Structure of Ero1p, Source of Disulfide Bonds for Oxidative Protein Folding in the Cell

The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balance in the ER. In this paper, we show that Pdi1p is the key regulator of Ero1p activity. Reduced Pdi1p resulted in the activation of Ero1p by direct reduction of Ero1p regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was inactivated by both autonomous oxidation and Pdi1p-mediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative levels of reduced and oxidized glutathione in the ER to control Ero1p activity and ensure that cells generate the minimum number of disulfide bonds needed for efficient oxidative protein folding.

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Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

10.26434/chemrxiv.12770702.v1 ◽

2020 ◽

Author(s):

Reem Mousa ◽

Taghreed Hidmi ◽

Sergei Pomyalov ◽

Shifra Lansky ◽

Lareen Khouri ◽

...

Keyword(s):

Crystal Structure ◽

Protein Folding ◽

Disulfide Bonds ◽

Crystal Structure Analysis ◽

Folding Rate ◽

Oxidative Folding ◽

Functional Studies ◽

Oxidative Protein Folding ◽

And Function

The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of in vitro oxidative folding of many proteins.In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the in vitro oxidative folding of many complex disulfide-rich proteins.

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Oxidative protein folding in eukaryotes

The Journal of Cell Biology ◽

10.1083/jcb.200311055 ◽

2004 ◽

Vol 164 (3) ◽

pp. 341-346 ◽

Cited By ~ 660

Author(s):

Benjamin P. Tu ◽

Jonathan S. Weissman

Keyword(s):

Protein Folding ◽

Molecular Oxygen ◽

Disulfide Bonds ◽

Terminal Electron Acceptor ◽

Oxidative Protein Folding ◽

Disulfide Formation ◽

Open Questions ◽

The Many ◽

Related Proteins ◽

Protein Disulfide

The endoplasmic reticulum (ER) provides an environment that is highly optimized for oxidative protein folding. Rather than relying on small molecule oxidants like glutathione, it is now clear that disulfide formation is driven by a protein relay involving Ero1, a novel conserved FAD-dependent enzyme, and protein disulfide isomerase (PDI); Ero1 is oxidized by molecular oxygen and in turn acts as a specific oxidant of PDI, which then directly oxidizes disulfide bonds in folding proteins. While providing a robust driving force for disulfide formation, the use of molecular oxygen as the terminal electron acceptor can lead to oxidative stress through the production of reactive oxygen species and oxidized glutathione. How Ero1p distinguishes between the many different PDI-related proteins and how the cell minimizes the effects of oxidative damage from Ero1 remain important open questions.

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Oxidative Protein-Folding Systems in Plant Cells

International Journal of Cell Biology ◽

10.1155/2013/585431 ◽

2013 ◽

Vol 2013 ◽

pp. 1-15 ◽

Cited By ~ 17

Author(s):

Yayoi Onda

Keyword(s):

Protein Folding ◽

Disulfide Bonds ◽

De Novo ◽

Protein Body ◽

Structural Diversity ◽

Carrier Proteins ◽

Exchange Reactions ◽

Disulfide Exchange ◽

Protein Storage ◽

Oxidative Protein Folding

Plants are unique among eukaryotes in having evolved organelles: the protein storage vacuole, protein body, and chloroplast. Disulfide transfer pathways that function in the endoplasmic reticulum (ER) and chloroplasts of plants play critical roles in the development of protein storage organelles and the biogenesis of chloroplasts, respectively. Disulfide bond formation requires the cooperative function of disulfide-generating enzymes (e.g., ER oxidoreductase 1), which generate disulfide bonds de novo, and disulfide carrier proteins (e.g., protein disulfide isomerase), which transfer disulfides to substrates by means of thiol-disulfide exchange reactions. Selective molecular communication between disulfide-generating enzymes and disulfide carrier proteins, which reflects the molecular and structural diversity of disulfide carrier proteins, is key to the efficient transfer of disulfides to specific sets of substrates. This review focuses on recent advances in our understanding of the mechanisms and functions of the various disulfide transfer pathways involved in oxidative protein folding in the ER, chloroplasts, and mitochondria of plants.

