scholarly journals Catalysis of Protein Disulfide Bond Isomerization in a Homogeneous Substrate†

Biochemistry ◽  
2005 ◽  
Vol 44 (36) ◽  
pp. 12168-12178 ◽  
Author(s):  
Elizabeth A. Kersteen ◽  
Seth R. Barrows ◽  
Ronald T. Raines
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bond Isomerization ◽  
Protein Disulfide

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

scholarly journals Distinct roles and actions of protein disulfide isomerase family enzymes in catalysis of nascent-chain disulfide bond formation

iScience ◽  
2021 ◽  
pp. 102296
Author(s):  
Chihiro Hirayama ◽  
Kodai Machida ◽  
Kentaro Noi ◽  
Tadayoshi Murakawa ◽  
Masaki Okumura ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Protein Disulfide Isomerase ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Disulfide Isomerase ◽  
Nascent Chain ◽  
Protein Disulfide ◽  
Protein Disulfide Isomerase Family

scholarly journals Mapping the endothelial cell S-Sulfhydrome highlights the crucial role of integrin sulfhydration in vascular function

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
D R Bibli ◽  
D R Hu ◽  
D R Looso ◽  
D R Weigert ◽  
D R Wittig ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Vascular Disease ◽  
Disulfide Bond ◽  
Mesenteric Arteries ◽  
Human Endothelial Cells ◽  
Β3 Integrin ◽  
Static Conditions ◽  
Protein Disulfide

Abstract Background In vascular endothelial cells, cysteine metabolism by the cystathionine-γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the “S-sulfhydrome” i.e. the spectrum of proteins targeted by H2Sn in human endothelial cells. Methods LC-MS/MS was used to identify S-sulfhydrated cysteines in endothelial cell proteins and β3 integrin intra-protein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements and flow-induced vasodilatation in endothelial cell-specific CSE knock out mice and a small collective of patients with endothelial dysfunction. Results Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low), (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression, and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell “S-sulfhydrome” consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that S-sulfhydration affected intra-protein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between β3 integrin and Gα13, resulting in the constitutive activation of RhoA and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation and a failure to detect β3 integrin S-sulfhydration, all of which were rescued following the administration of an H2S supplement. Conclusions Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

scholarly journals Prediction Method of Protein Disulfide Bond Based on Pattern Selection

Engineering ◽  
2013 ◽  
Vol 05 (10) ◽  
pp. 409-412 ◽  
Author(s):  
Pengfei Sun ◽  
Yuanquan Cui ◽  
Tiankai Chen ◽  
Ying Zhao
Keyword(s):  
Disulfide Bond ◽  
Prediction Method ◽  
Pattern Selection ◽  
Protein Disulfide

Protein Disulfide Bond Formation in Prokaryotes

2003 ◽  
Vol 72 (1) ◽  
pp. 111-135 ◽  
Author(s):  
Hiroshi Kadokura ◽  
Federico Katzen ◽  
Jon Beckwith
Keyword(s):  
Disulfide Bond ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Protein Disulfide

scholarly journals Furin Cleavage Potentiates the Membrane Fusion-Controlling Intersubunit Disulfide Bond Isomerization Activity of Leukemia Virus Env

2006 ◽  
Vol 80 (11) ◽  
pp. 5540-5551 ◽  
Author(s):  
Mathilda Sjöberg ◽  
Michael Wallin ◽  
Birgitta Lindqvist ◽  
Henrik Garoff
Keyword(s):  
Membrane Fusion ◽  
Disulfide Bond ◽  
Fusion Reaction ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Furin Cleavage ◽  
Membrane Fusion Protein ◽  
Peptide Complexes ◽  
Disulfide Bond Isomerization

ABSTRACT The membrane fusion protein of murine leukemia virus is a trimer of a disulfide-linked peripheral-transmembrane (SU-TM) subunit complex. The intersubunit disulfide bond is in SU linked to a disulfide bond isomerization motif, CXXC, with which the virus controls its fusion reaction (M. Wallin, M. Ekström, and H. Garoff, EMBO J. 23:54-65, 2004). Upon receptor binding the isomerase rearranges the intersubunit disulfide bond into a disulfide bond isomer within the motif. This facilitates SU dissociation and fusion activation in the TM subunit. In the present study we have asked whether furin cleavage of the Env precursor potentiates the isomerase to be triggered. To this end we accumulated the late form of the precursor, gp90, in the cell by incubation in the presence of a furin-inhibiting peptide. The isomerization was done by NP-40 incubation or by a heat pulse under alkylation-free conditions. The cells were lysed in the presence of alkylator, and the precursor was immunoprecipitated, gel isolated, deglycosylated, and subjected to complete trypsin digestion. Disulfide-linked peptide complexes were separated by sodium dodecyl sulfate-tricine-polyacrylamide gel electrophoresis under nonreducing conditions. This assay revealed the size of the characteristic major disulfide-linked peptide complex that differentiates the two isomers of the disulfide bond between Cys336 (or Cys339) and Cys563, i.e., the bond corresponding to the intersubunit disulfide bond. The analyses showed that the isomerase was five- to eightfold more resistant to triggering in the precursor than in the mature, cleaved form. This suggests that the isomerase becomes potentiated for triggering by a structural change in Env that is induced by furin cleavage in the cell.

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