ERp44 plays a critical role in protein quality control in the early secretory pathway by retrieving ER enzymes and immature secretory proteins from the Golgi to the ER. This function is regulated by Zn2+- and pH-dependent conformational changes in ERp44. However, the detailed mechanisms by which Zn2+ governs client binding in the Golgi and client release in the ER remain to be elucidated. Here, we report cryo-EM structures of complexes between Zn2+-bound ERp44 and two representative clients, ERAP1 and Ero1α. These structures reveal that, beyond the mixed disulfide-mediated interactions, Zn2+ is coordinated by conserved histidine residues of ERp44 together with residues in client loops, forming two distinct types of Zn2+-mediated tethers that stabilize the complexes. Consistently, removal of Zn2+ promotes complex dissociation more effectively than reduction of the mixed disulfide bonds. In the physiological ER environment, millimolar concentrations of glutathione, together with picomolar levels of labile Zn²⁺maintained by the ER-localized Zn2+ transporter ZIP7, promote complex dissociation by removing Zn2+ from the complex. The present findings demonstrate that Zn2+ plays dual roles in protein quality control by allosterically activating ERp44 and directly mediating client binding.

The biogenesis of secretory proteins proceeds under sequential quality control checkpoints operating along the exocytic pathway. Unlike other chaperones that reside primarily in the endoplasmic reticulum, ERp44 cycles through the Golgi to control the assembly of polymeric proteins and the localization of a few endoplasmic reticulum resident enzymes (ERAP1, Prx4, Ero1α, and SUMF1). To gain information about its pathophysiological role, we generated ERp44-deficient models. ERp44 KO mice are smaller than control siblings, and show skeletal malformations and delayed bone development, with reduced collagen deposition. Similar skeletal defects were also observed in ERp44 knocked down zebrafish embryos, supporting a conserved role for ERp44 in skeletal development. In cellular models, ERp44 downregulation dramatically affects collagen type 1 deposition, causing intracellular procollagen 1 accumulation. We thus conclude that the levels of ERp44 are crucial for efficient collagen deposition.

KDEL receptors (KDELRs) prevent the secretion of soluble chaperones and enzymes meant to reside in the endoplasmic reticulum. While a single KDELR exists in yeast (ERD2), three variants are present in mammals, displaying high sequence similarity. All three can prevent the secretion of KDEL-bearing clients. However, their diverse tissue distribution and the high phylogenetic conservation of the differences suggest functional specialization. Accordingly, we show here that while KDELR2 plays a major role in client retrieval, KDELR1 and KDELR3 regulate the production of AGR2, a key assistant of mucin folding, in opposite ways. AGR2 transcripts increase dramatically upon silencing KDELR3 but decrease when KDELR1 is downregulated. Silencing ERp44, but no other ER residents, phenocopies KDELR3 knockdown, suggesting that AGR2 regulation depends on ERp44-KDELR3 interactions. Our findings identify a novel regulatory circuit, distinct from the unfolded protein response, that controls the molecular composition of the early secretory pathway based on specific interactions between KDELRs and ER residents.

Zinc homeostasis is crucial for various biological processes, including gene regulation, signal transduction, and proteostasis. ZIP7 is a membrane transporter that exports zinc ions (Zn2+) from the lumen of the endoplasmic reticulum (ER) to the cytosol, and its dysfunction causes ER stress, although the underlying mechanism remains unclear. Here, we show that ZIP7 inhibition increases the labile Zn2+ concentration in the ER to micromolar levels, approximately 106 times higher than its steady-state level. Such abnormally high Zn2+ concentrations disrupt the function and trafficking of the Zn2+-dependent chaperone ERp44 at the ER-Golgi interface. In vitro assays using recombinant proteins demonstrated that Zn2+ inhibits the Ero1α-PDI oxidative system, and that ERp44 enhances this inhibitory effect. Consequently, the ER redox environment becomes more reducing, severely impairing the oxidative folding of key membrane receptors such as Notch1 and EGFR. These findings reveal the essential role of zinc homeostasis in redox-dependent proteostasis within the ER.

KDEL receptors (KDELRs) prevent the secretion of soluble chaperones and enzymes meant to reside in the endoplasmic reticulum. While a single KDELR exists in yeast (ERD2), three variants are present in mammals, displaying high sequence similarity (73-83%). However, the phylogenetic conservation of the differences and the diverse tissue distribution of the three KDELRs suggest functional specialization. Here we show that, while all three receptors can prevent the secretion of KDEL-bearing clients, KDELR1 and KDELR3 regulate the production of AGR2, a key assistant of mucin folding, in opposite ways. AGR2 transcripts increase dramatically upon silencing KDELR3 but decrease when KDELR1 is downregulated. Silencing ERp44, but not other ER residents, phenocopies KDELR3 knockdown, suggesting that AGR2 regulation depends on ERp44-KDELR3 interactions. Our findings identify a novel regulatory circuit that controls the molecular composition of the early secretory pathway based on specific interactions between KDELRs and ER residents.

