Browsing tag: białka Dsb

Strategie badań tiolowych oksydoreduktaz

Strategies for the analysis of thioloxidorductases
E. K. Jagusztyn-Krynicka, A. M. Banaś, M. J. Grzeszczuk

1. Wprowadzenie. 2. Analizy funkcjonowania białek Dsb in vivo. 2.1. Wyznaczanie stanu redoks białek. 2.2. Analiza fenotypowa zmutowanych szczepów. 3. Analizy funkcjonalne białek Dsb in vitro. 3.1. Test redukcji insuliny. 3.2 Określanie potencjału redoks. 3.3. Analiza aktywności oksydacyjnej i izomeryzacyjnej. 3.4. Określanie wartości pKa nukleofilowej cysteiny motywu CXXC. 3.5. Analizy oddziaływań pomiędzy DsbA a DsbB. 3.6. Struktury białek. 3.7. Identyfikacja substratów białek Dsb. 4. Podsumowanie

Abstract: Bacterial Dsb (disulfide bond) enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between thiol groups of cysteine residues. This process is critical for the correct folding and structural stability of many secreted and membrane proteins. The rapidly expanding number of sequenced bacterial genomes has revealed the enormous diversity among bacterial Dsb systems. While the Escherichia coli oxidative protein folding has been studied in great details, the mechanism of the Dsb systems functioning in other bacteria are rather poorly understood. Herein, we present the current methodology, both in vivo and in vitro experimental techniques, which allow us to understand the functioning of the Dsb proteins and has broaden our knowledge in the field of biochemistry and microbiology of this posttranslational protein modification. Many bacterial virulence factors are extracytoplasmic Dsb-dependent proteins. Thus, this system plays an important role in bacterial pathogenesis and the proteins of the Dsb network represent possible targets for new drugs.

1. Introduction. 2. Analysis of the Dsb functioning in vivo. 2.1. Determination of the in vivo redox state. 2.2. Phenotypic assay of the mutated strains. 3. Analysis of the Dsb functioning in vitro. 3.1. Insulin reduction assay. 3.2. Determination of the redox potential. 3.3. Assay of the oxidative and isomerase activity. 3.4. Determination of the pKa value of the cysteine residue 3.5. Determination of the interaction between DsbA and DsbB. 3.6. Protein structures. 3.7. Searching for Dsb protein substrates. 4. Conclusions

Proces biogenezy cytochromów c w komórkach bakteryjnych – rola białek Dsb (disulfide bond)

Cytochrome c biogenesis in prokaryotic cells – the role of Dsb proteins
P. Roszczenko, M. Grzeszczuk, E. K. Jagusztyn-Krynicka

1. Białka Dsb (disulfide bond). 2. Różnorodność procesu biogenezy cytochromów c w komórkach bakteryjnych. 2.1. Transport i redukcja apocytochromu. 2.2. Transport i przyłączanie hemu do zredukowanego apocytochromu. 3. Podsumowanie

Abstract: The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges, a post-translational modification of many extracytoplasmic proteins, leading to stabilization of their tertiary and quaternary structures. In Gram-negative bacteria this process takes place in the periplasm whereas in Gram-positive bacteria it occurs in the analogous space between the cytoplasmic membrane and the cell wall. In E. coli (Ec) the Dsb system operates in two partially coinciding metabolic pathways: the oxidation (DsbA and DsbB) and the isomerization/reduction (DsbC and DsbD). In the highly oxidizing environment of the periplasm, there is also a need for selected proteins to be kept in a reduced form. Assembly of c-type cytochromes, essential for energy metabolism, is a case of point. Two distinct different systems for cytochrome-c maturation was found in bacteria: system I known as Ccm (cytochrome c maturation) and system II known as Ccs system (cytochrome c synthesis). They comprise two kind of proteins: those contributing to transport and reduction of disulfide bond of CXXCH of apocytochrome c and those involved in handling of heme and playing a role in its ligation to the apocytochrome. The cytochrome c maturation process requires ligation of heme to reduced thiols of the Cys-X-X-Cys-His motif of the apocytochrome. Since DsbA, the main periplasmic dithiol-oxidase randomly introduces disulfide bonds into apocytochromes, bacterial evolved a special redox system to revert these disulfides, in highly oxidizing environment, into reduced cysteine residues. Thiol-oxidoreductases, CcmG proteins, previously designated as DsbE, play a key role in this process. Here we discuss the variety of two cytochrome c biogenesis systems and discuss some of the current problems in understanding how the process works putting special emphasis on the recent achievements concerning the process driving by CcmGs.

