Search

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.

Summary 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., Graphical Abstract, Highlights • ERp44 governs a pH-regulated assembly control cycle in the early secretory pathway • Accessibility of ERp44’s active site and –RDEL ER retrieval motif is pH dependent • Unmasking of ERp44’s active site likely involves protonation of cysteine 29 • ERp44 captures client proteins at cisGolgi-equivalent pH for retrieval to the ER

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception. In the extracellular space, certain Ig-L chains form fibrils causing systemic amyloidosis. On the other hand, Ig variants lacking the first constant domain condense in dilated cisternae of the early secretory compartment, called Russell Bodies (RB), frequently observed in plasma cell dyscrasias, autoimmune diseases and chronic infections. RB biogenesis can be recapitulated in lymphoid and non-lymphoid cells by expressing mutant Ig-μ, providing powerful models to investigate the pathophysiology of endoplasmic reticulum storage disorders. Here we analyze the aggregation propensity and the biochemical features of the intra- and extra-cellular Ig deposits in human cells, revealing β-aggregated features for RB.

In the endoplasmic reticulum (ER) of human cells, ERO1a and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1a is essential to avoid ER hyperoxidation. To investigate how ERO1a oxidizes PDI selectively among more than 20 ER resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding b-hairpin of ERO1a specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b"-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a"-domain. ERO1a associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1a to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1! with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1a-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1a activity may suggest its physiological role in ER redox and protein homeostasis.

Due vasi Centuripe a Palermo sono stati analizzati utilizzando XRF portatile complementare e FTIR a riflessione totale spettroscopie. Le analisi consentono di chiarire la tavolozza e la tecnica della decorazione (pittura a tempera con un legante organico), e mostrano la presenza di pigmenti sia antichi che moderni (a causa di qualche recente riverniciatura). Uno dei risultati più interessanti è stata l'identificazione di due fasi del solfato di calcio nello strato sottostante i dipinti: a causa delle condizioni che trasformano il gesso in bassanite, la presenza di il gesso o, in alternativa, la bassanite in ciascuna parte analizzata possono rappresentare un criterio per distinguere l'originale dipinti di quelli restaurati / contraffatti. Two Centuripe vases in Palermo were analyzed using complementary portable XRF and Total Reflectance FTIR spectroscopies. The analyses allow to clarify the palette and the technique of decoration (tempera painting with an organic binder), and show the presence of both ancient and modern pigments (due to some recent repainting). One of the most interesting results was the identification of two phases of the calcium sulfate in the stratum underlying the paintings: due to the conditions that make the gypsum transform into bassanite, the presence of gypsum or alternatively bassanite in each analyzed part can represent a criterium for distinguishing the original paintings from the restored/ counterfeit ones.

Il cratere attico a fondo bianco del pittore Phiale (440 a.C.), esposto al Museo Archeologico "Pietro Griffo" di Agrigento è un manufatto di grande raffinatezza. Raffigura il mito di Perseo e Andromeda ed è molto interessante sia per l'iconografia che per la tecnica pittorica generalmente documentata su lekythoi e vasi più piccoli. La presente ricerca interdisciplinare si è concentrata sulla tecnica pittorica di questo cratere ed è stata condotta direttamente al Museo mediante tecniche spettroscopiche non invasive e non distruttive (fluorescenza a raggi X e TRFTIR). Lo studio, di cui presentiamo qui i risultati preliminari, intende definire la natura del terreno bianco e pigmenti (secondo bianco, rosso, nero con marrone e arancio) e la tecnica di realizzazione del vaso The Attic white-ground crater of the Phiale Painter (440 BC), exhibited at the Archaeological Museum “Pietro Griffo” of Agrigento is an artifact of great refinement. It depicts the myth of Perseus and Andromeda and it is very interesting for both the iconography and the painting technique generally documented on lekythoi and smaller vases. The present interdisciplinary research has focused on the painting technique of this crater and was conducted directly in the Museum by means of non-invasive and non-destructive spectroscopic techniques (x-ray fluorescence and TRFTIR). The study, of which we present here the preliminary results, intends to define the nature of the white ground and pigments (second-white, red, black with brown and orange) and the technique of realization of the vase.

