Biogenesis of secretory immunoglobulin M requires intermediate non-native disulfide bonds and engagement of the protein disulfide isomerase ERp44.

Antibodies of the immunoglobulin M (IgM) class represent the frontline of humoral immune responses. They are secreted as planar polymers in which flanking µ2 L2 "monomeric" subunits are linked by two disulfide bonds, one formed by the penultimate cysteine (C575) in the tailpiece of secretory µ chains (µs tp) and the second by C414 in the Cµ3. The latter bond is not present in membrane IgM. Here, we show that C575 forms a non-native, intra-subunit disulfide bond as a key step in the biogenesis of secretory IgM. The abundance of this unexpected intermediate correlates with the onset and extent of polymerization. The rearrangement of the C-terminal tails into a native quaternary structure is guaranteed by the engagement of protein disulfide isomerase ERp44, which attacks the non-native C575 bonds. The resulting conformational changes promote polymerization and formation of C414 disulfide linkages. This unusual assembly pathway allows secretory polymers to form without the risk of disturbing the role of membrane IgM as part of the B cell antigen receptor.

Profound architectural and functional readjustments of the secretory pathway in decidualization of endometrial stromal cells.

Certain cell types must expand their exocytic pathway to guarantee efficiency and fidelity of protein secretion. A spectacular case is offered by decidualizing human endometrial stromal cells (EnSCs). In the midluteal phase of the menstrual cycle, progesterone stimulation induces proliferating EnSCs to differentiate into professional secretors releasing proteins essential for efficient blastocyst implantation. Here, we describe the architectural rearrangements of the secretory pathway of a human EnSC line (TERT-immortalized human endometrial stromal cells (T-HESC)). As in primary cells, decidualization entails proliferation arrest and the coordinated expansion of the entire secretory pathway without detectable activation of unfolded protein response (UPR) pathways. Decidualization proceeds also in the absence of ascorbic acid, an essential cofactor for collagen biogenesis, despite also the secretion of some proteins whose folding does not depend on vitamin C is impaired. However, even in these conditions, no overt UPR induction can be detected. Morphometric analyses reveal that the exocytic pathway does not increase relatively to the volume of the cell. Thus, differently from other cell types, abundant production is guaranteed by a coordinated increase of the cell size following arrest of proliferation.

The pivotal role of ERp44 in patrolling protein secretion.

Interactions between protein ligands and receptors are the main language of intercellular communication; hence, how cells select proteins to be secreted or presented on the plasma membrane is a central concern in cell biology. A series of checkpoints are located along the secretory pathway, which ensure the fidelity of such protein signals (quality control). Proteins that pass the checkpoints operated in the endoplasmic reticulum (ER) by the binding immunoglobulin protein (BiP; also known as HSPA5 and GRP78) and the calnexin-calreticulin systems, must still overcome additional scrutiny in the ER-Golgi intermediate compartment (ERGIC) and the Golgi. One of the main players of this process in all metazoans is the ER-resident protein 44 (ERp44); by cycling between the ER and the Golgi, ERp44 controls the localization of key enzymes designed to act in the ER but that are devoid of suitable localization motifs. ERp44 also patrols the secretion of correctly assembled disulfide-linked oligomeric proteins. Here, we discuss the mechanisms driving ERp44 substrate recognition, with important consequences on the definition of 'thiol-mediated quality control'. We also describe how pH and zinc gradients regulate the functional cycle of ERp44, coupling quality control and membrane trafficking along the early secretory compartment.

A pH-regulated quality control cycle for surveillance of secretory protein assembly.

To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44's active cysteine, simultaneously unmask the substrate binding site and -RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles.

A peptide extension dictates IgM assembly.

Professional secretory cells can produce large amounts of high-quality complex molecules, including IgM antibodies. Owing to their multivalency, polymeric IgM antibodies provide an efficient first-line of defense against pathogens. To decipher the mechanisms of IgM assembly, we investigated its biosynthesis in living cells and faithfully reconstituted the underlying processes in vitro. We find that a conserved peptide extension at the C-terminal end of the IgM heavy (Ig-µ) chains, termed the tailpiece, is necessary and sufficient to establish the correct geometry. Alanine scanning revealed that hydrophobic amino acids in the first half of the tailpiece contain essential information for generating the correct topology. Assembly is triggered by the formation of a disulfide bond linking two tailpieces. This induces conformational changes in the tailpiece and the adjacent domain, which drive further polymerization. Thus, the biogenesis of large and topologically challenging IgM complexes is dictated by a local conformational switch in a peptide extension.

