Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.

The first report of the full-length structure of the collagen-like polypeptide [(Pro-Pro-Gly) 10 ] 3 is given. This structure was obtained from crystals grown in a microgravity environment, which diffracted up to 1.3 Å, using synchrotron radiation. The final model, which was refined to an R factor of 0.18, is the highestresolution description of a collagen triple helix reported to date. This structure provides clues regarding a series of aspects related to collagen triple helix structure and assembly. The strict dependence of proline puckering on the position inside the Pro-Pro-Gly triplets and the correlation between backbone and side chain dihedral angles support the propensity-based mechanism of triple helix stabilization/destabilization induced by hydroxyproline. Furthermore, the analysis of [(Pro-Pro-Gly) 10 ] 3 packing, which is governed by electrostatic interactions, suggests that charges may act as locking features in the axial organization of triple helices in the collagen fibrils.

No abstract

No abstract

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein–protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

The interplay between side-chain and main-chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1-4 and 1-5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis-trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.

, is expected to exert strong influence in this field. To obtain further insights into the features of this unique structural motif, we report several molecular dynamics simulations of various GNNQQNY aggregates. Our analyses show that even pairs of ␤-sheets composed of a limited number of ␤-strands are stable in the 20-ns time interval considered, which suggests that steric zipper interactions at a ␤-sheet-␤-sheet interface strongly contribute to the stability of these aggregates. Moreover, although the basic features of side chain-side chain interactions are preserved in the simulation, the backbone structure undergoes significant variations. Upon equilibration, a significant twist of the ␤-strands that compose the ␤-sheets is observed. These results demonstrate that the occurrence of steric zipper interactions is compatible with flat and twisted ␤-sheets. Molecular dynamics simulations carried out on two pairs of ␤-sheets, separated in the crystal state by a hydrated interface, lead to interesting results. The two pairs of sheets, while twisting, associate through stable peptide-peptide interactions. These findings provide insight into the mechanism that leads to the formation of higher aggregates. On these bases, it is possible to reconcile the crystallographic and the EM data on the size of the basic GNNQQNY fiber unit.amyloid fiber ͉ prion ͉ neurodegenerative diseases T he insurgence of severe neurodegenerative diseases, which include Alzheimer's disease, type-II diabetes, systemic amyloidosis, and transmissible spongiform encephalopathies, is commonly associated with the presence of amyloid fibers (1-4). Intriguingly, the soluble, often globular, precursors of these ordered deposits do not share any sequence͞structure similarity. Extensive structural investigations carried out on the soluble forms of proteins involved in neurodegenerative diseases have provided a large amount of information on these forms (5-7). In contrast, limited information has been collected for the insoluble aggregates (8-10). Indeed, the intimate nature of these aggregates prevents their detailed characterization.The presence in the fiber diffraction pattern of these aggregates of strong reflections at 4.8-Å and at 10-to 11-Å resolution has led to the commonly accepted notion that a cross-␤-spine represents the basic structural motif of amyloid fibers (10, 11). Nevertheless, the complete structural definition of these aggregates is still highly debated. Over the years, several models, essentially variations on a common theme (cross-␤-spine), have been proposed (12).The use of model peptides whose solid aggregates display most of the features associated with the amyloid fibers has proved to be an important strategy used to overcome these difficulties (7, 13). Particularly impressive are the results achieved in the structural characterization of the peptide GNNQQNY derived from the prion-determining domain of the yeast protein Sup35 (14). The solid aggregates of this peptide show all of the properties associated with amyloid fibrils:...

No abstract