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Oxidative Protein Folding: Many Different Ways to Introduce Disulfide Bonds

Antioxidants and Redox Signaling ◽

10.1089/ars.2006.8.731 ◽

2006 ◽

Vol 8 (5-6) ◽

pp. 731-733 ◽

Cited By ~ 7

Author(s):

Hiroshi Kadokura

Keyword(s):

Protein Folding ◽

Disulfide Bonds ◽

Oxidative Protein Folding

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Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

10.26434/chemrxiv.12770702 ◽

2020 ◽

Author(s):

Reem Mousa ◽

Taghreed Hidmi ◽

Sergei Pomyalov ◽

Shifra Lansky ◽

Lareen Khouri ◽

...

Keyword(s):

Crystal Structure ◽

Protein Folding ◽

Disulfide Bonds ◽

Crystal Structure Analysis ◽

Folding Rate ◽

Oxidative Folding ◽

Functional Studies ◽

Oxidative Protein Folding ◽

And Function

The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of in vitro oxidative folding of many proteins.In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the in vitro oxidative folding of many complex disulfide-rich proteins.

Download Full-text

Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

10.21203/rs.3.rs-73016/v1 ◽

2020 ◽

Author(s):

Reem Mousa ◽

Taghreed Hidmi ◽

Sergei Pomyalov ◽

Shifra Lansky ◽

Lareen Khouri ◽

...

Keyword(s):

Protein Folding ◽

Disulfide Bonds ◽

Systematic Investigation ◽

Crystal Structure Analysis ◽

Folding Rate ◽

Oxidative Protein Folding ◽

Wide Range ◽

Natural Inhibitor

Abstract The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and is viewed as a folding model for a wide range of disulfide-rich proteins. Hirudin’s folding pathway is notorious for its highly heterogeneous intermediates and scrambled isomers, which plague its folding rate and yield in vitro. Aiming to overcome these limitations, we undertook a systematic investigation of diselenide bridges at native and non-native positions and investigated their effect on hirudin’s folding, structure and activity. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the corresponding hirudin analogs, while reducing the complexity and heterogeneity of the process. Moreover, crystal structure analysis confirmed that the diselenide substitutions maintained the overall structure of the protein and left the function virtually unchanged. The choice of hirudin as a study model has implications beyond its specific folding mechanism, demonstrating the high potential of diselenide substitutions in the design, preparation and characterization of disulfide-rich proteins.

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Identification of Redox Partners of the Thiol-Disulfide Oxidoreductase SdbA inStreptococcus gordonii

Journal of Bacteriology ◽

10.1128/jb.00030-19 ◽

2019 ◽

Vol 201 (10) ◽

Cited By ~ 1

Author(s):

Naif A. Jalal ◽

Lauren Davey ◽

Scott A. Halperin ◽

Song F. Lee

Keyword(s):