Progressive quality control of secretory proteins in the early secretory compartment by ERp44.

ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes.

Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface.

Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

Pathogenesis of ER storage disorders: modulating Russell body biogenesis by altering proximal and distal quality control.

In many protein storage diseases, detergent-insoluble proteins accumulate in the early secretory compartment (ESC). Protein condensation reflects imbalances between entry into (synthesis/translocation) and exit from (secretion/degradation) ESC, and can be also a consequence of altered quality control (QC) mechanisms. Here we exploit the inducible formation of Russell bodies (RB), dilated ESC cisternae containing mutant Ig-micro chains, as a model to mechanistically dissect protein condensation. Depending on the presence or absence of Ig-L chains, mutant Ig-micro chains lacking their first constant domain (Ch1) accumulate in rough or smooth RB (rRB and sRB), dilations of the endoplasmic reticulum (ER) and ER-Golgi intermediate compartment (ERGIC), respectively, reflecting the proximal and distal QC stations in the stepwise biogenesis of polymeric IgM. Either weakening ERp44-dependent distal QC or facilitating ER-associated degradation (ERAD) inhibits RB formation. Overexpression of PDI or ERp44 inhibits muDeltaCh1 secretion. However, PDI inhibits while ERp44 promotes muDeltaCh1 condensation. Both Ero1alpha silencing and overexpression prevent RB formation, demonstrating a strict redox dependency of the phenomenon. Altogether, our findings identify key controllers of protein condensation along the ESC as potential targets to handle certain storage disorders.

Physiology and pathology of proteostasis in the early secretory compartment.

The endoplasmic reticulum (ER), the port of entry for proteins into the secretory pathway, is a multifunctional organelle emerging as a central integrator of numerous signalling pathways. The mechanisms that control proteostasis are integral part of this signalling network, providing cues for morphological and functional cell remodelling, proliferation, inflammation and cell death. The complexity of ER responses is exploited during physiological and pathological tissue development, cell differentiation and lifespan control. This essay outlines some of the mechanisms that link proteostasis within the early secretory compartment to signalling in development and disease.

Protein quality control in the early secretory pathway.

Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)-Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. 'Public' and 'private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion.

CREB3L1 and CREB3L2 control Golgi remodelling during decidualization of endometrial stromal cells.

Upon progesterone stimulation, Endometrial Stromal Cells (EnSCs) undergo a differentiation program into secretory cells (decidualization) to release in abundance factors crucial for embryo implantation. We previously demonstrated that decidualization requires massive reshaping of the secretory pathway and, in particular, of the Golgi complex. To decipher the underlying mechanisms, we performed a time-course transcriptomic analysis of in vitro decidualizing EnSC. Pathway analysis shows that Gene Ontology terms associated with vesicular trafficking and early secretory pathway compartments are the most represented among those enriched for upregulated genes. Among these, we identified a cluster of co-regulated genes that share CREB3L1 and CREB3L2 binding elements in their promoter regions. Indeed, both CREB3L1 and CREB3L2 transcription factors are up-regulated during decidualization. Simultaneous downregulation of CREB3L1 and CREB3L2 impairs Golgi enlargement, and causes dramatic changes in decidualizing EnSC, including Golgi fragmentation, collagen accumulation in dilated Endoplasmic Reticulum cisternae, and overall decreased protein secretion. Thus, both CREB3L1 and CREB3L2 are required for Golgi reshaping and efficient protein secretion, and, as such, for successful decidualization.

A virtuous cycle operated by ERp44 and ERGIC-53 guarantees proteostasis in the early secretory compartment.

