Electrostatic interactions contribute to the control of intramolecular thiol–disulfide isomerization in a protein

The roles of structural factors and of electrostatic interactions with the environment on the outcome of thiol–disulfide exchange reactions were investigated in a mutated immunoglobulin domain (I27*) under mechanical stress....

Thiol switches in membrane proteins - Extracellular redox regulation in cell biology

Redox-mediated signal transduction depends on the enzymatic production of second messengers such as hydrogen peroxide, nitric oxide and hydrogen sulfite, as well as specific, reversible redox modifications of cysteine-residues in proteins. So-called thiol switches induce for instance conformational changes in specific proteins that regulate cellular pathways e.g., cell metabolism, proliferation, migration, gene expression and inflammation. Reduction, oxidation and disulfide isomerization are controlled by oxidoreductases of the thioredoxin family, including thioredoxins, glutaredoxins, peroxiredoxins and protein dsisulfide isomerases. These proteins are located in different cellular compartments, interact with substrates and catalyze specific reactions. Interestingly, some of these proteins are released by cells. Their extracellular functions and generally extracellular redox control have been widely underestimated. Here, we give an insight into extracellular redox signaling, extracellular thiol switches and their regulation by secreted oxidoreductases and thiol-isomerases, a topic whose importance has been scarcely studied so far, likely due to methodological limitations. We focus on the secreted redox proteins and characterized thiol switches in the ectodomains of membrane proteins, such as integrins and the metalloprotease ADAM17, which are among the best-characterized proteins and discuss their underlying mechanisms and biological implications.

Applications of catalyzed cytoplasmic disulfide bond formation

Disulfide bond formation is an essential post-translational modification required for many proteins to attain their native, functional structure. The formation of disulfide bonds, otherwise known as oxidative protein folding, occurs in the endoplasmic reticulum and mitochondrial inter-membrane space in eukaryotes and the periplasm of prokaryotes. While there are differences in the molecular mechanisms of oxidative folding in different compartments, it can essentially be broken down into two steps, disulfide formation and disulfide isomerization. For both steps, catalysts exist in all compartments where native disulfide bond formation occurs. Due to the importance of disulfide bonds for a plethora of proteins, considerable effort has been made to generate cell factories which can make them more efficiently and cheaper. Recently synthetic biology has been used to transfer catalysts of native disulfide bond formation into the cytoplasm of prokaryotes such as Escherichia coli. While these engineered systems cannot yet rival natural systems in the range and complexity of disulfide-bonded proteins that can be made, a growing range of proteins have been made successfully and yields of homogenously folded eukaryotic proteins exceeding g/l yields have been obtained. This review will briefly give an overview of such systems, the uses reported to date and areas of future potential development, including combining with engineered systems for cytoplasmic glycosylation.

An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth

The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance.

Redox diversity in ERAD-mediated protein retrotranslocation from the endoplasmic reticulum: a complex puzzle

Misfolded and incorrectly assembled proteins in the secretory pathway are eliminated by ubiquitylation and proteasomal degradation in a process known as ER-associated degradation (ERAD). Retrotranslocation of diverse substrates including misfolded proteins and viruses occurs through channels in the ER membrane, which are also utilized for host cell penetration by A/B class protein toxins such as cholera toxin, ricin or K28. According to the current view, disulfide-bonded proteins must either be reduced or rearranged to ensure translocation competence and entry into the cytosol from the ER. As the underlying mechanisms are still largely mysterious, we here focus on the redox status and disulfide isomerization of ERAD substrates and the role of oxidoreductases in the essential process of ER-to-cytosol retrotranslocation.

Regulation of a Phage Endolysin by Disulfide Caging

ABSTRACT In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, LyzP1, of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz103, the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz103 does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz103 is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, LyzP1 and Lyz103 define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz103 is the first enzyme found to be regulated by disulfide bond caging of its active site.