Marta Moretti

Numb activates the E3 ligase Itch to control Gli1 function through a novel degradation signal

Hedgehog pathway regulates tissue patterning and cell proliferation. Gli1 transcription factor is the major effector of Hedgehog signaling and its deregulation is often associated to medulloblastoma formation. Proteolytic processes represent a critical mechanism by which this pathway is turned off. Here, we characterize the regulation of an ubiquitin-mediated mechanism of Gli1 degradation, promoted by the coordinated action of the E3 ligase Itch and the adaptor protein Numb. We show that Numb activates the catalytic activity of Itch, releasing it from an inhibitory intramolecular interaction between its homologous to E6-AP C-terminus and WW domains. The consequent activation of Itch, together with the recruitment of Gli1 through direct binding with Numb, allows Gli1 to enter into the complex, resulting in Gli1 ubiquitination and degradation. This process is mediated by a novel Itch-dependent degron, composed of a combination of two PPXYs and a phospho-serine/proline motifs, localized in Gli1 C-terminal region, indicating the role of two different WW docking sites in Gli1 ubiquitination. Remarkably, Gli1 protein mutated in these modules is no longer regulated by Itch and Numb, and determines enhanced Gli1-dependent medulloblastoma growth, migration and invasion abilities, as well as in vitro transforming activity. Our data reveal a novel mechanism of regulation of Gli1 stability and function, which influences Hedgehog/Gli1 oncogenic potential.

Molecular organization of the cullin E3 ligase adaptor KCTD11

The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (lKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that lKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and lKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than lKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and lKCTD11 and their individual domains in Escherichia coli yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both lKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for lKCTD11 BTB and for its complex with Cul3. These analyses indicate that a stable lKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in lKCTD11-Cul3 recognition.

Androgens and Hypertension in Men and Women: a Unifying View

Purpose of ReviewThis review was designed to revaluate the androgen role on the mechanisms of hypertension and cardiovascular risks in both men and women. Sex steroids are involved in the regulation of blood pressure, but pathophysiological mechanism is not well understood. Androgens have an important effect on metabolism, adipose and endothelial cell function, and cardiovascular risk in both men and women. A focal point in this contest is represented by the possible gender-specific regulation of different tissues and in particular of the adipose cell. Available data confirm that androgen deficiency is linked to increased prevalence of hypertension and cardiovascular diseases. Adipocyte dysfunction seems to be the main involved mechanism. Androgen replacement reduces inflammation state in man, protecting by metabolic syndrome progression. In women, androgen excess has been considered as promoting factor of cardiovascular risk. However, recent data suggest that excessive androgen production has little effect per se in inducing hypertension in young women of reproductive age. Also in postmenopausal women, data on relative androgen excess and hypertension are missing, while adrenal androgen deficiency has been associated to increased mortality.Recent FindingsMolecular mechanisms linking androgen dysregulation to hypertension are almost Unknown, but they seem to be related to increased visceral fat, promoting a chronic inflammatory state through different mechanisms. One of these may involve the recruitment and over-activation of NF-kB, a ubiquitous transcription factor also expressed in adipose cells, where it may cause the production of cytokines and other immune factors. The NF-kB signalling pathway may also influence brown adipogenesis leading to the preferential enlargement of visceral adipocytes. Chronic inflammation and adipocyte dysfunction may alter endothelial function leading to hypertension.SummaryBoth in men and in women, particularly in the post-menopausal period, hypoandrogenism seems to be a major determinant of the increased prevalence of hypertension. The relationship between androgen signalling and NF-kB might explain the pathophysiological mechanism leading to the development of endothelium dysfunction and hypertension.

Delta-cell-specific expression of hedgehog pathway Ptch1 receptor in murine and human endocrine pancreas

Hedgehog pathway plays an important role during pancreas development, when its inactivation is crucial to assure expression of pancreatic marker genes involved in the organ formation and to assure an appropriate organogenesis. Patched1 (Ptch1) is a transmembrane receptor of hedgehog pathway which has a key role in this process. In fact, heterozygous Ptch1 mutant (ptc+/−) mice are affected by an impaired glucose tolerance accompanied by reduced islet function. In the light that the cell distribution of Ptch1 receptor within the endocrine pancreas has not yet been established, we aimed at identifying the pancreatic endocrine cell subset(s) expressing such molecule.

Foxm1 controls a pro-stemness microRNA network in neural stem cells

Cerebellar neural stem cells (NSCs) require Hedgehog-Gli (Hh-Gli) signalling for their maintenance and Nanog expression for their self-renewal. To identify novel molecular features of this regulatory pathway, we used next-generation sequencing technology to profile mRNA and microRNA expression in cerebellar NSCs, before and after induced differentiation (Diff-NSCs). Genes with higher transcript levels in NSCs (vs. Diff-NSCs) included Foxm1, which proved to be directly regulated by Gli and Nanog. Foxm1 in turn regulated several microRNAs that were overexpressed in NSCs: miR-130b, miR-301a, and members of the miR-15~16 and miR-17~92 clusters and whose knockdown significantly impaired the neurosphere formation ability. Our results reveal a novel Hh-Gli-Nanog-driven Foxm1-microRNA network that controls the self-renewal capacity of NSCs.