The composition of the secretome depends on the combined action of cargo receptors that facilitate protein transport and sequential checkpoints that restrict it to native conformers. Acting after endoplasmic reticulum (ER)-resident chaperones, ERp44 retrieves its clients from downstream compartments. To guarantee efficient quality control, ERp44 should exit the ER as rapidly as its clients, or more. Here, we show that appending ERp44 to different cargo proteins increases their secretion rates. ERp44 binds the cargo receptor ER-Golgi intermediate compartment (ERGIC)-53 in the ER to negotiate preferential loading into COPII vesicles. Silencing ERGIC-53, or competing for its COPII binding with 4-phenylbutyrate, causes secretion of Prdx4, an enzyme that relies on ERp44 for intracellular localization. In more acidic, zinc-rich downstream compartments, ERGIC-53 releases its clients and ERp44, which can bind and retrieve non-native conformers via KDEL receptors. By coupling the transport of cargoes and inspector proteins, cells ensure efficiency and fidelity of secretion.

Endoplasmic reticulum oxidoreductase 1 alpha modulates prostate cancer hallmarks.

BACKGROUND: Therapies available for late stage prostate cancer (PCa) patients are limited and mostly palliative. The necessary development of unexplored therapeutic options relies on a deeper knowledge of molecular mechanisms leading to cancer progression. Redox signals are known to modulate the intensity and duration of oncogenic circuits; cues originating from the endoplasmic reticulum (ER) and downstream exocytic organelles are relevant in secretory tumors, including PCa. Ero 1α is a master regulator of redox homeostasis and oxidative folding. METHODS: We assessed Ero 1α mRNA expression by bioinformatic analysis of three public datasets and protein expression levels in PCa cell lines representing different degrees of tumor progression and different human prostate specimens. Transient Ero 1α knockdown was achieved by RNA interference (siRNA). Consequences of Ero 1α downregulation were monitored by PCa proliferation, migration and invasion properties. RESULTS: Ero 1α mRNA and protein levels are upregulated in PCa cell lines compared to non-tumorigenic cells (P=0.0273). Ero 1α expression increases with the grade of malignancy, reaching the highest level in the androgen resistant PC3. In patients' samples from 3 datasets, Ero 1α mRNA expression correlates with pathological Gleason scores. Ero 1α knockdown inhibits proliferation (P=0.0081), migration (P=0.0085) and invasion (P=0.0007) of PC3 cells and alters the levels of integrin ß1 (P=0.0024). CONCLUSIONS: Results indicate that Ero 1α levels correlate with PCa aggressiveness; Ero 1α silencing inhibits key steps over the PCa metastatic process. Therefore, Ero 1α has the potential to be exploited as a novel biomarker and a therapeutic target in PCa.

Crystal structure of human ERp44 shows a dynamic functional modulation by its carboxy-terminal tail.

ERp44 mediates thiol-dependent retention in the early secretory pathway, forming mixed disulphides with substrate proteins through its conserved CRFS motif. Here, we present its crystal structure at a resolution of 2.6 A. Three thioredoxin domains-a, b and b'-are arranged in a clover-like structure. A flexible carboxy-terminal tail turns back to the b' and a domains, shielding a hydrophobic pocket in domain b' and a hydrophobic patch around the CRFS motif in domain a. Mutational and functional studies indicate that the C-terminal tail gates the CRFS area and the adjacent hydrophobic pocket, dynamically regulating protein quality control.

How to Avoid a No-Deal ER Exit.

Efficiency and fidelity of protein secretion are achieved thanks to the presence of different steps, located sequentially in time and space along the secretory compartment, controlling protein folding and maturation. After entering into the endoplasmic reticulum (ER), secretory proteins attain their native structure thanks to specific chaperones and enzymes. Only correctly folded molecules are allowed by quality control (QC) mechanisms to leave the ER and proceed to downstream compartments. Proteins that cannot fold properly are instead retained in the ER to be finally destined to proteasomal degradation. Exiting from the ER requires, in most cases, the use of coated vesicles, departing at the ER exit sites, which will fuse with the Golgi compartment, thus releasing their cargoes. Protein accumulation in the ER can be caused by a too stringent QC or by ineffective transport: these situations could be deleterious for the organism, due to the loss of the secreted protein, and to the cell itself, because of abnormal increase of protein concentration in the ER. In both cases, diseases can arise. In this review, we will describe the pathophysiology of protein folding and transport between the ER and the Golgi compartment.