Presentazione dei dati relativi alle pitture parietali del III e IV secolo d.C. scoperte dal Progetto Ostia Marina a Ostia antica.

Il contributo divulga la recente scoperta di un intonaco parietale recentemente rinvenuto nella città ellenistico romana di Phoinike, in Albania. Si tratta di un sistema completo riferibile al cd. Primo Stile pompeiano, che apre interessant scenari di ricerca sulla decorazione parietale in ambito epirota e illirico in età ellenistica.

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.

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception. In the extracellular space, certain Ig-L chains form fibrils causing systemic amyloidosis. On the other hand, Ig variants lacking the first constant domain condense in dilated cisternae of the early secretory compartment, called Russell Bodies (RB), frequently observed in plasma cell dyscrasias, autoimmune diseases and chronic infections. RB biogenesis can be recapitulated in lymphoid and non-lymphoid cells by expressing mutant Ig-μ, providing powerful models to investigate the pathophysiology of endoplasmic reticulum storage disorders. Here we analyze the aggregation propensity and the biochemical features of the intra- and extra-cellular Ig deposits in human cells, revealing β-aggregated features for RB.

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.

Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are generally dislocated across the membrane to be degraded by cytosolic proteasomes. To investigate how the quality control machinery handles individual subunits that are part of covalent oligomers, we have analyzed the fate of transport-competent Ig light (L) chains that form disulfide bonds with short-lived mu heavy chains. When expressed alone, L chains are secreted. In cells producing excess mu, most L chains are retained in the ER as covalent mu-L or mu2-L2 complexes. While mu chains present in these complexes are degraded by proteasomes, L chains are stable. Few L chains are secreted; most reassociate with newly synthesized mu chains. Therefore, interchain disulfide bonds are reduced in the ER lumen before the dislocation of mu chains in a site from which freed L chains can be rapidly reinserted in the assembly line. The ER can thus sustain the simultaneous formation and reduction of disulfide bonds.

Background: Ero1α and peroxiredoxin 4 contribute to disulfide formation in the early secretory compartment (ESC), but lack known retention signals. Results: Retention and localization of Ero1α and peroxiredoxin 4 are maintained through multistep and pH-dependent interactions with PDI and ERp44 in ESC. Conclusion: PDI and ERp44 dynamically localize Ero1α and peroxiredoxin 4 in ESC. Significance: The levels and localization of four interactors allow differential ESC redox control., 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.

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.

The endoplasmic reticulum (ER) is involved in many functions, including protein folding, redox homeostasis,and Ca2+ storage and signaling. To perform these multiple tasks, the ER is composed of distinct,specialized subregions, amongst which mitochondrial-associated ER membranes (MAM) emerge as key signalinghubs. How these multiple functions are integrated with one another in living cells remains unclear.Results: Here we show that Ero1a, a key controller of oxidative folding and ER redox homeostasis, is enriched inMAM and regulates Ca2+ fluxes. Downregulation of Ero1a by RNA interference inhibits mitochondrial Ca2+fluxes and modifies the activity of mitochondrial Ca2+ uniporters. The overexpression of redox active Ero1aincreases passive Ca2+ efflux from the ER, lowering [Ca2+]ER and mitochondrial Ca2+ fluxes in response to IP3agonists. Innovation: The unexpected observation that Ca2+ fluxes are affected by either increasing or decreasingthe levels of Ero1a reveals a pivotal role for this oxidase in the early secretory compartment and implies a strictcontrol of its amounts. Conclusions: Taken together, our results indicate that the levels, subcellular localization,and activity of Ero1a coordinately regulate Ca2+ and redox homeostasis and signaling in the early secretorycompartment. Antioxid. Redox Signal. 16, 1077–1087.

