α-Crystallin B (CRYAB or HspB5) is a chaperone member of the small heat-shock protein family that prevents aggregation of many cytosolic client proteins by means of its ATP-independent holdase activity. Surprisingly, several reports show that CRYAB exerts a protective role also extracellularly, and it has been recently demonstrated that CRYAB is secreted from human retinal pigment epithelial cells by an unconventional secretion pathway that involves multi-vesicular bodies. Here we show that autophagy is crucial for this unconventional secretion pathway and that phosphorylation at serine 59 residue regulates CRYAB secretion by inhibiting its recruitment to the autophagosomes. In addition, we found that autophagosomes containing CRYAB are not able to fuse with lysosomes. Therefore, CRYAB is capable to highjack and divert autophagosomes toward the exocytic pathway, inhibiting their canonical route leading to the lysosomal compartment. Potential implications of these findings in the context of disease-associated mutant proteins turn-over are discussed.

Frizzled 4 belongs to the superfamily of G protein coupled receptors. The unstructured cytosolic tail of the receptor is essential for its activity. The mutation L501fsX533 in the fz4 gene results in a new COOH-tail of the receptor and causes a form of Familial exudative vitreoretinopathy. Here we show that the mutated tail is structured. Two amphipathic helices, displaying affinity for membranes and resembling the structure of Influenza Hemagglutinin fusion peptide, constitute the new fold. This tail induces the aggregation of the receptor in the Endoplasmic Reticulum and it is sufficient to block the export to the Golgi of a chimeric VSVG protein containing the mutated tail. Affecting the tail's structure, net charge or amphipathicity relocates the mutated Fz4 receptor to the Plasma Membrane. Such disorder-to-order structural transition was never described in GPCRs and opens a new scenario on the possible effect of mutations on unstructured regions of proteins.

SummaryE3 ubiquitin ligases give specificity to the ubiquitylation process by selectively binding substrates. Recently, their function has emerged as a crucial modulator of T-cell tolerance and immunity. However, substrates, partners and mechanism of action for most E3 ligases remain largely unknown. In this study, we identified the human T-cell co-receptor CD8 a-chain as binding partner of the ligand of Numb proteins X1 (LNX1p80 isoform) and X2 (LNX2). Both LNX mRNAs were found expressed in T cells purified from human blood, and both proteins interacted with CD8a in human HPB-ALL T cells. By using an in vitro assay and a heterologous expression system we showed that the interaction is mediated by the PDZ (PSD95-DlgA-ZO-1) domains of LNX proteins and the cytosolic C-terminal valine motif of CD8a. Moreover, CD8a redistributed LNX1 or LNX2 from the cytosol to the plasma membrane, whereas, remarkably, LNX1 or LNX2 promoted CD8a ubiquitylation, downregulation from the plasma membrane, transport to the lysosomes, and degradation. Our findings highlight the function of LNX proteins as E3 ligases and suggest a mechanism of regulation for CD8a localization at the plasma membrane by ubiquitylation and endocytosis.

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) activates the unfolded protein response, also known as the ER stress response. We previously demonstrated that ER stress induces transcription of the ER Golgi intermediate compartment protein ERGIC-53. To investigate the molecular events that regulate unfolded protein response-mediated induction of the gene, we have analyzed the transcriptional regulation of ERGIC-53. We found that the ERGIC-53 promoter contains a single cis-acting element that mediates induction of the gene by thapsigargin and other ER stress-causing agents. This ER stress response element proved to retain a novel structure and to be highly conserved in mammalian ERGIC-53 genes. The ER stress response element identified contains a 5-end CCAAT sequence that constitutively binds NFY/CBF and, 9 nucleotides away, a 3-end region (5-CCCTGTTGGCCATC-3) that is equally important for ER stressmediated induction of the gene. This sequence is the binding site for endogenous YY1 at the 5-CCCTGTTGG-3 part and for undefined factors at the CCATC 3-end. ATF6␣-YY1, but not XBP1, interacted with the ERGIC-53 regulatory region and activated ERGIC-53 ER stress response element-dependent transcription. A molecular model for the transcriptional regulation of the ERGIC-53 gene is proposed.

BackgroundThe importance of non-coding RNAs (ncRNAs) as fine regulators of eukaryotic gene expression has emerged by several studies focusing on microRNAs (miRNAs). miRNAs represent a newly discovered family of non coding-RNAs. They are thought to be crucial players of human hematopoiesis and related tumorigenesis and to represent a potential tool to detect the early stages of cancer. More recently, the expression regulation of numerous long ncRNAs has been linked to cell growth, differentiation and cancer although the molecular mechanism of their function is still unknown.NB4 cells are promyelocytic cells that can be induced to differentiation upon retinoic acid (ATRA) treatment and represent a feasible model to study changes of non coding RNAs expression between cancer cells and their terminally differentiated counterpart.Findingswe screened, by microarray analysis, the expression of 243 miRNAs and 492 human genes transcribing for putative long ncRNAs different from miRNAs in NB4 cells before and after ATRA induced differentiation. Our data show that 8 miRNAs, and 58 long ncRNAs were deregulated by ATRA induced NB4 differentiation.Conclusionour data suggest that ATRA-induced differentiation lead to deregulation of a large number of the ncRNAs that can play regulatory roles in both tumorigenesis and differentiation.