Translational research in immune and inflammatory diseases; what are the challenges, expected advances, and innovative therapies?

Despite very different aetiologies and clinical expressions, advancing knowledge in the physiopathology and treatment of immune and inflammatory diseases (IID) prompts us to consider them as a whole. These are chronic, often incapacitating and painful illnesses that progress and destroy organs. Management by discipline too often leads to erroneous diagnoses and sometimes inappropriate treatment. More integrated translational research would further understanding of the complex relationships between cytokines and organ damage, which vary with the conditions and patients, making it possible to develop new biomarkers and personalize treatment. The research in France has very many strengths but its organization is fragmented. Better coordinated research into IID, which could be based on creating a strategic valorization field (domaine de valorisation stratégique, DVS) and thematic multi-organization institute (Institut thématique multi-organismes ITMO), would advance patient management.

RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane.

We have used a c-Src-GFP fusion protein to address the spatial control of Src activation and the nature of Src-associated intracellular structures during stimulus-induced transit to the membrane. Src is activated during transit, particularly in RhoB-containing cytoplasmic endosomes associated with the perinuclear recycling compartment. Knocking out RhoB or expressing a dominant-interfering Rab11 mutant suppresses both catalytic activation of Src and translocation of active kinase to peripheral membrane structures. In addition, the Src- and RhoB-containing endosomes harbor proteins involved in actin polymerization and filament assembly, for example Scar1, and newly polymerized actin can associate with these endosomes in a Src-dependent manner. This implies that Src may regulate an endosome-associated actin nucleation activity. In keeping with this, Src controls the actin dependence of RhoB endosome movement toward the plasma membrane. This work identifies RhoB as a component of "outside-in" signaling pathways that coordinate Src activation with translocation to transmembrane receptors.

Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A.

Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bbeta, as TCTP-interacting partners. In vitro and in vivo studies confirmed that TCTP bound specifically eEF1Bbeta and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bbeta. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.

The p53-inducible TSAP6 gene product regulates apoptosis and the cell cycle and interacts with Nix and the Myt1 kinase.

The p53 tumor suppressor protein plays a crucial role in tumorigenesis by controlling cell-cycle progression and apoptosis. We have previously described a transcript designated tumor suppressor activated pathway-6 (TSAP6) that is up-regulated in the p53-inducible cell line, LTR6. Cloning of the murine and human full-length TSAP6 cDNA revealed that it encodes a 488-aa protein with five to six transmembrane domains. This gene is the murine and human homologue of the recently published rat pHyde. Antibodies raised against murine and human TSAP6 recognize a 50- to 55-kDa band induced by p53. Analysis of the TSAP6 promoter identified a functional p53-responsive element. Functional studies demonstrated that TSAP6 antisense cDNA diminished levels of the 50- to 55-kDa protein and decreased significantly the levels of p53-induced apoptosis. Furthermore, TSAP6 small interfering RNA inhibited apoptosis in TSAP6-overexpressing cells. Yeast two-hybrid analysis followed by GST/in vitro-transcribed/translated pull-down assays and in vivo coimmunoprecipitations revealed that TSAP6 associated with Nix, a proapoptotic Bcl-2-related protein and the Myt1 kinase, a negative regulator of the G(2)/M transition. Moreover, TSAP6 enhanced the susceptibility of cells to apoptosis and cooperated with Nix to exacerbate this effect. Cell-cycle studies indicated that TSAP6 could augment Myt1 activity. Overall, these data suggest that TSAP6 may act downstream to p53 to interface apoptosis and cell-cycle progression.

Nuclear localization of CDC25B1 and serine 146 integrity are required for induction of mitosis.

CDC25B phosphatases are essential regulators that control cyclin-dependent kinases activities at the entry into mitosis. In this study, we demonstrate that serine 146 is required for two crucial features of CDC25B1. It is essential for CDC25B1 to function as a mitotic inducer and to prevent CDC25B1 export from the nucleus. We also show that serine 146 is phosphorylated in vitro by CDK1-cyclin B. However, phosphorylation of CDC25B does not stimulate its phosphatase activity, and mutation of serine 146 had no effect on its catalytic activity. Serine 146 phosphorylation is proposed to be a key event in the regulation of the CDC25B function in the initiation of mammalian mitosis.

c-Src-mediated phosphorylation of hnRNP K drives translational activation of specifically silenced mRNAs.

hnRNPK and hnRNP E1/E2 mediate translational silencing of cellular and viral mRNAs in a differentiation-dependent way by binding to specific regulatory sequences. The translation of 15-lipoxygenase (LOX) mRNA in erythroid precursor cells and of the L2 mRNA of human papilloma virus type 16 (HPV-16) in squamous epithelial cells is silenced when either of these cells is immature and is activated in maturing cells by unknown mechanisms. Here we address the question of how the silenced mRNA can be translationally activated. We show that hnRNP K and the c-Src kinase specifically interact with each other, leading to c-Src activation and tyrosine phosphorylation of hnRNP K in vivo and in vitro. c-Src-mediated phosphorylation reversibly inhibits the binding of hnRNP K to the differentiation control element (DICE) of the LOX mRNA 3' untranslated region in vitro and specifically derepresses the translation of DICE-bearing mRNAs in vivo. Our results establish a novel role of c-Src kinase in translational gene regulation and reveal a mechanism by which silenced mRNAs can be translationally activated.