Biology subjects

Reznik, N.

Publications and source records attributed to Reznik, N..

4 recordsLinked to original sources

bioRxiv2022

CWLP and PRP940 form plasma-membrane nanodomain complexes with aquaporins, interact with PP2A and contribute to dehydration tolerance

The C-type hybrid-proline-rich protein (HyPRP) AtCWLP and its homolog AtPRP940 are referred as cell wall (CW)-plasma-membrane (PM) linker proteins, but little is known about their functions. Here we show that N-terminal proline-rich domains of CWLP and PRP940, containing glycosylated hydroxyproline residues, contact the CW, while their C-terminal 8CM domains function as PM-scaffolds. Both proteins are detected in PM nanodomains (PM-ND) and show co-localization and co-immunoprecipitation with aquaporins PIP2;1 and PIP2;7. Inhibition of actin polymerization by latrunculin B promotes CWLP-endosome appearance, while blocking the actomyosin-based transport by a truncated form of myosin XI-K relaxes lateral boundaries of CWLP-PIP2;1 PD-NDs. Mass spectrometry data indicate that CWLP co-purifies with dynamins implicated in fission of endocytic PD-ND invaginations. Lack of co-localization and co-immunoprecipitation with aquaporin-binding flotillin (FLOT2) indicates that CWLP and PRP940 mark a new distinct type of PM-ND. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that CWLP and PRP940 interact with multiple aquaporins and several protein phosphatase PP2A-B regulatory subunits. By preventing irreversible separation of CW and PM, and likely assisting PP2A-mediated dephosphorylation of aquaporins and closure of their water channels, overexpression of CWLP confers tolerance to plasmolysis, dehydration and freezing in Arabidopsis and to water shortage in potato plants. Summary StatementArabidopsis Hybrid-Proline-Rich Proteins CWLP and PRP940 occur in association with dynamins, recruit PP2A protein phosphatases to aquaporin water channels in plasma-membrane (PM) nanodomains and elevate tolerance to cellular dehydration.

plant biology↗

bioRxiv2022

The Colon Mucosal Sialylglycome Is Redox-Regulated by the Golgi Enzyme QSOX1

Mucus shields the intestinal epithelium from pathogens and provides a supportive environment for commensal bacteria. Mucus is composed of enormous, heavily glycosylated proteins called mucins, which become disulfide crosslinked in a multi-step biosynthetic pathway culminating in the Golgi apparatus and secretory granules of goblet cells. We observed that knockout mice lacking the Golgi-localized disulfide catalyst QSOX1 produced poorly protective colon mucus, were hypersensitive to induced colitis, and had an altered microbiome. The initial hypothesis arising from these observations was that QSOX1 catalyzes disulfide crosslinking of mucins. Contrary to this hypothesis, the disulfide-mediated polymerization of mucins and related glycoproteins proceeded normally without QSOX1. Instead, we found that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases and thereby promotes effective sialylation of the colon glycome. Our findings reveal that enzymatic control of Golgi redox state impacts glycan elaboration in goblet cells, and that this pathway is crucial for maintaining mucosal function.

cell biology↗

bioRxiv2022

Intestinal Mucin Is a Chaperone of Multivalent Copper

Mucus protects the body by many mechanisms, but a role in managing toxic transition metals was not previously known. Here we report that secreted mucins, the major mucus glycoproteins coating the respiratory and intestinal epithelia, are specific copper-binding proteins. Most remarkably, the intestinal mucin, MUC2, has two juxtaposed copper binding sites, one that accommodates Cu2+ and the other Cu1+, which can be formed in situ by reduction with vitamin C. Copper is an essential trace metal because it is a cofactor for a variety of enzymes catalyzing electron transfer reactions, but copper damages macromolecules when unregulated. We observed that MUC2 protects against copper toxicity while permitting nutritional uptake into cells. These findings introduce mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones and potentially important players in physiological copper homeostasis.

biochemistry↗

bioRxiv2018

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. Such electrophilic fragments were considered non-selective 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 ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination 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.

biochemistry↗