Protein Folding ◽

Disulfide Bonds ◽

Extracellular Dna ◽

Folding Pathway ◽

Content Type ◽

Cellular Processes ◽

Oxidative Protein Folding ◽

Redox Partners ◽

Disulfide Oxidoreductase ◽

Substrate Proteins

ABSTRACTWe previously identified a novel thiol-disulfide oxidoreductase, SdbA, inStreptococcus gordoniithat formed disulfide bonds in substrate proteins and played a role in multiple phenotypes. In this study, we used mutational, phenotypic, and biochemical approaches to identify and characterize the redox partners of SdbA. Unexpectedly, the results showed that SdbA has multiple redox partners, forming a complex oxidative protein-folding pathway. The primary redox partners of SdbA that maintain its active site in an oxidized state are a surface-exposed thioredoxin family lipoprotein called SdbB (Sgo_1171) and an integral membrane protein annotated as CcdA2. Inactivation ofsdbBandccdA2simultaneously, but not individually, recapitulated thesdbAmutant phenotype. ThesdbB-ccdA2mutant had defects in a range of cellular processes, including autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release. AtlS, the natural substrate of SdbA produced by thesdbB-ccdA2mutant lacked activity and an intramolecular disulfide bond. The redox state of SdbA in thesdbB-ccdA2mutant was found to be in a reduced form and was restored whensdbBandccdA2were knocked back into the mutant. In addition, we showed that SdbB formed a disulfide-linked complex with SdbA in the cell. Recombinant SdbB and CcdA2 exhibited oxidase activity and reoxidized reduced SdbAin vitro. Collectively, our results demonstrate thatS. gordoniiuses multiple redox partners for oxidative protein folding.IMPORTANCEStreptococcus gordoniiis a commensal bacterium of the human dental plaque. Previously, we identified an enzyme, SdbA, that forms disulfide bonds in substrate proteins and plays a role in a number of cellular processes inS. gordonii. Here, we identified the redox partners of SdbA. We showed that SdbA has multiple redox partners, SdbB and CcdA2, forming a complex oxidative protein-folding pathway. This pathway is essential for autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release inS. gordonii. These cellular processes are considered to be important for the success ofS. gordoniias a dental plaque organism. This is the first example of an oxidative protein-folding pathway in Gram-positive bacteria that consists of an enzyme that uses multiple redox partners to function.

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Revisiting the Formation of a Native Disulfide Bond: Consequences for Protein Regeneration and Beyond

Molecules ◽

10.3390/molecules25225337 ◽

2020 ◽

Vol 25 (22) ◽

pp. 5337

Author(s):

Mahesh Narayan

Keyword(s):

Protein Folding ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Active Form ◽

Rnase A ◽

Oxidative Folding ◽

Regeneration Rate ◽

Oxidative Protein Folding ◽

Pancreatic Ribonuclease A ◽

The Impact

Oxidative protein folding involves the formation of disulfide bonds and the regeneration of native structure (N) from the fully reduced and unfolded protein (R). Oxidative protein folding studies have provided a wealth of information on underlying physico-chemical reactions by which disulfide-bond-containing proteins acquire their catalytically active form. Initially, we review key events underlying oxidative protein folding using bovine pancreatic ribonuclease A (RNase A), bovine pancreatic trypsin inhibitor (BPTI) and hen-egg white lysozyme (HEWL) as model disulfide bond-containing folders and discuss consequential outcomes with regard to their folding trajectories. We re-examine the findings from the same studies to underscore the importance of forming native disulfide bonds and generating a “native-like” structure early on in the oxidative folding pathway. The impact of both these features on the regeneration landscape are highlighted by comparing ideal, albeit hypothetical, regeneration scenarios with those wherein a native-like structure is formed relatively “late” in the R→N trajectory. A special case where the desired characteristics of oxidative folding trajectories can, nevertheless, stall folding is also discussed. The importance of these data from oxidative protein folding studies is projected onto outcomes, including their impact on the regeneration rate, yield, misfolding, misfolded-flux trafficking from the endoplasmic reticulum (ER) to the cytoplasm, and the onset of neurodegenerative disorders.

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Organocatalysts of oxidative protein folding inspired by protein disulfide isomerase

Organic & Biomolecular Chemistry ◽

10.1039/c4ob01738b ◽

2014 ◽

Vol 12 (43) ◽

pp. 8598-8602 ◽

Cited By ~ 7

Author(s):

John C. Lukesh III ◽

Kristen A. Andersen ◽

Kelly K. Wallin ◽

Ronald T. Raines

Keyword(s):

Protein Folding ◽

Disulfide Bonds ◽

Protein Disulfide Isomerase ◽

Native Protein ◽

Disulfide Isomerase ◽

Oxidative Protein Folding ◽

Hydrophobic Moiety ◽

Protein Disulfide Bonds ◽

Protein Disulfide

Organocatalysts derived from ethylenetriamine and containing a hydrophobic moiety effect the isomerization of non-native protein disulfide bonds to native ones.

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