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Small molecule modulators of the Endoplasmic Reticulum glycoprotein folding quality control (ERQC) machinery have broad-spectrum antiviral activity against a number of enveloped viruses and have the potential to rescue secretion of misfolded but active glycoproteins in rare diseases. In vivo assays of candidate inhibitors in mammals are expensive and cannot be afforded at the preliminary stages of drug development programs. The strong conservation of the ERQC machinery across eukaryotes makes transgenic plants an attractive system for low-cost, easy and fast proof-of-concept screening of candidate ERQC inhibitors. The Arabidopsis thaliana immune response is mediated by glycoproteins, the folding of which is controlled by ERQC. We have used the plant response to bacterial peptides as a means of assaying an ERQC inhibitor in vivo. We show that the treatment of the plant with the iminosugar N B-DNJ, which is a known ER α-glucosidase inhibitor in mammals, influences the immune response of the plant to the bacterial peptide elf18 but not to the flagellin-derived flg22 peptide. In the N B-DNJ-treated plant, the responses to elf18 and flg22 treatments closely follow the ones observed for the ER α-glucosidase II impaired plant, At psl5-1 . We propose Arabidopsis thaliana as a promising platform for the development of low-cost proof-of-concept in vivo ERQC modulation.

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER‐localized eukaryotic glycoprotein secretion checkpoint, UDP‐glucose glycoprotein glucosyl‐transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re‐glucosylating one of its N‐linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT‐mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop‐2‐Q118E, E227K and L186P mutants, which cause gelatinous drop‐like corneal dystrophy (GDLD). Compared with the wild‐type Trop‐2, which is correctly localized at the plasma membrane, these Trop‐2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop‐2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9‐mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop‐2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1−/−cells. UGGT1 also efficiently reglucosylated Trop‐2‐Q118E‐EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad‐spectrum rescue‐of‐secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. [ABSTRACT FROM AUTHOR]

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucoseaccepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxinlike (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of themolecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold. [ABSTRACT FROM AUTHOR]

Plants offer a simpler and cheaper alternative to mammalian animal models for the study of endoplasmic reticulum glycoprotein folding quality control (ERQC). In particular, the Arabidopsis thaliana (At) innate immune response to bacterial peptides provides an easy means of assaying ERQC function in vivo. A number of mutants that are useful to study ERQC in planta have been described in the literature, but only for a subset of these mutants the innate immune response to bacterial elicitors has been measured beyond monitoring plant weight and some physio-pathological parameters related to the plant immune response. In order to probe deeper into the role of ERQC in the plant immune response, we monitored expression levels of the Phosphate-induced 1 (PHI-1) and reticulin-oxidase homologue (RET-OX) genes in the At ER α-Glu II rsw3 and the At UGGT uggt1-1 mutant plants, in response to bacterial peptides elf18 and flg22. The elf18 response was impaired in the rsw3 but not completely abrogated in the uggt1-1 mutant plants, raising the possibility that the latter enzyme is partly dispensable for EF-Tu receptor (EFR) signaling. In the rsw3 mutant, seedling growth was impaired only by concomitant application of the At ER α-Glu II NB-DNJ inhibitor at concentrations above 500 nM, compatibly with residual activity in this mutant. The study highlights the need for extending plant innate immune response studies to assays sampling EFR signaling at the molecular level. [ABSTRACT FROM AUTHOR]

UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (Ct UGGT), two Ct UGGT double-cysteine mutants, and its TRXL2 domain truncation (Ct UGGT-ΔTRXL2). Ct UGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name "Parodi limit" the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro , in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70–80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a "one-size-fits-all adjustable spanner" UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain. [Display omitted] • UGGT MD simulations widen the conformational range observed in crystal structures • The UGGT TRXL2 domain is essential for enzymatic activity • A misfold site is closer than the Parodi limit to a glycan in UGGT clients in vitro • N-Glycan distributions suggest evolution optimizes glycoprotein surface coverage Modenutti et al. carry out molecular dynamics simulations of UGGT, the enzyme surveying correct folding of glycoproteins, and propose a "one-size-fits all adjustable spanner" UGGT:substrate recognition model. The UGGT TRXL2 domain is essential for its function. The size of UGGT likely dictates restraints on the evolution of N-linked glycosylation sites. [ABSTRACT FROM AUTHOR]