Immunoglobulin M (IgM) antibodies are an essential and conserved part of adaptive immunity. IgMs assemble into pentamers and hexamers that bind to antigens with high avidity. Pentamers incorporate a small protein called J-chain (JC) that is important for their transcytosis via the poly-immunoglobulin receptor (pIgR). IgM antibodies can efficiently activate complement and interact with different Fc receptors (FcμR, Fcα/μR, pIgR) that trigger distinct effector functions and biodistribution. Even if these features have made the clinical use of IgM attractive over the past decades, there are currently no approved therapeutic IgMs on the market. In this review, we summarize the recent advances in the knowledge of IgM biogenesis and structure and discuss the therapeutic opportunities of IgM over IgG arising from high avidity, target clustering, binding to distinct IgM receptors, complement activation, transcytosis, and protein engineering opportunities. In addition, we summarize possibilities and outstanding challenges in the production of therapeutic IgM, including available technologies for IgM purification. Finally, we review recent preclinical and clinical data showing that IgM outperforms IgG in various in vitro assays but still fails to pass through clinical trials successfully. Challenges remain for IgM development, such as the need for a better understanding of IgM biology to facilitate a smoother transition from the preclinic to successful clinical trials.

KDEL receptors (KDELRs) prevent the secretion of soluble chaperones and enzymes meant to reside in the endoplasmic reticulum. While a single KDELR exists in yeast (ERD2), three variants are present in mammals, displaying high sequence similarity. All three can prevent the secretion of KDEL-bearing clients. However, their diverse tissue distribution and the extremely high phylogenetic conservation of the differences suggest functional specialization. Accordingly, we show here that while KDELR2 plays a significant role in client retrieval, KDELR1 and KDELR3 regulate the production of AGR2, a key assistant of mucin folding, in opposite ways. AGR2 transcripts increase dramatically upon silencing KDELR3 but decrease when KDELR1 is downregulated. Silencing ERp44, but not other ER residents, phenocopies KDELR3 knockdown, suggesting that AGR2 regulation depends on ERp44-KDELR3 interactions. Our findings identify a novel regulatory circuit, independent from the unfolded protein response, that controls the molecular composition of the early secretory pathway based on specific interactions between KDELRs and ER residents.

KDEL receptors (KDELRs) prevent the secretion of soluble chaperones and enzymes meant to reside in the endoplasmic reticulum. While a single KDELR exists in yeast (ERD2), three variants are present in mammals, displaying high sequence similarity (73-83%). However, the phylogenetic conservation of the differences and the diverse tissue distribution of the three KDELRs suggest functional specialization. Here we show that, while all three receptors can prevent the secretion of KDEL-bearing clients, KDELR1 and KDELR3 regulate the production of AGR2, a key assistant of mucin folding, in opposite ways. AGR2 transcripts increase dramatically upon silencing KDELR3 but decrease when KDELR1 is downregulated. Silencing ERp44, but not other ER residents, phenocopies KDELR3 knockdown, suggesting that AGR2 regulation depends on ERp44-KDELR3 interactions. Our findings identify a novel regulatory circuit that controls the molecular composition of the early secretory pathway based on specific interactions between KDELRs and ER residents.

The unfolded protein response can switch from a pro-survival to a maladaptive, pro-apoptotic mode. During ER stress, IRE1α sensors dimerize, become phosphorylated, and activate XBP1 splicing, increasing folding capacity in the ER protein factory. The steps that turn on the IRE1α endonuclease activity against endogenous mRNAs during maladaptive ER stress are still unknown. Here, we show that although necessary, IRE1α dimerization is not sufficient to trigger phosphorylation. Random and/or guided collisions among IRE1α dimers are needed to elicit cross-phosphorylation and endonuclease activities. Thus, reaching a critical concentration of IRE1α dimers in the ER membrane is a key event. Formation of stable IRE1α clusters is not necessary for RNase activity. However, clustering could modulate the potency of the response, promoting interactions between dimers and decreasing the accessibility of phosphorylated IRE1α to phosphatases. The stepwise activation of IRE1α molecules and their low concentration at the steady state prevent excessive responses, unleashing full-blown IRE1 activity only upon intense stress conditions.

