Oxidative Folding and Structural Analyses of a Kunitz-Related Inhibitor and Its Disulfide Intermediates: Functional Implications

Bronsoms, Sílvia; Pantoja-Uceda, David; Gabrijelcic-Geiger, Dusica; Sanglas, Laura; Aviles, Francesc X.; Santoro, Jorge; Sommerhoff, Christian P.; Arolas, Joan L.

doi:10.1016/j.jmb.2011.10.018

Cited by 17 publications

(12 citation statements)

References 63 publications

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“…In the case of the TdPI protein such a misfolded structure can be very similar to the native one (compared, e.g., by the rmsd value), but covalent loops should be definitely more labile. In fact, the oxidative and reductive folding of this protein was studied previously (29). In particular, it was shown that in oxidative conditions the protein rarely achieves its native state, forming most often a nonnative structure with three (of four) cysteine bonds.…”

Section: Proteins With the Hopf Linkmentioning

confidence: 99%

See 1 more Smart Citation

Topological knots and links in proteins

Dąbrowski-Tumański

Sułkowska

2017

Proc. Natl. Acad. Sci. U.S.A.

118

100

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Twenty years after their discovery, knots in proteins are now quite well understood. They are believed to be functionally advantageous and provide extra stability to protein chains. In this work, we go one step further and search for links-entangled structures, more complex than knots, which consist of several components. We derive conditions that proteins need to meet to be able to form links. We search through the entire Protein Data Bank and identify several sequentially nonhomologous chains that form a Hopf link and a Solomon link. We relate topological properties of these proteins to their function and stability and show that the link topology is characteristic of eukaryotes only. We also explain how the presence of links affects the folding pathways of proteins. Finally, we define necessary conditions to form Borromean rings in proteins and show that no structure in the Protein Data Bank forms a link of this type.folding | catenanes | slipknot | lasso | disulphide bridge K notted proteins have been identified in all kingdoms of life, in organisms separated even by 1 billion years of evolution (1-5). High conservation (5) of knotted motifs and their location (usually) in enzymatic active sites indicates that knots are crucial for protein function. Over 1,300 knotted or slipknotted (shoelace-type) structures, including the trefoil (31), figure-eight (41), three-twist (52), and Stevedore's (61) knots (4, 6, 7), have been deposited in the Protein Data Bank (PDB) to date according to KnotProt (8).Mathematically, a knot is defined as an embedding of a circle into a 3D space. A link is a generalization of a knot, defined as an embedding of a finite set of circles. The simplest examples of links are, e.g., the Hopf link and the Solomon link ( Fig. 1, Center). Links have been found in DNA (9, 10) and have been synthesized in template synthesis (11,12). In proteins, the first attempts to identify links were made by Mislow (13,14). In his approach, however, the link-forming loops were defined either by including interaction with a metal ion (noncovalent loop) or by at least two disulfide bonds for each (covalent) loop. The links formed by covalent loops, each closed by one disulfide bridge only, were considered "unlikely to lead to knots or links" (ref. 14, p. 4,202) by Mislow and therefore hardly examined. Moreover, all of the structures were scanned only by "visual examination of their 3D structures" (ref. 14, p. 4,202). To date, the only known simple protein links are designed p53 protein catenanes (15) (with the backbones of both chains artificially closed, forming linked loops) and a thermophilic two-chain complex (16) (with linked loops formed by the backbones closed via disulfide bridges). However, the discovery of a wide class of complex lasso proteins (17, 18), in which a chain pierces a covalently closed loop (Fig. 1), opens a unique possibility of defining and identifying links. Such links (or more formally, pretzelanes) are defined using covalent loops closed by disulfide bridges in a single-protein cha...

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Section: Proteins With the Hopf Linkmentioning

confidence: 99%

“…Therefore, it is highly probable that the folding trap observed in ref. 29 was the topologically trivial structure.…”

Section: Proteins With the Hopf Linkmentioning

confidence: 99%

Topological knots and links in proteins

Dąbrowski-Tumański

Sułkowska

2017

Proc. Natl. Acad. Sci. U.S.A.

