Mia40 Combines Thiol Oxidase and Disulfide Isomerase Activity to Efficiently Catalyze Oxidative Folding in Mitochondria

Abstract The release of protein disulfide isomerase by activated platelets was hypothesized on the basis of reported intermolecular and intramolecular thiol-disulfide exchange and disulfide reduction involving released thrombospondin in the supernatant solution of activated platelets (Danishefsky, Alexander, Detwiler: Biochemistry, 23:4984, 1984; Speziale, Detwiler: J Biol Chem, 265:17859, 1990; Speziale, Detwiler: Arch Biochem Biophys 286:546, 1991). Protein disulfide isomerase activity, measured by catalysis of the renaturation of ribonuclease inactivated by randomization of disulfide bonds, was detected in the supernatant solution after platelet activation. The activity was inhibited by peptides known to inhibit protein disulfide isomerase; the peptides also inhibited formation of disulfide-linked thrombospondin- thrombin complexes. The reaction catalyzed by the supernatant solution showed a pH dependence distinct from that of the uncatalyzed reaction. The activity was excluded by a 50-Kd dialysis membrane, and it was eluted in the void volume of a gel-filtration column, indicating that it was associated with a macromolecule. The activity was not removed by centrifugation at 100,000 g for 150 minutes indicating that it was not associated with membrane microvesicles. Possible functions for the release of protein disulfide isomerase by activated platelets are discussed.

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Genetic Analysis of Disulfide Isomerization in Escherichia coli: Expression of DsbC Is Modulated by RNase E-Dependent mRNA Processing

Journal of Bacteriology ◽

10.1128/jb.186.3.654-660.2004 ◽

2004 ◽

Vol 186 (3) ◽

pp. 654-660 ◽

Cited By ~ 10

Author(s):

Xiaoming Zhan ◽

Junjun Gao ◽

Chaitanya Jain ◽

Michael J. Cieslewicz ◽

James R. Swartz ◽

...

Keyword(s):

Escherichia Coli ◽

Disulfide Bonds ◽

Critical Role ◽

Chemical Mutagenesis ◽

Selection Strategy ◽

Rnase E ◽

Disulfide Isomerase ◽

Isomerase Activity ◽

Disulfide Isomerization ◽

Disulfide Isomerase Activity

ABSTRACT We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity. The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell. Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain. Following chemical mutagenesis, a total of eight E. coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated. This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC. In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product. Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci. The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37°C in wild-type cells but was two- to threefold longer in some of the stronger mutants. These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression.

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Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase, PmScsC

10.1101/421420 ◽

2018 ◽

Author(s):

Emily J. Furlong ◽

Fabian Kurth ◽

Lakshmanane Premkumar ◽

Andrew E. Whitten ◽

Jennifer L. Martin

Keyword(s):

Crystal Structure ◽

Catalytic Domain ◽

Copper Tolerance ◽

Disulfide Isomerase ◽

Isomerase Activity ◽

Catalytic Domains ◽

X Ray Scattering ◽

Low Concentrations ◽

Protein Disulfide ◽

Disulfide Isomerase Activity

AbstractSuppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance – a key virulence trait of the uropathogen. Each protomer of the enzyme has an N-terminal trimerisation stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, we characterise two PmScsC variants, PmScsCΔN and PmScsCΔLinker. PmScsCΔN, is an N-terminally truncated form of the protomer with two helices of the trimerisation stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsCΔN reported here reveals – as expected – a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant PmScsCΔLinker was designed to remove the 11 amino acid linker and we show that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsCΔLinker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentration, whereas PmScsCΔLinker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase our understanding of disulfide isomerase activity, showing how (i) oligomerisation, (ii) spacing between, and (iii) dynamic motion of, catalytic domains in PmScsC all contribute to its native function.

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