Significance: Fidelity of intercellular communication depends on unambiguous interactions between protein ligands and membrane receptors. Most proteins destined to the extracellular space adopt the required three-dimensional shape as they travel through the endoplasmic reticulum (ER), Golgi complex, and other organelles of the exocytic pathway. However, some proteins, many of which are involved in inflammation, avoid this classical secretory route and follow unconventional pathways to leave the cell. Recent Advances: Stringent quality control systems operate in the ER and cis-Golgi, restricting transport to native conformers, devoid of non-native disulfides and/or reactive thiols. However, some proteins released by living cells require reduced cysteines to exert their extracellular function(s). Remarkably, these proteins lack the secretory signal sequence normally required by secretory proteins for translocation into the ER lumen. Critical Issues: Why do interleukin-1β, high mobility group box 1, and other proinflammatory proteins avoid the ER-Golgi route to reach the intercellular space? These proteins require reactive cysteines for exerting their function. Therefore, eluding thiol-mediated quality control along the exocytic pathway is likely one of the main reasons why extracellular proteins that need to be reduced utilize unconventional pathways of secretion, where a quality control aimed at oxidating native cysteines is not present. Future Directions: Particularly under stress conditions, cells release redox-active enzymes and nonprotein thiol compounds that exert an extracellular control of redox-sensitive protein activity, shaping inflammatory responses. This post-secretion, redox-dependent editing of protein messages is still largely undefined. Understanding the underlying mechanistic events will hopefully provide new tools to control inflammation. Antioxid. Redox Signal. 41, 693-705.

Upon hormonal stimulation, uterine endometrial stromal cells undergo a dramatic morpho-functional metamorphosis that allows them to secrete large amounts of matrix proteins, cytokines, and growth factors. This step, known as decidualization, is crucial for embryo implantation. We previously demonstrated how the secretory pathway is remodelled during this process. Here we show that hormonal stimulation rapidly induces the expression of many mitochondrial genes, encoded in both the mitochondrial and the nuclear genomes. Altogether, the mitochondrial network quadruples its size and establishes more contacts with the ER. This new organization results in the increased respiratory capacity of decidualized cells. These findings reveal how achieving an efficient secretory phenotype requires a radical metabolic rewiring.

Abstract Polymeric IgM immunoglobulins have high avidity for antigen and complement, and dominate primary antibody responses. They are produced either as assemblies of six µ2L2 subunits (i.e., hexamers), or as pentamers of two µ2L2 subunits and an additional protein termed J-chain (JC), which allows transcytosis across epithelia. The molecular mechanism of IgM assembly with the desired stoichiometry remained unknown. Here, we show in vitro and in cellula that JC outcompetes the sixth IgM subunit during assembly. Before insertion into IgM, JC exists as an ensemble of largely unstructured, protease-sensitive species with heterogeneous, non-native disulfide bonds. The J-chain interacts with the hydrophobic β-sheets selectively exposed by nascent pentamers. Completion of an amyloid-like core triggers JC folding and drives disulfide rearrangements that covalently stabilize JC-containing pentamers. In cells, the quality control factor ERp44 surveys IgM assembly and prevents the secretion of aberrant conformers. This mechanism allows the efficient production of high-avidity IgM for systemic or mucosal immunity.

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Aquaporin11 (AQP11) is an endoplasmic reticulum (ER) resident peroxiporin. It allows H2O2 transport from the lumen to the cytosol, guaranteeing redox homeostasis and signaling in and between the two organelles. Interestingly, Aqp11-/- mice develop a fatal, early onset polycystic kidney disease (PKD) similar to Autosomal Dominant PKD, a condition frequently associated with mutations of polycystin-1 (PC-1) in human patients. Here we investigated the molecular mechanisms of AQP11-associated PKD. Using different cell models, we show that transient downregulation of AQP11 selectively prevents the biogenesis of overexpressed PC-1. Expression of catalase in the ER lumen rescues the phenotype, demonstrating a direct role of (H2O2)ER in controlling the complex maturation of PC-1. Analysis of endogenous Pc-1 revealed an additional regulatory role at the pre-translational level. Taken together, our results show that AQP11 controls the complex biogenesis of PC-1 at multiple levels governing H2O2 intra and inter-organellar fluxes, with important implications in the pathogenesis and onset of PKD.

Protein disulfide isomerase-A1 (PDIA1) is a master regulator of oxidative protein folding and proteostasis in the endoplasmic reticulum (ER). However, PDIA1 can reach the extracellular space, impacting thrombosis and other pathophysiological phenomena. Whether PDIA1 is externalized via passive release or active secretion is not known. To investigate how PDIA1 negotiates its export, we generated a tagged variant that undergoes N-glycosylation in the ER (Glyco-PDIA1). Addition of N- glycans does not alter its enzymatic functions. Upon either deletion of its KDEL ER-localization motif or silencing of KDEL receptors, Glyco-PDIA1 acquires complex glycans in the Golgi and is secreted. In control cells, however, Glyco-PDIA1 is released with endoglycosidase-H sensitive glycans, implying that it does not follow the classical ER-Golgi route, nor does it encounter glycanases in the cytosol. Extracellular Glyco-PDIA1 is more abundant than actin, lactate dehydrogenase or other proteins released by damaged or dead cells, suggesting active transport through a Golgi-independent route. The strategy we describe herein can be extended to dissect how select ER-residents reach the extracellular space.

Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria -ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induc-tion of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1a covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1a interaction requires the C-terminal active site of ERO1a and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1a complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca2+ flux to maintain bioenergetics in both organ-elles, while limiting oxidative stress. Therefore, the PERK-ERO1a complex is a key molecular machinery that allows quick metabolic adaptation to ER stress.