Roberto Sitia

A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence.

Interleukin 1 (IL‐1) is a major soluble mediator of inflammation. Two human IL‐1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20‐30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL‐1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum‐‐Golgi route. Drugs which block the intracellular transport of IL‐6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL‐1 beta. Secretion of IL‐1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL‐1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL‐1 beta are made but none is secreted. In these cells IL‐1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL‐1 beta are part of the protein secretory pathway. We conclude that IL‐1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.

Quality control in the endoplasmic reticulum protein factory

The endoplasmic reticulum (ER) is a factory where secretory proteins are manufactured, and where stringent quality-control systems ensure that only correctly folded proteins are sent to their final destinations. The changing needs of the ER factory are monitored by integrated signalling pathways that constantly adjust the levels of folding assistants. ER chaperones and signalling molecules are emerging as drug targets in amyloidoses and other protein-conformational diseases.

Protein degradation in the endoplasmic reticulum

Etat actuel des connaissances sur le processus de degradation des proteines au sein du reticulum endoplasmique: interet porte aux proteines membranaires et proteines secretees

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.

Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation

T lymphocytes are defective in cystine uptake and thus require exogenous thiols for activation and function. Here we show that monocyte-derived human dendritic cells (DCs) release cysteine in the extracellular space. Cysteine generation is increased by lipopolysaccharide and tumor necrosis factor α, and by contact with T cells specifically recognizing soluble or alloantigens. These stimuli also induce thioredoxin (TRX) accumulation in DCs. However, only the contact with antigen-specific T cells triggers TRX secretion by the antigen-presenting cells. Fewer extracellular thiols are recovered after DC–T cell interactions when cystine uptake or TRX activity are inhibited. In addition, glutamate (Glu) and anti-TRX-inactivating antibodies inhibit antigen-dependent T lymphocyte proliferation. These findings indicate that, during antigen presentation, DCs uptake cystine and release cysteine and TRX, thus providing a reducing microenvironment that facilitates immune response.

Sequential Waves of Functionally Related Proteins Are Expressed When B Cells Prepare for Antibody Secretion

Upon encounter with antigen, B lymphocytes differentiate into Ig-secreting plasma cells. This step involves a massive development of secretory organelles, most notably the endoplasmic reticulum. To analyze the relationship between organelle reshaping and Ig secretion, we performed a dynamic proteomics study of B lymphoma cells undergoing in vitro terminal differentiation. By clustering proteins according to temporal expression patterns, it appeared that B cells anticipate their secretory role in a multistep process. Metabolic capacity and secretory machinery expand first to accommodate the mass production of IgM that follows.

Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells

Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)‐sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N‐terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re‐accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady‐state Ca2+ concentration was obtained using Sr2+, a well‐known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady‐state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3‐fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10‐fold drop in a few minutes.

A novel pathway for secretory proteins

In eukaryotes, most proteins which are transported to the extracellular space, into mitochondria or into chloroplasts are synthesized as precursor polypeptides containing cleavable N-terminal signal or targeting sequences. We have searched the literature for proteins that are exported from the cytosol without being proteolytically processed. Some of these proteins contain uncleaved signal or targeting sequences. However, among secretory proteins there is a class that does not possess hydrophobic signal sequences and appears to leave the cell by a secretory pathway clearly distinct from the classical route through the endoplasmic reticulum and Golgi apparatus.

Manipulation of oxidative protein folding and PDI redox state in mammalian cells

In the endoplasmic reticulum (ER), disulfide bonds are simultaneously formed in nascent proteins and removed from incorrectly folded or assembled molecules. In this compartment, the redox state must be, therefore, precisely regulated. Here we show that both human Ero1‐Lα and Ero1‐Lβ (hEROs) facilitate disulfide bond formation in immunoglobulin subunits by selectively oxidizing PDI. Disulfide bond formation is controlled by hEROs, which stand at a crucial point of an electron‐flow starting from nascent secretory proteins and passing through PDI. The redox state of ERp57, another ER‐resident oxidoreductase, is not affected by over‐expression of Ero1‐Lα, suggesting that parallel and specific pathways control oxidative protein folding in the ER. Mutants in the Ero1‐Lα CXXCXXC motif act as dominant negatives by limiting immunoglobulin oxidation. PDI‐dependent oxidative folding in living cells can thus be manipulated by using hERO variants.

Endoplasmic Reticulum Oxidoreductin 1-Lβ (ERO1-Lβ), a Human Gene Induced in the Course of the Unfolded Protein Response

Oxidative conditions must be generated in the endoplasmic reticulum (ER) to allow disulfide bond formation in secretory proteins. A family of conserved genes, termed EROfor ER oxidoreductins, plays a key role in this process. We have previously described the human gene ERO1-L, which complements several phenotypic traits of the yeast thermo-sensitive mutant ero1-1 (Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery, M. R., Bulleid, N. J., and Sitia, R. (2000)J. Biol. Chem. 275, 4827–4833). Here, we report the cloning and characterization of a novel human member of this family,ERO1-Lβ. Immunofluorescence, endoglycosidase sensitivity, and in vitro translation/translocation assays reveal that the products of the ERO1-Lβ gene are primarily localized in the ER of mammalian cells. The ability to allow growth at 37 °C and to alleviate the “unfolded protein response” when expressed inero1-1 cells indicates that ERO1-Lβ is involved also in generating oxidative conditions in the ER. ERO1-L and ERO1-Lβ display different tissue distributions. Furthermore, only ERO1-Lβ transcripts are induced in the course of the unfolded protein response. Our results suggest a complex regulation of ER redox homeostasis in mammalian cells.