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-μ chains, as a model to mechanistically dissect protein condensation. Depending on the presence or absence of Ig-L chains, mutant Ig-μ chains lacking their first constantdomain (CH1) accumulate in rough or smooth RB (rRB and sRB), dilations of the endoplasmic reticulum (ER) and ERGolgi 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 μ!CH1 secretion. However, PDI inhibits while ERp44 promotes μ!CH1 condensation. Both Ero1! 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. Key words: early secretory compartment, endoplasmic reticulum, ERGIC, protein condensation

Owing to the impossibility of reaching the Golgi for secretion or the cytosol for degradation, mutant Ig-mu chains that lack the first constant domain (muDeltaCH1) accumulate as detergent-insoluble aggregates in dilated endoplasmic reticulum cisternae, called Russell bodies. The presence of similar structures hallmarks many ER storage diseases, but their pathogenic role(s) remain obscure. Exploiting inducible cellular systems, we show here that Russell bodies form when the synthesis of muDeltaCH1 exceeds the degradation capacity. Condensation occurs in different sub-cellular locations, depending on the interacting molecules present in the host cell: if Ig light chains are co-expressed, detergent-insoluble muDeltaCH1-light chain oligomers accumulate in large ribosome-coated structures (rough Russell bodies). In absence of light chains, instead, aggregation occurs in smooth tubular vesicles and is controlled by N-glycan-dependent interactions with ER-Golgi intermediate compartment 53 (ERGIC-53). In cells containing smooth Russell bodies, ERGIC-53 co-localizes with muDeltaCH1 aggregates in a Ca2+ -dependent fashion. Our findings identify a novel ERGIC-53 substrate, and indicate that interactions with light chains or ERGIC-53 seed muDeltaCH1 condensation in different stations of the early secretory pathway.

Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1 alpha, indicating that cytosolic GSH and lumenal Ero1 alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1 alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins.

B lymphocytes are small cells that express antigen receptors and secrete little if any IgM. Upon encounter with antigen, they differentiate into short-lived plasma cells, which secrete large amounts of polymeric IgM. Plasma cell differentiation entails a massive development of the endoplasmic reticulum to sustain high levels of Ig production. Recent findings suggest a role for the unfolded protein response in orchestrating the architectural and functional changes during terminal plasma cell differentiation.

The tailpiece of secretory Ig-μ-chains (μ s tp) is highly conserved throughout evolution: in particular, a carboxy-terminal cysteine residue (Cys575) and a glycan linked to Asn563 are found in all species sequenced so far. Here we show that the μ s tp oligosaccharide moieties are important for the binding of J-chains and for the process of IgM polymerization. In the absence of the μ s tp glycans, pentamers cannot be assembled and polymers containing six or more subunits are secreted. Despite their increased valency, these molecules have a lower association rate with antigen than wild-type polymers. Unexpectedly, the C-terminal oligosaccharides also affect kinetic parameters on unpolymerized subunits. Thus, monomers lacking the C-terminal sugars because of either site-directed mutagenesis or selective enzymatic deglycosylation with endoglycosidase H, have a lower k on for the antigen. Taken together, our results indicate that the C-terminal μ -chain glycans can shape the structure of μ s2 L 2 subunits and their further assembly into polymers.

Plasma cells secrete IgM only in the polymeric form: the C-terminal cysteine of the mu heavy chain (Cys575) is responsible for both intracellular retention and assembly of IgM subunits. Polymerization is not quantitative, and part of IgM is degraded intracellularly. Neither chloroquine nor brefeldin A (BFA) inhibits degradation, suggesting that this process occurs in a pre-Golgi compartment. Degradation of IgM assembly intermediates requires Cys575: the monomeric IgMala575 mutant is stable also when endoplasmic reticulum (ER) to Golgi transport is blocked by BFA. Addition of the 20 C-terminal residues of mu to the lysosomal protease cathepsin D is sufficient to induce pre-Golgi retention and degradation of the chimeric protein: the small amounts of molecules which exit from the ER are mostly covalent dimers. By contrast, when retained by the KDEL sequence, cathepsin D is stable in the ER, indicating that retention is not sufficient to cause degradation. Replacing the C-terminal cysteine with serine restores transport through the Golgi. As all chimeric cathepsin D constructs display comparable protease activity in vitro, their different fates are not determined by gross alterations in folding. Thus, also out of its normal context, the mu chain Cys575 plays a crucial role in quality control, mediating assembly, retention and degradation.

Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi.