The disulfide catalyst QSOX1 maintains the colon mucosal barrier by regulating Golgi glycosyltransferases.

Mucus is made of enormous mucin glycoproteins that polymerize by disulfide crosslinking in the Golgi apparatus. QSOX1 is a catalyst of disulfide bond formation localized to the Golgi. Both QSOX1 and mucins are highly expressed in goblet cells of mucosal tissues, leading to the hypothesis that QSOX1 catalyzes disulfide-mediated mucin polymerization. We found that knockout mice lacking QSOX1 had impaired mucus barrier function due to production of defective mucus. However, an investigation on the molecular level revealed normal disulfide-mediated polymerization of mucins and related glycoproteins. Instead, we detected a drastic decrease in sialic acid in the gut mucus glycome of the QSOX1 knockout mice, leading to the discovery that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases. Sialylation defects in the colon are known to cause colitis in humans. Here we show that QSOX1 redox control of sialylation is essential for maintaining mucosal function.

Disulfide bond formation and redox regulation in the Golgi apparatus.

Formation of disulfide bonds in secreted and cell-surface proteins involves numerous enzymes and chaperones abundant in the endoplasmic reticulum (ER), the first and main site for disulfide bonding in the secretory pathway. Although the Golgi apparatus is the major station after the ER, little is known about thiol-based redox activity in this compartment. QSOX1 and its paralog QSOX2 are the only known Golgi-resident enzymes catalyzing disulfide bonding. The localization of disulfide catalysts in an organelle downstream of the ER in the secretory pathway has long been puzzling. Recently, it has emerged that QSOX1 regulates particular glycosyltransferases, thereby influencing a central activity of the Golgi. Surprisingly, a few important disulfide-mediated multimerization events occurring in the Golgi were found to be independent of QSOX1. These multimerization events depend, however, on the low pH of the Golgi lumen and secretory granules. We compare and contrast disulfide-mediated multimerization in the ER vs. the Golgi to illustrate the variety of mechanisms controlling covalent supramolecular assembly of secreted proteins.

Intestinal mucin is a chaperone of multivalent copper.

Mucus protects the epithelial cells of the digestive and respiratory tracts from pathogens and other hazards. Progress in determining the molecular mechanisms of mucus barrier function has been limited by the lack of high-resolution structural information on mucins, the giant, secreted, gel-forming glycoproteins that are the major constituents of mucus. Here, we report how mucin structures we determined enabled the discovery of an unanticipated protective role of mucus: managing the toxic transition metal copper. Using two juxtaposed copper binding sites, one for Cu2+ and the other for Cu1+, the intestinal mucin, MUC2, prevents copper toxicity by blocking futile redox cycling and the squandering of dietary antioxidants, while nevertheless permitting uptake of this important trace metal into cells. These findings emphasize the value of molecular structure in advancing mucosal biology, while introducing mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones.

Conformational switches and redox properties of the colon cancer-associated human lectin ZG16.

Zymogen granule membrane protein 16 (ZG16) is produced in organs that secrete large quantities of enzymes and other proteins into the digestive tract. ZG16 binds microbial pathogens, and lower ZG16 expression levels correlate with colorectal cancer, but the physiological function of the protein is poorly understood. One prominent attribute of ZG16 is its ability to bind glycans, but other aspects of the protein may also contribute to activity. An intriguing feature of ZG16 is a CXXC motif at the carboxy terminus. Here, we describe crystal structures and biochemical studies showing that the CXXC motif is on a flexible tail, where it contributes little to structure or stability but is available to engage in redox reactions. Specifically, we demonstrate that the ZG16 cysteine thiols can be oxidized to a disulfide by quiescin sulfhydryl oxidase 1, which is a sulfhydryl oxidase present together with ZG16 in the Golgi apparatus and in mucus, as well as by protein disulfide isomerase. ZG16 crystal structures also draw attention to a nonproline cis peptide bond that can isomerize within the protein and to the mobility of glycine-rich loops in the glycan-binding site. An understanding of the properties of the ZG16 CXXC motif and the discovery of internal conformational switches extend existing knowledge relating to the glycan-binding activity of the protein.

Phenotypic Screen Identifies JAK2 as a Major Regulator of FAT10 Expression.

FAT10 is a ubiquitin-like protein suggested to target proteins for proteasomal degradation. It is highly upregulated upon pro-inflammatory cytokines, namely, TNFα, IFNγ, and IL6, and was found to be highly expressed in various epithelial cancers. Evidence suggests that FAT10 is involved in cancer development and may have a pro-tumorigenic role. However, its biological role is still unclear, as well as its biochemical and cellular regulation. To identify pathways underlying FAT10 expression in the context of pro-inflammatory stimulation, which characterizes the cancerous environment, we implemented a phenotypic transcriptional reporter screen with a library of annotated compounds. We identified AZ960, a potent JAK2 inhibitor, which significantly downregulates FAT10 under pro-inflammatory cytokines induction, in an NFκB-independent manner. We validated JAK2 as a major regulator of FAT10 expression via knockdown, and we suggest that the transcriptional effects are mediated through pSTAT1/3/5. Overall, we have elucidated a pathway regulating FAT10 transcription and discovered a tool compound to chemically downregulate FAT10 expression, and to further study its biology.

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains.

The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion. Important open questions are how multiple mucin 2 dimers become covalently linked to one another and how mucin 2 multimerization compares with analogous processes in related polymers such as respiratory tract mucins and the hemostasis protein von Willebrand factor. Here we report the x-ray crystal structure of the mucin 2 multimerization module, found to form a dimer linked by two intersubunit disulfide bonds. The dimer structure calls into question the current model for intestinal mucin assembly, which proposes disulfide-mediated trimerization of the same module. Key residues making interactions across the dimer interface are highly conserved in intestinal mucin orthologs, supporting the physiological relevance of the observed quaternary structure. With knowledge of the interface residues, it can be demonstrated that many of these amino acids are also present in other mucins and in von Willebrand factor, further indicating that the stable dimer arrangement reported herein is likely to be shared across this functionally broad protein family. The mucin 2 module structure thus reveals the manner by which both mucins and von Willebrand factor polymerize, drawing deep structural parallels between macromolecular assemblies critical to mucosal epithelia and the vasculature.

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.