1. Dsb proteins. 2. Diversity of cytochrome c biogenesis. 2.1 Transport and reduction of apocytochrome c. 2.2 Heme translocation and ligation into reduced apocytochrome. 3. Conclusions

Różnorodność szlaków utleniania białek Dsb (disulfide bond) w świecie mikroorganizmów

Diversity of the Dsb (disulfide bond) oxidative protein folding pathways in microbial world
A. Dobosz, K. M. Bocian-Ostrzycka, E. K. Jagusztyn-Krynicka

1. Wprowadzenie. 2. Modelowy system szlaku utleniania Dsb Escherichia coli. 2.1. Charakterystyka białek EcDsbA i EcDsbB. 2.2. Szlaki utleniania i izomeryzacji/redukcji E. coli. 2.3. Rola motywu CXXC i pętli cis-Pro151 białka EcDsbA. 3. Różnorodność bakteryjnych szlaków oksydacji białek. 3.1. Zwielokrotniona liczba białek DsbA. 3.2. Różnorodność strukturalna monomerycznych białek DsbA. 3.3. Dimeryczne oksydoreduktazy DsbA. 3.4. Obecność lub brak białka pełniącego funkcję EcDsbB. 4. Specyficzność substratowa oksydoreduktaz DsbA. 4.1. Metody poszukiwania i lokalizacja substratów. 4.2. Czynniki wirulencji. 5. Inne systemy Dsb. 6. Podsumowanie

Abstract: The introduction of structural disulfide bonds is crucial to the stability and activity of many extra-cytoplasmic proteins. The disulfide bond formation is a rate-limiting step in the folding process of a protein. However, most microorganisms encode a machinery to catalyse this oxidative protein folding step. In prototypic Escherichia coli oxidative pathway, the introduction of disulfide bridges into folding proteins is mediated by the thioredoxin family members – Dsb system proteins. Correct oxidative protein folding in the E. coli envelope depends on both EcDsbA and EcDsbB. Periplasmic oxidoreductase EcDsbA is a key disulfide bond formation catalyst, which is maintained in its active form by membrane-bound protein EcDsbB. To date, over 300 EcDsbA homologues from different bacterial organisms have been identified. Nevertheless, the structure, function and interactions of EcDsbA still remain the best studied. The rapidly expanding number of sequenced bacterial genomes has revealed dramatic differences between the model E. coli oxidative pathway and the pathway in other microorganisms. In this article, we review current knowledge about EcDsbA and focus on the diversity of the disulfide bond generation pathways functioning in the microbial world.

1. Introduction. 2. The classical Escherichia coli thiol oxidizing pathway. 2.1. Characteristics of EcDsbA and EcDsbB proteins. 2.2. E. coli oxidative and isomerization/reduction pathways. 2.3. The role of EcDsbA CXXC motif and cis-Pro151 loop. 3. Variety of bacterial oxidative pathways. 3.1. Multiplied number of DsbAs. 3.2. Structural diversity of monomeric DsbA proteins. 3.3. Dimeric DsbA oxidoreductases. 3.4. The presence or absence of a protein acting as EcDsbB. 4. Substrate specificity of DsbA oxidoreductases. 4.1. Procedures for DsbA substrates identification and methods of location analysis. 4.2. Virulence associated with DsbA substrates. 5. Other Dsb systems. 6. Conclusions

POSTĘPY MIKROBIOLOGII 2020, 59, 2

POSTĘPY MIKROBIOLOGII 2020, 59, 2

POSTĘPY MIKROBIOLOGII
2020, 59, 2

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