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.

Plasma cells require autophagy for sustainable immunoglobulin production.

The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5(-/-) differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.

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.

Evolution, role in inflammation, and redox control of leaderless secretory proteins.

Members of the interleukin (IL)-1 family are key determinants of inflammation. Despite their role as intercellular mediators, most lack the leader peptide typically required for protein secretion. This lack is a characteristic of dozens of other proteins that are actively and selectively secreted from living cells independently of the classical endoplasmic reticulum-Golgi exocytic route. These proteins, termed leaderless secretory proteins (LLSPs), comprise proteins directly or indirectly involved in inflammation, including cytokines such as IL-1ß and IL-18, growth factors such as fibroblast growth factor 2 (FGF2), redox enzymes such as thioredoxin, and proteins most expressed in the brain, some of which participate in the pathogenesis of neurodegenerative disorders. Despite much effort, motifs that promote LLSP secretion remain to be identified. In this review, we summarize the mechanisms and pathophysiological significance of the unconventional secretory pathways that cells use to release LLSPs. We place special emphasis on redox regulation and inflammation, with a focus on IL-1ß, which is secreted after processing of its biologically inactive precursor pro-IL-1ß in the cytosol. Although LLSP externalization remains poorly understood, some possible mechanisms have emerged. For example, a common feature of LLSP pathways is that they become more active in response to stress and that they involve several distinct excretion mechanisms, including direct plasma membrane translocation, lysosome exocytosis, exosome formation, membrane vesiculation, autophagy, and pyroptosis. Further investigations of unconventional secretory pathways for LLSP secretion may shed light on their evolution and could help advance therapeutic avenues for managing pathological conditions, such as diseases arising from inflammation.

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.

The unconventional secretion of IL-1ß: Handling a dangerous weapon to optimize inflammatory responses.

Interleukin 1ß (IL-1ß) is a major mediator of inflammation, with a causative role in many diseases. Unlike most other cytokines, however, it lacks a secretory signal sequence, raising intriguing mechanistic, functional and evolutionary questions. Despite decades of strenuous efforts in many laboratories, how IL-1ß is secreted is still a matter of intense debate. Here, we summarize the different mechanisms and pathways that have been proposed for IL-1ß secretion. At least two of them, namely the endolysosomal vesicle-based and gasdermin D-dependent pathways (types III and I in the recent Rabouille's classification of unconventional protein secretion), can be triggered in monocytes, the main source of IL-1ß in humans, according to the type and strength of the pro-inflammatory stimuli. As during the escalation of human conflicts, monocytes deploy secretory mechanisms of increasing efficiency and dangerousness, shifting from the specific and controlled type III pathway to the much faster release of type I. Thus, the different mechanisms are activated depending on the severity of the conditions, from the self-limiting type III pathways in response of low pathogen load or small trauma, to the uncontrolled responses that underlie autoinflammatory disorders and sepsis.

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19.

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.

Structural basis of pH-dependent client binding by ERp44, a key regulator of protein secretion at the ER-Golgi interface.

ERp44 retrieves some endoplasmic reticulum (ER)-resident enzymes and immature oligomers of secretory proteins from the Golgi. Association of ERp44 with its clients is regulated by pH-dependent mechanisms, but the molecular details are not fully understood. Here we report high-resolution crystal structures of human ERp44 at neutral and weakly acidic pH. These structures reveal key regions in the C-terminal tail (C tail) missing in the original crystal structure, including a regulatory histidine-rich region and a subsequent extended loop. The former region forms a short α-helix (α16), generating a histidine-clustered site (His cluster). At low pH, the three Trx-like domains of ERp44 ("a," "b," and "b'") undergo significant rearrangements, likely induced by protonation of His157 located at the interface between the a and b domains. The α16-helix is partially unwound and the extended loop is disordered in weakly acidic conditions, probably due to electrostatic repulsion between the protonated histidines in the His cluster. Molecular dynamics simulations indicated that helix unwinding enhances the flexibility of the C tail, disrupting its normal hydrogen-bonding pattern. The observed pH-dependent conformational changes significantly enlarge the positively charged regions around the client-binding site of ERp44 at low pH. Mutational analyses showed that ERp44 forms mixed disulfides with specific cysteines residing on negatively charged loop regions of Ero1α. We propose that the protonation states of the essential histidines regulate the ERp44-client interaction by altering the C-tail dynamics and surface electrostatic potential of ERp44.

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.

Atypical IgM on T cells predict relapse and steroid dependence in idiopathic nephrotic syndrome.

The clinical heterogeneity of idiopathic nephrotic syndrome in childhood may reflect different mechanisms of disease that are as yet unclear. Here, we evaluated the association between an atypical presence of IgM on the surface of T cells (T-cell IgM) and the response to steroid therapy in a total of 153 pediatric patients with idiopathic nephrotic syndrome in different phases of disease. At disease onset, T-cell IgM median levels were significantly elevated and predictive of risk of relapse in 47 patients. They were also significantly increased comparing 58 steroid-dependent to 8 infrequently relapsing and 14 frequently relapsing patients, especially during relapse, whereas they were within the normal range in 7 genetic steroid-resistant patients. T-cell IgM in vivo was not affected by the amount of total circulating IgM, nor by concomitant acute infections or oral immunosuppression. However, it was affected by rituximab treatment in 21 steroid-dependent patients. By in vitro experiments, elevated T-cell IgM was not influenced by total circulating IgM levels or by the presence of other circulating factors, and there was no distinctive antigen-specificity or atypical IgM polymerization. Rather, we found that increased T-cell IgM correlates with reduced IgM sialylation, which influences T-cell response to steroid inhibition and T-cell production of podocyte-damaging factors. Thus, the atypical presence of IgM on the surface of T cells may predispose a subset of steroid-sensitive pediatric patients with idiopathic nephrotic syndrome to a poor response to steroid therapy since disease onset.