118

100

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“…In some animals, however, these inhibitors evolved to serve as venom (Mourao and Schwartz, 2013). For arachnids, Kunitz-domain-containing proteins either interfere with ion channel activity (Chen et al, 2012;Dai et al, 2012;Valdes and Moal, 2014;Santibañez-Lopez et al, 2018) or inhibit critical host proteins (Decrem et al, 2008;Bronsoms et al, 2011;Andreotti et al, 2012;Louw et al, 2013;Zhang et al, 2017). Tick saliva is essentially a form of venom evolved to facilitate bloodfeeding, and although Ixodes spp.…”

Section: Implications Of Individual Protein Classes On Viral Behaviormentioning

confidence: 99%

“…Kunitz-domain serine protease inhibitors are known to inhibit coagulation (Dai et al, 2012;Chen et al, 2013;Louw et al, 2013;Assumpção et al, 2015;Zhang et al, 2017), angiogenesis (Drewes et al, 2012;Soares et al, 2016), and may reduce inflammation (Bronsoms et al, 2011). Their primary purpose is to ensure successful feeding by inhibiting blood coagulation.…”

Section: Implications Of Individual Protein Classes On Viral Behaviormentioning

confidence: 99%

Tick-Borne Encephalitis Virus Infection Alters the Sialome of Ixodes ricinus Ticks During the Earliest Stages of Feeding

Hart

Ribeiro

Kazimírová

et al. 2020

Front. Cell. Infect. Microbiol.

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Ticks are hematophagous arthropods that transmit a number of pathogens while feeding. Among these is tick-borne encephalitis virus (TBEV), a flavivirus transmitted by Ixodes ricinus ticks in the temperate zone of Europe. The infection results in febrile illness progressing to encephalitis and meningitis with a possibility of fatality or long-term neurological sequelae. The composition of tick saliva plays an essential role in the initial virus transmission during tick feeding. Ticks secrete a diverse range of salivary proteins to modulate the host response, such as lipocalins to control the itch and inflammatory response, and both proteases and protease inhibitors to prevent blood coagulation. Here, the effect of viral infection of adult females of Ixodes ricinus was studied with the goal of determining how the virus alters the tick sialome to modulate host tissue response at the site of infection. Uninfected ticks or those infected with TBEV were fed on mice and removed and dissected one-and 3-h post-attachment. RNA from the salivary glands of these ticks, as well as from unfed ticks, was extracted and subjected to next-generation sequencing to determine the expression of key secreted proteins at each timepoint. Genes showing statistically significant up-or down-regulation between infected and control ticks were selected and compared to published literature to ascertain their function. From this, the effect of tick viral infection on the modulation of the tick-host interface was determined. Infected ticks were found to differentially express a number of uncategorized genes, proteases, Kunitz-type serine protease inhibitors, cytotoxins, and lipocalins at different timepoints. These virus-induced changes to the tick sialome may play a significant role in facilitating virus transmission during the early stages of tick feeding.

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“…As shown in vitro for the well-studied Bovine Pancreatic Trypsin Inhibitor (BPTI), the folding of disulfide-bonded proteins is often acquired through the accumulation of disulfide intermediates [18,19]. For some disulfide-rich proteins, oxidative folding generates heterogeneous populations of intermediates containing native but also non-native disulfide-bonded species, which require isomerization to reach the natively-folded oxidized state [20,21]. Thus, proteins with disulfide bonds are especially prone to aggregation due to possible mispairing of cysteine residues or undesirable intermolecular disulfide bonds.…”

Section: Introductionmentioning

confidence: 99%

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

et al. 2013

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BackgroundDisulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.ResultsHere we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization.ConclusionsOur results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB-/gor- strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

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Oxidative Folding and Structural Analyses of a Kunitz-Related Inhibitor and Its Disulfide Intermediates: Functional Implications

Cited by 17 publications

References 63 publications

Topological knots and links in proteins

Topological knots and links in proteins

Tick-Borne Encephalitis Virus Infection Alters the Sialome of Ixodes ricinus Ticks During the Earliest Stages of Feeding

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli

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