Current and future roles of mucins in cholangiocarcinoma-recent evidences for a possible interplay with bile acids.

Cholangiocarcinoma (CCA) is rare but highly malignant tumour. The diagnosis is difficult due to its silent clinical character and the inefficiency of currently available diagnostic markers. An enhanced understanding of the molecular pathways involved in CCA carcinogenesis would herald targeted, individualized therapies, as well as new early diagnostic tool with improvement of patient survival. Recently, two mucin proteins, MUC4 and MUC5 have gained interest for their involvement in tumour growth and progression and possible use as diagnostic and prognostic cancer biomarkers. Moreover, a number of studies have demonstrated an association between biliary or serum bile acids (BAs) and some forms of cancers including CCA. More importantly, BAs have been shown to participate in tumour progression by inducing alterations in the expression of oncogenic mucins. This review summarizes the most important findings regarding the possible use of mucin glycoproteins and BAs in the diagnosis and prognostication of CCA and discuss evidences suggesting a role of BAs in regulating the expression of transmembrane and secreted mucins.

Innovative approach to safely induce controlled lipolysis by superparamagnetic iron oxide nanoparticles-mediated hyperthermic treatment.

During last years, evidence has been provided on the involvement of overweight and obesity in the pathogenesis and aggravation of several life-threatening diseases. Here, we demonstrate that, under appropriate administration conditions, polyhedral iron oxide nanoparticles are efficiently and safely taken up by 3T3 cell line-derived adipocytes (3T3 adipocytes) in vitro. Since these nanoparticles proved to effectively produce heat when subjected to alternating magnetic field, 3T3 adipocytes were submitted to superparamagnetic iron oxide nanoparticles-mediated hyperthermia treatment (SMHT), with the aim of modulating their lipid content. Notably, the treatment resulted in a significant delipidation persisting for at least 24h, and in the absence of cell death, damage or dedifferentiation. Interestingly, transcript expression of adipose triglyceride lipase (ATGL), a key gene involved in canonical lipolysis, was not modulated upon SMHT, suggesting the involvement of a novel/alternative mechanism in the effective lipolysis observed. By applying the same experimental conditions successfully used for 3T3 adipocytes, SMHT was able to induce delipidation also in primary cultures of human adipose-derived adult stem cells. The success of this pioneering approach in vitro opens promising perspectives for the application of SMHT in vivo as an innovative safe and physiologically mild strategy against obesity, potentially useful in association with balanced diet and healthy lifestyle.

ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation.

The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis.

Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca(2+) channels.

Our previous studies revealed that L-type voltage-dependent Ca(2+) channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular domains of Cav1.2α1 to identify putative binding sites between the channel and hyaluronic acid or another class of polyanionic glycans, such as heparin/heparan sulfates. None of the tested peptides showed detectable interaction with hyaluronic acid, but two peptides derived from the first pore-forming domain of Cav1.2α1 subunit bound to heparin. At 25 °C the binding of the peptide P7 (MGKMHKTCYN) was at ~50 µM, and that of the peptide P8 (GHGRQCQNGTVCKPGWDGPKHG) was at ~21 µM. The Cav1.2α1 first pore forming segment that contained both peptides maintained a high affinity for heparin (~23 µM), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin-agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity.

Evolution of mitochondria reconstructed from the energy metabolism of living bacteria.

The ancestors of mitochondria, or proto-mitochondria, played a crucial role in the evolution of eukaryotic cells and derived from symbiotic α-proteobacteria which merged with other microorganisms - the basis of the widely accepted endosymbiotic theory. However, the identity and relatives of proto-mitochondria remain elusive. Here we show that methylotrophic α-proteobacteria could be the closest living models for mitochondrial ancestors. We reached this conclusion after reconstructing the possible evolutionary pathways of the bioenergy systems of proto-mitochondria with a genomic survey of extant α-proteobacteria. Results obtained with complementary molecular and genetic analyses of diverse bioenergetic proteins converge in indicating the pathway stemming from methylotrophic bacteria as the most probable route of mitochondrial evolution. Contrary to other α-proteobacteria, methylotrophs show transition forms for the bioenergetic systems analysed. Our approach of focusing on these bioenergetic systems overcomes the phylogenetic impasse that has previously complicated the search for mitochondrial ancestors. Moreover, our results provide a new perspective for experimentally re-evolving mitochondria from extant bacteria and in the future produce synthetic mitochondria.

New insights into functional roles of the polypyrimidine tract-binding protein.

Polypyrimidine Tract Binding Protein (PTB) is an intensely studied RNA binding protein involved in several post-transcriptional regulatory events of gene expression. Initially described as a pre-mRNA splicing regulator, PTB is now widely accepted as a multifunctional protein shuttling between nucleus and cytoplasm. Accordingly, PTB can interact with selected RNA targets, structural elements and proteins. There is increasing evidence that PTB and its paralog PTBP2 play a major role as repressors of alternatively spliced exons, whose transcription is tissue-regulated. In addition to alternative splicing, PTB is involved in almost all steps of mRNA metabolism, including polyadenylation, mRNA stability and initiation of protein translation. Furthermore, it is well established that PTB recruitment in internal ribosome entry site (IRES) activates the translation of picornaviral and cellular proteins. Detailed studies of the structural properties of PTB have contributed to our understanding of the mechanism of RNA binding by RNA Recognition Motif (RRM) domains. In the present review, we will describe the structural properties of PTB, its paralogs and co-factors, the role in post-transcriptional regulation and actions in cell differentiation and pathogenesis. Defining the multifunctional roles of PTB will contribute to the understanding of key regulatory events in gene expression.