the endoplasmic reticulum. 7. Comparison of these results with previously published data on disulphide bonding in tendon cells suggest that the rate of inter-chain disulphide-bond formation is significantly slower in cartilage cells.

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TriPer, an optical probe tuned to the endoplasmic reticulum tracks H2O2 consumption by glutathione

10.1101/088351 ◽

2016 ◽

Author(s):

Eduardo Pinho Melo ◽

Carlos Lopes ◽

Peter Gollwitzer ◽

Stephan Lortz ◽

Sigurd Lenzen ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Biology ◽

Genetic Manipulation ◽

Bond Formation ◽

Ectopic Expression ◽

Optical Probe ◽

Disulphide Bond ◽

In Vivo Studies ◽

Disulphide Bond Formation

AbstractThe fate of 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. 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 β-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 lumen of the ER, enhanced further the luminal H2O2 signal and eroded β-cell viability. Tracking ER H2O2 in live cells points to an unanticipated role for glutathione in H2O2 turnover.Significance statementThe presence of millimolar glutathione in the lumen of the endoplasmic reticulum has been difficult to understand purely in terms of modulation of protein-based disulphide bond formation in secreted proteins. Over the years hints have suggested that glutathione might have a role in reducing the heavy burden of hydrogen peroxide (H2O2) produced by the luminal enzymatic machinery for disulphide bond formation. However, limitations in existing in vivo H2O2 probes have rendered them all but useless in the thiol-oxidizing ER, precluding experimental follow-up of glutathione’s role ER H2O2 metabolism.Here we report on the development and mechanistic characterization of an optical probe, TriPer that circumvents the limitations of previous sensors by retaining specific responsiveness to H2O2 in thiol-oxidizing environments. Application of this tool to the ER of an insulin-producing pancreatic b-cells model system revealed that ER glutathione antagonizes locally-produced H2O2 resulting from the oxidative folding of pro-insulin.This study presents an interdisciplinary effort intersecting cell biology and chemistry: An original redox chemistry concept leading to development of a biological tool, broadly applicable for in vivo studies of H2O2 metabolism in the ER. More broadly, the concept developed here sets a precedent for applying a tri-cysteine relay system to discrimination between various oxidative reactants, in complex redox milieux.

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