scholarly journals Oxidative Protein-Folding Systems in Plant Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
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.

scholarly journals Balanced Ero1 activation and inactivation establishes ER redox homeostasis

2012 ◽  
Vol 196 (6) ◽  
pp. 713-725 ◽  
Author(s):  
Sunghwan Kim ◽  
Dionisia P. Sideris ◽  
Carolyn S. Sevier ◽  
Chris A. Kaiser
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Direct Reduction ◽  
Redox Homeostasis ◽  
Feedback Regulation ◽  
Redox Balance ◽  
Oxidative Protein Folding ◽  
Reduced And Oxidized Glutathione ◽  
Minimum Number ◽  
Substrate Proteins

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.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

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

<p>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 <i>in vitro</i> oxidative folding of many proteins.</p><p>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.</p><p>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. </p>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 <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

scholarly journals Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2714 ◽  
Author(s):  
Florian Gauthier ◽  
Jean-Rémi Bertrand ◽  
Jean-Jacques Vasseur ◽  
Christelle Dupouy ◽  
Françoise Debart
Keyword(s):  
Disulfide Bonds ◽  
Chemotherapeutic Drugs ◽  
Exchange Reactions ◽  
Disulfide Exchange ◽  
Physiological Conditions ◽  
Drug Conjugates ◽  
Reducing Conditions ◽  
Redox Responsive ◽  
Covalent Interactions ◽  
Premature Release

Co-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2′-position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with their complementary strands and the duplexes were shown to form an A-helix sufficiently stable under physiological conditions. The enzymatic stability of Dox-siRNAs in human serum was enhanced compared to the unmodified siRNA, especially when two Dox are attached to siRNA. The release of native Dox and RNA from the bioconjugate was demonstrated under reducing conditions suggesting efficient linker disintegration. These results demonstrate the feasibility of making siRNA-drug conjugates via disulfide-based self-immolative linkers for potential therapeutic applications.

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

Cell ◽  
2004 ◽  
Vol 117 (5) ◽  
pp. 601-610 ◽  
Author(s):  
Einav Gross ◽  
David B Kastner ◽  
Chris A Kaiser ◽  
Deborah Fass
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Oxidative Protein Folding

scholarly journals Oxidative protein folding in eukaryotes

2004 ◽  
Vol 164 (3) ◽  
pp. 341-346 ◽  
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.

scholarly journals Structural and mechanistic aspects of S-S bonds in the thioredoxin-like family of proteins

2019 ◽  
Vol 400 (5) ◽  
pp. 575-587 ◽  
Author(s):  
Sérgio F. Sousa ◽  
Rui P.P. Neves ◽  
Sodiq O. Waheed ◽  
Pedro A. Fernandes ◽  
Maria João Ramos
Keyword(s):  
Disulfide Bonds ◽  
Protein Disulfide Isomerase ◽  
Critical Role ◽  
Exchange Reactions ◽  
Disulfide Exchange ◽  
Active Site Cysteine ◽  
The Family ◽  
Protein Disulfide ◽  
Extracellular Environment ◽  
Insight Into

Abstract Disulfide bonds play a critical role in a variety of structural and mechanistic processes associated with proteins inside the cells and in the extracellular environment. The thioredoxin family of proteins like thioredoxin (Trx), glutaredoxin (Grx) and protein disulfide isomerase, are involved in the formation, transfer or isomerization of disulfide bonds through a characteristic thiol-disulfide exchange reaction. Here, we review the structural and mechanistic determinants behind the thiol-disulfide exchange reactions for the different enzyme types within this family, rationalizing the known experimental data in light of the results from computational studies. The analysis sheds new atomic-level insight into the structural and mechanistic variations that characterize the different enzymes in the family, helping to explain the associated functional diversity. Furthermore, we review here a pattern of stabilization/destabilization of the conserved active-site cysteine residues presented beforehand, which is fully consistent with the observed roles played by the thioredoxin family of enzymes.

scholarly journals Protein storage vacuoles form de novo during pea cotyledon development

1995 ◽  
Vol 108 (1) ◽  
pp. 299-310 ◽  
Author(s):  
B. Hoh ◽  
G. Hinz ◽  
B.K. Jeong ◽  
D.G. Robinson
Keyword(s):  
Membrane Protein ◽  
De Novo ◽  
Storage Proteins ◽  
Protein Body ◽  
Membrane System ◽  
Intermediate Stage ◽  
Integral Membrane Protein ◽  
Protein Storage ◽  
Protein Storage Vacuoles ◽  
Protein Storage Vacuole

We have investigated the formation of protein storage vacuoles in peas (Pisum sativum L.) in order to determine whether this organelle arises de novo during cotyledon development. A comparison of different stages in cotyledon development indicates that soluble protease activities decline and the amounts of storage proteins and the integral membrane protein of the protein body, alpha-TIP, increase during seed maturation. On linear sucrose density gradients we have been able to distinguish between two separate vesicle populations: one enriched in alpha-TIP, and one in TIP-Ma 27, a membrane protein characteristic of vegetative vacuoles. Both vesicle populations possess, however, PPase and V-ATPase activities. Conventionally fixed cotyledonary tissue at an intermediate stage in cotyledon development reveals the presence of a complex tubular-cisternal membrane system that seems to surround the pre-existing vacuoles. The latter gradually become compressed as a result of dilation of the former membrane system. This was confirmed immunocytochemically with the TIP-Ma 27 antiserum. Deposits of the storage proteins vicilin and legumin in the lumen, and the presence of alpha-TIP in the membranes of the expanding membrane system provide evidence of its identity as a precursor to the protein storage vacuole.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

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

<p>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 <i>in vitro</i> oxidative folding of many proteins.</p><p>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.</p><p>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. </p>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 <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

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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