Guanabenz inhibits TLR9 signaling through a pathway that is independent of eIF2α dephosphorylation by the GADD34/PP1c complex

Endoplasmic reticulum (ER) stress triggers or amplifies inflammatory signals and cytokine production in immune cells. Upon the resolution of ER stress, the inducible phosphatase 1 cofactor GADD34 promotes the dephosphorylation of the initiation factor eIF2α, thereby enabling protein translation to resume. Several aminoguanidine compounds, such as guanabenz, perturb the eIF2α phosphorylation-dephosphorylation cycle and protect different cell or tissue types from protein misfolding and degeneration. We investigated how pharmacological interference with the eIF2α pathway could be beneficial to treat autoinflammatory diseases dependent on proinflammatory cytokines and type I interferons (IFNs), the production of which is regulated by GADD34 in dendritic cells (DCs). In mouse and human DCs and B cells, guanabenz prevented the activation of Toll-like receptor 9 (TLR9) by CpG oligodeoxynucleotides or DNA-immunoglobulin complexes in endosomes. In vivo, guanabenz protected mice from CpG oligonucleotide-dependent cytokine shock and decreased autoimmune symptom severity in a chemically induced model of systemic lupus erythematosus. However, we found that guanabenz exerted its inhibitory effect independently of GADD34 activity on eIF2α and instead decreased the abundance of CH25H, a cholesterol hydroxylase linked to antiviral immunity. Our results therefore suggest that guanabenz and similar compounds could be used to treat type I IFN-dependent pathologies and that CH25H could be a therapeutic target to control these diseases.publishe

Induction of GADD34 Is Necessary for dsRNA-Dependent Interferon-β Production and Participates in the Control of Chikungunya Virus Infection

Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection

Splicing of antigenic peptides by the proteasome

Antigens presented by MHC class I molecules to CD8+ T lymphocytes consist of peptides of 8-11 amino acids, corresponding to continuous fragments of intracellular proteins. However, some antigenic peptides were found to be created by the splicing of two distinct fragments of the respective parental proteins. Peptide splicing represents a newly described mode of production of antigenic peptides whereby two noncontiguous peptide fragments are joined together after the excision of an intervening segment. Three spliced antigenic peptides have been described. The first example is derived from fibroblast growth factor-5 (FGF-5) and is made up of two fragments of five and four residues. Another is a peptide produced from melanosomal protein gp100 by splicing of two fragments of three and six residues. The third example is a human minor histocompatibility antigenic peptide created by a polymorphism in the SP110 gene. This epitope is composed of two noncontiguous fragments of four and six residues which are spliced together in the reverse order to that in which they appear in the parental protein. In the two latter cases, splicing involves the excision of a short intervening segment of four or six residues and was shown to occur in the proteasome by transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by the N terminus of a peptide fragment present at the active site. In a first part of this work, we studied the mechanism of production of the FGF-5 spliced peptide, for which the length of the intervening segment (40 aa) is much longer than the two other described examples. We confirmed that the proteasome also produces this spliced peptide by transpeptidation and we showed that reducing the length of the intervening segment increased the production of the spliced peptide. We also tested the possibility of trans-splicing (i.e., splicing of fragments from two distinct substrates) and observed that this event can occur in the cell between two peptide precursors with the same efficiency as cis-splicing. However, trans-splicing between two distinct proteins was far less efficient than splicing of peptide fragments from a single protein substrate and unlikely contributes to the production of the spliced peptides derived from FGF-5 and gp100. The production of the three spliced peptides by the standard proteasome and the immunoproteasome was then compared in details, using a cellular approach and an in vitro approach. We showed that both proteasome types can produce spliced peptides although they differ in their efficiency of production of each peptide. The FGF-5 and gp100 peptides are produced more efficiently by the standard proteasome, while the SP110 peptide is better produced by the immunoproteasome. By showing that splicing depends on the efficiency of production of the splicing partners, these results support the transpeptidation model of peptide splicing. In collaboration with Dr. Paul Robbins from the National Cancer Institute in Bethesda, we have identified a new spliced antigenic peptide, which is derived from the melanosomal protein tyrosinase. This original peptide is composed of two spliced and rearranged peptide fragments, each one containing an aspartate residue resulting from asparagine deamidation. The processing of this antigenic peptide involves retrotranslocation of tyrosinase molecules from the endoplasmic reticulum (ER) to the cytoplasm, deamidation of presumably glycosylated asparagines by peptide-N-glycanase, cleavage and splicing by the standard proteasome, and transport of the resulting peptide into the ER via the TAP transporter. The potential role of an additional, yet unidentified, protease during this process is also discussed in this work.(BIOL 3) -- UCL, 201

L'inhibition du protéasome comme thérapie anticancéreuse (exemple du Bortézomib)

