Bruton's tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5′RACE and 3′RACE we identified three IBTKα, IBTKβ and IBTKγ mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk.

BackgroundDown syndrome (DS) is a complex disorder caused by the trisomy of either the entire, or a critical region of chromosome 21 (21q22.1-22.3). Despite representing the most common cause of mental retardation, the molecular bases of the syndrome are still largely unknown.MethodsTo better understand the pathogenesis of DS, we analyzed the genome-wide transcription profiles of lymphoblastoid cell lines (LCLs) from six DS and six euploid individuals and investigated differential gene expression and pathway deregulation associated with trisomy 21. Connectivity map and PASS-assisted exploration were used to identify compounds whose molecular signatures counteracted those of DS lymphoblasts and to predict their therapeutic potential. An experimental validation in DS LCLs and fetal fibroblasts was performed for the most deregulated GO categories, i.e. the ubiquitin mediated proteolysis and the NF-kB cascade.ResultsWe show, for the first time, that the level of protein ubiquitination is reduced in human DS cell lines and that proteasome activity is increased in both basal conditions and oxidative microenvironment. We also provide the first evidence that NF-kB transcription levels, a paradigm of gene expression control by ubiquitin-mediated degradation, is impaired in DS due to reduced IkB-alfa ubiquitination, increased NF-kB inhibitor (IkB-alfa) and reduced p65 nuclear fraction. Finally, the DSCR1/DYRK1A/NFAT genes were analysed. In human DS LCLs, we confirmed the presence of increased protein levels of DSCR1 and DYRK1A, and showed that the levels of the transcription factor NFATc2 were decreased in DS along with a reduction of its nuclear translocation upon induction of calcium fluxes.ConclusionsThe present work offers new perspectives to better understand the pathogenesis of DS and suggests a rationale for innovative approaches to treat some pathological conditions associated to DS.

ERGIC-53 is a lectin-like transport receptor protein, which recirculates between the ER and the Golgi complex and is required for the intracellular transport of a restricted number of glycoproteins. We show in this article that ERGIC-53 accumulates during the heat shock response. However, at variance with the unfolded protein response, which results in enhanced transcription of ERGIC-53 mRNA, heat shock leads only to enhanced translation of ERGIC-53 mRNA. In addition, the half-life of the protein does not change during heat shock. Therefore, distinct signal pathways of the cell stress response modulate the ERGIC-53 protein level. Heat shock also affects the recycling pathway of ERGIC-53. The protein rapidly redistributes in a more peripheral area of the cell, in a vesicular compartment that has a lighter sedimentation density on sucrose gradient in comparison to the compartment that contains the majority of ERGIC-53 at 37°C. This effect is specific, as no apparent reorganization of the endoplasmic reticulum, intermediate compartment and Golgi complex is morphologically detectable in the cells exposed to heat shock. Moreover, the anterograde transport of two unrelated reporter proteins is not affected. Interestingly, MCFD2, which interacts with ERGIC-53 to form a complex required for the ER-to-Golgi transport of specific proteins, is regulated similarly to ERGIC-53 in response to cell stress. These results support the view that ERGIC-53 alone, or in association with MCFD2, plays important functions during cellular response to stress conditions.