TL;DR
Methods for investigating glycan-related degrons that are key determinants for the ERAD of misfolded glycoproteins in plants are described.
Maintenance of proteostasis is essential for cellular and organismal homeostasis, and disruption of protein quality control (QC) networks underlies numerous human diseases. The endoplasmic reticulum (ER) functions as a central organelle for the synthesis, folding, maturation, and trafficking of secretory and membrane proteins, and serves as a central hub of intracellular proteostasis. Recent studies have established that the ER membrane serves not only as a site of protein translocation but also as a dynamic platform integrating translational regulation, RNA surveillance, and multiple QC pathways. During ER-associated translation, cells continuously monitor ribosome dynamics, mRNA integrity, nascent-chain folding, and transmembrane protein insertion processes to prevent the accumulation of aberrant proteins. These surveillance systems include the PKR-like ER kinase (PERK)-mediated integrated stress response (ISR), regulated IRE1-dependent decay (RIDD), nonsense-mediated mRNA decay (NMD), RNA silencing, ribosome-associated QC (RQC), ubiquitin-fold modifier 1 conjugation (UFMylation), ER-phagy, and ER stress-induced pre-emptive QC (ERpQC). Although these pathways were originally characterized independently, increasing evidence indicates that they function cooperatively on or near the ER membrane to coordinate translational attenuation, mRNA degradation, ribosome recycling, nascent-chain elimination, and organelle remodeling. In particular, UFMylation has emerged as a central mechanism linking ER-associated RQC, translocation-associated QC (TAQC), and ER-phagy. Dysfunction of these ER-localized translational QC pathways contributes to neurodegeneration, inflammation, fibrosis, cancer, and aging-related disorders. In this review, we summarize recent advances in ER-localized translational control and discuss how integrated QC networks on the ER membrane maintain proteostasis and influence disease pathogenesis.
Glycosylation is the most common post-translational modification in the human proteome, with over half of all human proteins bearing covalently attached glycans. These glycan structures direct protein folding through ER quality control machinery, shield polypeptides from proteolytic degradation, regulate circulatory half-life via the asialoglycoprotein receptor, and serve as molecular signals for immune recognition and intracellular trafficking. For biopharmaceuticals, which constitute a rapidly growing share of approved drugs, glycan profiles are critical quality attributes that directly determine clinical efficacy and safety. Yet achieving the correct glycosylation on a therapeutic protein remains one of the field’s central challenges, as glycan biosynthesis is non-template-driven and highly sensitive to expression system and manufacturing conditions. This review connects the biological functions of glycosylation to the practical strategies of glycoengineering, examining how sequence design, expression system selection, and downstream enzymatic remodeling are used to optimize therapeutic glycoproteins. Clinical case studies spanning monoclonal antibodies, cytokines, and enzyme replacement therapies illustrate how glycan engineering translates into improved patient outcomes. We conclude by surveying emerging technologies poised to make precisely glycosylated therapeutics more accessible.
Glycosylation is critical for viral-host cell interactions in influenza A virus (IAV) infection, but we lack a comprehensive understanding of how IAV infection shapes the host glycoproteome. Here, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic, glycomic, and glycoproteomic characterisation of the dynamic subcellular responses to an in vitro time course infection of human A549 cells. IAV infection resulted in only modest changes to the subcellular proteome, but robust and significant changes to the host secreted and organelle glycome and glycoproteome. Infection caused reductions in sialylation across the N- and O-glyco(proteo)me; increased oligomannose, paucimannose, and phosphorylated glycans; and shorter hybrid/complex glycans. Desialylation was greater when glycans were fucosylated; for sialic acid linked α2,3 or on the α3 arm; on larger, complex glycans; and on proteins likely to be more accessible to IAV neuraminidase (NA). Subtle activation of the unfolded protein response in infection was associated with a doubling of oligomannose N-glycosylation. Glycans were shorter in infection, implicating IAV-induced disruption of Golgi glycoprotein flux as a mechanism that reduces host glycoprotein sialylation and promotes virion release, independent of NA activity.
About one-third of the eukaryotic proteome transits the secretory pathway to reach its correct cellular or extracellular destination. At the earliest stage, transport from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) or Golgi apparatus is mediated by coat protein complex II (COPII). COPII coats consist of inner and outer layers formed by Sec23–Sec24 heterodimers and Sec13–Sec31 heterotetramers, respectively, which initially assemble at ER exit sites (ERES) to form transport carriers. Sec23-interacting protein (Sec23IP) links the inner and outer coats through its interactions with both Sec23A and Sec31A, positioning it as a key potential regulator of COPII function. However, the mechanisms controlling Sec23IP activity remain poorly understood. Here, we investigate how physiological stimuli regulate COPII function through the dynamic modification of Sec23IP by O-linked β-N-acetylglucosamine (O-GlcNAc), a reversible, intracellular form of glycosylation. We first validated Sec23IP as a bona fide O-GlcNAcylated protein. Rescue experiments in Sec23IP knockout cells with a nearly unglycosylatable mutant protein demonstrated the essential role of O-GlcNAcylation in the intrinsically disordered domain in protein transport and in recruiting Sec31A to ERES. Moreover, O-GlcNAcylation of Sec23IP increased during protein transport, coinciding with a reduction in its interaction with Sec31A. These results indicate that distinct site-specific O-GlcNAcylation of Sec23IP spatiotemporally modulates its association with Sec31A to fine-tune ERES recruitment and COPII assembly/disassembly. Our work provides new insight into Sec23IP regulation and suggests that O-GlcNAc on other COPII proteins may govern carrier formation, uncoating, and transport.
Targeted protein degradation mediated by antibodies has emerged as a promising strategy for degrading extracellular or membrane-bound proteins. Proteolysis-Targeting Antibodies (PROTABs) are bispecific antibodies specifically designed to induce the degradation of membrane proteins by tethering them to a cell surface E3 ligase, which promotes ubiquitination and subsequent degradation. Recent studies have demonstrated the potential of PROTABs to degrade oncogenic receptors, but their underlying mechanisms remain to be fully elucidated. Here, we investigated the mechanism of action of a HER2-targeting PROTAB comprising an anti-Zinc and RING finger protein 3 (ZNRF3) arm and an anti-receptor tyrosine-protein kinase erbB-2 (HER2) arm. We show that PROTAB induces rapid ternary complex formation, followed by receptor internalization and degradation, resulting in ~ 85% target depletion within 24 h. Mechanistically, ubiquitination enhances but is not strictly required for internalization, and degradation proceeds predominantly through the lysosomal pathway. Notably, ZNRF3 is not codegraded but instead accumulates at the cell surface, while the PROTAB antibody itself is largely recycled. Importantly, target degradation does not consistently translate into growth inhibition, highlighting the role of cellular context and target dependency. Together, these findings provide a mechanistic framework for PROTAB function and inform the rational design of next-generation antibody-based degraders.