Le système ubiquitine/protéasome est responsable du renouvellement sélectif de la plupart des protéines intracellulaires. Il joue un rôle important dans la régulation de divers processus cellulaires tels que la progression du cycle cellulaire, l'apoptose et la génération d'antigènes. Comme ces processus sont primordiaux pour la prolifération et la survie de toutes les cellules, et en particulier des cellules cancéreuses, l'inhibition du protéasome représente une voie thérapeutique potentielle particulièrement prometteuse. Des études pré-cliniques ayant démontré une activité cytotoxique encourageante, le bortézomib a été le premier inhibiteur du protéasome à être évalué lors des essais cliniques. Les résultats obtenus au cours d'un essai de phase II sur son efficacité et sa tolérance ont contribué à son approbation pour le traitement de patients atteints de myélome multiple récidivant et résistant aux traitements conventionnels.TOULOUSE3-BU Santé-Centrale (315552105) / SudocTOULOUSE3-BU Santé-Allées (315552109) / SudocSudocFranceF

Splicing of distant peptide fragments occurs in the proteasome by transpeptidation and produces the spliced antigenic peptide derived from fibroblast growth factor-5

Peptide splicing is a newly described mode of production of antigenic peptides presented by MHC class I molecules, whereby two noncontiguous fragments of the parental protein are joined together after excision of the intervening segment. Three spliced peptides have been described. In two cases, splicing involved the excision of a short intervening segment of 4 or 6 aa and was shown to occur in the proteasome by transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by the N terminus of the other peptide fragment. For the third peptide, which is derived from fibroblast growth factor-5 (FGF-5), the splicing mechanism remains unknown. In this case, the intervening segment is 40 aa long. This much greater length made the transpeptidation model more difficult to envision. Therefore, we evaluated the role of the proteasome in the splicing of this peptide. We observed that the spliced FGF-5 peptide was produced in vitro after incubation of proteasomes with a 49-aa-long precursor peptide. We evaluated the catalytic mechanism by incubating proteasomes with various precursor peptides. The results confirmed the transpeptidation model of splicing. By transfecting a series of mutant FGF-5 constructs, we observed that reducing the length of the intervening segment increased the production of the spliced peptide, as predicted by the transpeptidation model. Finally, we observed that trans-splicing (i.e., splicing of fragments from two distinct proteins) can occur in the cell, but with a much lower efficacy than splicing of fragments from the same protein

Secular shoreline response to large-scale estuarine shoal migration and welding

International audienceThe 14.5-km North-Médoc coast, southwest France, is a high-energy mesoto macro-tidal environment adjacent to the largest estuary in Europe. Over the last centuries, this coastline has locally suffered periods of severe erosion, threatening coastal infrastructures and requiring the progressive implementation of coastal structures and, more recently, localized beach nourishments. This contribution combines 84 years (1937-2021) of shoreline data from various sources, 118 years (1903-2021) of shallow water bathymetric surveys and historical photographs. Results show that, averaged in both time and space, the coast eroded by-0.6 m/yr over the last 84 years, but with a large alongshore and temporal variability. Erosion is locally peaking at-5.2 m/yr, while accretion is restricted to a remote 2.5-km and locally peaks at 5.4 m/yr. A salient characteristic of shoreline evolution is the alternation of rapid erosion ( 20 m/yr) periods over relatively short intervals (≈ 10 years) and across limited alongshore distances (e.g. couple o

Analysis of the processing of seven human tumor antigens by intermediate proteasomes.

We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3(114-122), MAGE-C2(42-50), MAGE-C2(336-344)) or the standard proteasome (Melan-A(26-35), tyrosinase(369-377), gp100(209-217)). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2(336-344) was only produced by intermediate proteasome β1i-β5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2(191-200) is produced only by intermediate proteasome β1i-β5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides

Protein phosphatase 1 subunit Ppp1r15a/GADD34 regulates cytokine production in polyinosinic:polycytidylic acid-stimulated dendritic cells.

International audienceIn response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response. Here we show that in response to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are expressed. In agreement with increased GADD34 levels, an extensive dephosphorylation of the translation initiation factor eIF2α was observed during DC activation. Unexpectedly, although DCs display an unusual resistance to protein synthesis inhibition induced in response to cytosolic dsRNA, GADD34 expression did not have a major impact on protein synthesis. GADD34, however, was shown to be required for normal cytokine production both in vitro and in vivo. These observations have important implications in linking further pathogen detection with the integrated stress response pathways

An antigen produced by splicing of noncontiguous peptides in the reverse order

CD8-positive T lymphocytes recognize peptides that are usually derived from the degradation of cellular proteins and are presented by class I molecules of the major histocompatibility complex. Here we describe a human minor histocompatibility antigen created by a polymorphism in the SP110 nuclear phosphoprotein gene. The antigenic peptide comprises two noncontiguous SP110 peptide segments spliced together in reverse order to that in which they occur in the predicted SP110 protein. The antigenic peptide could be produced in vitro by incubation of precursor peptides with highly purified 20S proteasomes. Cutting and splicing probably occur within the proteasome by transpeptidation