Aberrant disulphide bonding contributes to the ER retention of alpha1-antitrypsin deficiency variants.

Mutations in alpha1-antitrypsin (AAT) can cause the protein to polymerise and be retained in the endoplasmic reticulum (ER) of hepatocytes. The ensuing systemic AAT deficiency leads to pulmonary emphysema, while intracellular polymers are toxic and cause chronic liver disease. The severity of this process varies considerably between individuals, suggesting the involvement of mechanistic co-factors and potential for therapeutically beneficial interventions. We show in Hepa1.6 cells that the mildly polymerogenic I (Arg39Cys) AAT mutant forms aberrant inter- and intra-molecular disulphide bonds involving the acquired Cys39 and the only cysteine residue in the wild-type (M) sequence (Cys232). Substitution of Cys39 to serine partially restores secretion, showing that disulphide bonding contributes to the intracellular retention of I AAT. Covalent homodimers mediated by inter-Cys232 bonding alone are also observed in cells expressing the common Z and other polymerising AAT variants where conformational behaviour is abnormal, but not in those expressing M AAT. Prevention of such disulphide linkage through the introduction of the Cys232Ser mutation or by treatment of cells with reducing agents increases Z AAT secretion. Our results reveal that disulphide interactions enhance intracellular accumulation of AAT mutants and implicate the oxidative ER state as a pathogenic co-factor. Redox modulation, e.g. by anti-oxidant strategies, may therefore be beneficial in AAT deficiency-associated liver disease.

Sialylation of N-linked glycans influences the immunomodulatory effects of IgM on T cells.

Human serum IgM Abs are composed of heavily glycosylated polymers with five glycosylation sites on the µ (heavy) chain and one glycosylation site on the J chain. In contrast to IgG glycans, which are vital for a number of biological functions, virtually nothing is known about structure-function relationships of IgM glycans. Natural IgM is the earliest Ig produced and recognizes multiple Ags with low affinity, whereas immune IgM is induced by Ag exposure and is characterized by a higher Ag specificity. Natural anti-lymphocyte IgM is present in the serum of healthy individuals and increases in inflammatory conditions. It is able to inhibit T cell activation, but the underlying molecular mechanism is not understood. In this study, to our knowledge, we show for the first time that sialylated N-linked glycans induce the internalization of IgM by T cells, which in turn causes severe inhibition of T cell responses. The absence of sialic acid residues abolishes these inhibitory activities, showing a key role of sialylated N-glycans in inducing the IgM-mediated immune suppression.

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.

Assessing Heterogeneity of Osteolytic Lesions in Multiple Myeloma by ¹H HR-MAS NMR Metabolomics.

Multiple myeloma (MM) is a malignancy of plasma cells characterized by multifocal osteolytic bone lesions. Macroscopic and genetic heterogeneity has been documented within MM lesions. Understanding the bases of such heterogeneity may unveil relevant features of MM pathobiology. To this aim, we deployed unbiased ¹H high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) metabolomics to analyze multiple biopsy specimens of osteolytic lesions from one case of pathological fracture caused by MM. Multivariate analyses on normalized metabolite peak integrals allowed clusterization of samples in accordance with a posteriori histological findings. We investigated the relationship between morphological and NMR features by merging morphological data and metabolite profiling into a single correlation matrix. Data-merging addressed tissue heterogeneity, and greatly facilitated the mapping of lesions and nearby healthy tissues. Our proof-of-principle study reveals integrated metabolomics and histomorphology as a promising approach for the targeted study of osteolytic lesions.

A RIDDle solved: why an intact IRE1α/XBP-1 signaling relay is key for humoral immune responses.

As they commit to plasma cell differentiation, B lymphocytes must swiftly gear up to produce and secrete huge amounts of antibodies. To develop their secretory capacity, B cells exploit a signaling pathway that is employed by all eukaryotic cells in response to endoplasmic reticulum stress. An article by Benhamron et al. in this issue of the European Journal of Immunology, [Eur. J. Immunol. 2014. 44: 867-876] sheds new light on why an intact IRE1/XBP-1 signaling relay is central to orchestrate the full-blown expansion of the secretory machinery needed for massive antibody production.

Dynamic regulation of Ero1α and peroxiredoxin 4 localization in the secretory pathway.

In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.

Crystal structures of human Ero1α reveal the mechanisms of regulated and targeted oxidation of PDI.

In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1α activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1α were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1α modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine-tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1α with the PDI b'-domain through its substrate-binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells.

Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins.

To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-µ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

Preferential secretion of oxidation-sensitive proteins by unconventional pathways: Why is this important for inflammation?

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) and Golgi complex and other organelles of the exocytic pathway. However, some proteins, many of which involved in inflammation, avoid this classical secretory route and follow unconventional pathways to leave the cell. Recent advance 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 IL-1ß, HMGB1 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 non-protein 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.