Zinc regulates ERp44-dependent protein quality control in the early secretory pathway.

Zinc ions (Zn2+) are imported into the early secretory pathway by Golgi-resident transporters, but their handling and functions are not fully understood. Here, we show that Zn2+ binds with high affinity to the pH-sensitive chaperone ERp44, modulating its localization and ability to retrieve clients like Ero1α and ERAP1 to the endoplasmic reticulum (ER). Silencing the Zn2+ transporters that uptake Zn2+ into the Golgi led to ERp44 dysfunction and increased secretion of Ero1α and ERAP1. High-resolution crystal structures of Zn2+-bound ERp44 reveal that Zn2+ binds to a conserved histidine-cluster. The consequent large displacements of the regulatory C-terminal tail expose the substrate-binding surface and RDEL motif, ensuring client capture and retrieval. ERp44 also forms Zn2+-bridged homodimers, which dissociate upon client binding. Histidine mutations in the Zn2+-binding sites compromise ERp44 activity and localization. Our findings reveal a role of Zn2+ as a key regulator of protein quality control at the ER-Golgi interface.

Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-µ chains in the early secretory pathway.

The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-µ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant µ chains lacking the Cµ1 domain (µ∆). Our data reveal that µ∆ lacking 563 glycans (µ∆5) form larger intracellular aggregates than µ∆ and are not secreted. Like µ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, µ∆ lacking 402 glycans (µ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (µ∆4-5). These results suggest that the two C-terminal Ig-µ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers.

Regulation of Calcium Fluxes by GPX8, a Type-II Transmembrane Peroxidase Enriched at the Mitochondria-Associated Endoplasmic Reticulum Membrane.

Glutathione peroxidases (GPXs) are enzymes that are present in almost all organisms with the primary function of limiting peroxide accumulation. In mammals, two of the eight members (GPX7 and GPX8) reside in the endoplasmic reticulum (ER). A peculiar feature of GPX8 is the concomitant presence of a conserved N-terminal transmembrane domain (TMD) and a C-terminal KDEL-like motif for ER localization. AIMS: Investigating whether and how GPX8 impacts Ca2+ homeostasis and signaling. RESULTS: We show that GPX8 is enriched in mitochondria-associated membranes and regulates Ca2+ storage and fluxes. Its levels correlate with [Ca2+]ER, and cytosolic and mitochondrial Ca2+ fluxes. GPX7, which lacks a TMD, does not share these properties. Deleting or replacing the GPX8 TMD with an unrelated N-terminal membrane integration sequence abolishes all effects on Ca2+ fluxes, whereas appending the GPX8 TMD to GPX7 transfers the Ca2+-regulating properties. Innovation and Conclusion: The notion that the TMD of GPX8, in addition to its enzymatic activity, is essential for regulating Ca2+ dynamics reveals a novel level of integration between redox-related proteins and Ca2+ signaling/homeostasis. Antioxid. Redox Signal. 27, 583-595.

Proteostasis and "redoxtasis" in the secretory pathway: Tales of tails from ERp44 and immunoglobulins.

In multicellular organisms, some cells are given the task of secreting huge quantities of proteins. To comply with their duty, they generally equip themselves with a highly developed endoplasmic reticulum (ER) and downstream organelles in the secretory pathway. These professional secretors face paramount proteostatic challenges in that they need to couple efficiency and fidelity in their secretory processes. On one hand, stringent quality control (QC) mechanisms operate from the ER onward to check the integrity of the secretome. On the other, the pressure to secrete can be overwhelming, as for instance on antibody-producing cells during infection. Maintaining homeostasis is particularly hard when the products to be released contain disulfide bonds, because oxidative folding entails production of reactive oxygen species. How are redox homeostasis ("redoxtasis") and proteostasis maintained despite the massive fluxes of cargo proteins traversing the pathway? Here we describe recent findings on how ERp44, a multifunctional chaperone of the secretory pathway, can modulate these processes integrating protein QC, redoxtasis, and calcium signaling.