Bifunctional electrophiles cross-link thioredoxins with redox relay partners in cells.

Thioredoxin protects cells against oxidative damage by reducing disulfide bonds in improperly oxidized proteins. Previously, we found that the baker's yeast cytosolic thioredoxin Trx2 undergoes cross-linking to form several protein-protein complexes in cells treated with the bifunctional electrophile divinyl sulfone (DVSF). Here, we report that the peroxiredoxin Tsa1 and the thioredoxin reductase Trr1, both of which function in a redox relay network with thioredoxin, become cross-linked in complexes with Trx2 upon DVSF treatment. Treatment of yeast with other bifunctional electrophiles, including diethyl acetylenedicarboxylate (DAD), mechlorethamine (HN2), and 1,2,3,4-diepoxybutane (DEB), resulted in the formation of similar cross-linked complexes. Cross-linking of Trx2 and Tsa1 to other proteins by DVSF and DAD is dependent on modification of the active site Cys residues within these proteins. In addition, the human cytosolic thioredoxin, cytosolic thioredoxin reductase, and peroxiredoxin 2 form cross-linked complexes to other proteins in the presence of DVSF, although each protein shows different susceptibilities to modification by DAD, HN2, and DEB. Taken together, our results indicate that bifunctional electrophiles potentially disrupt redox homeostasis in yeast and human cells by forming cross-linked complexes between thioredoxins and their redox partners.

Spatially controlled glycocalyx engineering for growth factor patterning in embryoid bodies.

Growth factor (GF) patterning in stem cell spheroids, such as embryoid bodies (EBs), has been sought to guide their differentiation and organization into functional 3D tissue models and organoids. Current approaches relying on exposure of EBs to gradients of GFs suffer from poor molecular transport in the spheroid microenvironment and from high cost of production and low stability of recombinant GFs. We have developed an alternative method for establishing GF gradients in EBs utilizing stem cell surface engineering with membrane-targeting heparan sulfate-glycomimetic co-receptors for GFs. We have capitalized on the ability of amphiphilic lipid-functionalized glycopolymers with affinity for FGF2 to assemble into nanoscale vesicles with tunable dimensions and extracellular matrix penetrance. Upon size-dependent diffusion into EBs, the vesicles fused with the plasma membranes of stem cells, giving rise to concentric gradients of cells with enhanced FGF2-binding. The extracellular matrix-assisted cell surface remodeling process described is the first example of spatially-targeted glycocalyx engineering in multicellular systems to control GF localization. The glycopolymer structure, vesicle dimensions, and remodeling conditions determine the level of FGF2 adhesion and gradient slope. The increased chemical and thermal stability of the synthetic glycomimetics and the tunability of their GF-binding profile, which is defined by their glycosylation and may be extended to other recombinant or endogenous morphogens beyond FGF2, further increase the versatility of this method.

Mucin-mimetic glycan arrays integrating machine learning for analyzing receptor pattern recognition by influenza A viruses.

Influenza A viruses (IAVs) exploit host glycans in airway mucosa for entry and infection. Detection of changes in IAV glycan-binding phenotype can provide early indication of transmissibility and infection potential. While zoonotic viruses are monitored for mutations, the influence of host glycan presentation on viral specificity remains obscured. Here, we describe an array platform which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation and density in the mucinous glycocalyx may impact IAV recognition. H1N1 and H3N2 binding in arrays of α2,3- and α2,6-sialyllactose receptors confirmed their known sialic acid-binding specificities and revealed their different sensitivities to receptor presentation. Further, the transition of H1N1 from avian to mammalian cell culture improved the ability of the virus to recognize mucin-like displays of α2,6-sialic acid receptors. Support vector machine (SVM) learning efficiently characterized this shift in binding preference and may prove useful to study viral evolution to a new host.

Embryonic Stem Cell Engineering with a Glycomimetic FGF2/BMP4 Co-Receptor Drives Mesodermal Differentiation in a Three-Dimensional Culture.

Cell surface glycans, such as heparan sulfate (HS), are increasingly identified as co-regulators of growth factor signaling in early embryonic development; therefore, chemical tailoring of HS activity within the cellular glycocalyx of stem cells offers an opportunity to control their differentiation. The growth factors FGF2 and BMP4 are involved in mediating the exit of murine embryonic stem cells (mESCs) from their pluripotent state and their differentiation toward mesodermal cell types, respectively. Here, we report a method for remodeling the glycocalyx of mutant Ext1-/- mESCs with defective biosynthesis of HS to drive their mesodermal differentiation in an embryoid body culture. Lipid-functionalized synthetic HS-mimetic glycopolymers with affinity for both FGF2 and BMP4 were introduced into the plasma membrane of Ext1-/- mESCs, where they acted as functional co-receptors of these growth factors and facilitated signal transduction through associated MAPK and Smad signaling pathways. We demonstrate that these materials can be employed to remodel Ext1-/- mESCs within three-dimensional embryoid body structures, providing enhanced association of BMP4 at the cell surface and driving mesodermal differentiation. As a more complete understanding of the function of HS in regulating development continues to emerge, this simple glycocalyx engineering method is poised to enable precise control over growth factor signaling activity and outcomes of differentiation in stem cells.

Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker.

A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells.