Structure and non-structure of centrosomal proteins

Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php

Sequential functions of CPEB1 and CPEB4 regulate pathologic expression of vascular endothelial growth factor and angiogenesis in chronic liver disease.

BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF) regulates angiogenesis, yet therapeutic strategies to disrupt VEGF signaling can interfere with physiologic angiogenesis. In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal production or functions, we investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of portal hypertension and liver disease. METHODS: We obtained transjugular liver biopsies from patients with hepatitis C virus-associated cirrhosis or liver tissues removed during transplantation; healthy human liver tissue was obtained from a commercial source (control). We also performed experiments with male Sprague-Dawley rats and CPEB-deficient mice (C57BL6 or mixed C57BL6/129 background) and their wild-type littermates. Secondary biliary cirrhosis was induced in rats by bile duct ligation, and portal hypertension was induced by partial portal vein ligation. Liver and mesenteric tissues were collected and analyzed in angiogenesis, reverse transcription polymerase chain reaction, polyA tail, 3' rapid amplification of complementary DNA ends, Southern blot, immunoblot, histologic, immunohistochemical, immunofluorescence, and confocal microscopy assays. CPEB was knocked down with small interfering RNAs in H5V endothelial cells, and translation of luciferase reporters constructs was assessed. RESULTS: Activation of CPEB1 promoted alternative nuclear processing within noncoding 3'-untranslated regions of VEGF and CPEB4 messenger RNAs in H5V cells, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promoted cytoplasmic polyadenylation of VEGF messenger RNA, increasing its translation; the high levels of VEGF produced by these cells led to their formation of tubular structures in Matrigel assays. We observed increased levels of CPEB1 and CPEB4 in cirrhotic liver tissues from patients, compared with control tissue, as well as in livers and mesenteries of rats and mice with cirrhosis or/and portal hypertension. Mice with knockdown of CPEB1 or CPEB4 did not overexpress VEGF or have signs of mesenteric neovascularization, and developed less-severe forms of portal hypertension after portal vein ligation. CONCLUSIONS: We identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and nonredundant functions of CPEB1 and CPEB4. Regulation of CPEB4 by CPEB1 and the CPEB4 autoamplification loop induces pathologic angiogenesis. Strategies to block the activities of CPEBs might be developed to treat chronic liver and other angiogenesis-dependent diseases.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2011-29491 and SAF2014-55473-R to MF; BFU2011-30121, BFU2014-54122-P and Consolider RNAREG CSD2009-00080 to RM; PI13/00341 to JB; PI11/01562 and PI14/00125 to PN), Generalitat de Catalunya (SGR1436 to RM; SGR1108 to JB), Fundación Botín by Banco Santander through its Santander Universities Global Division (to RM), AECC Scientific Foundation (to RM and MF), Worldwide Cancer Research (to RM and MF) and RETIC Cancer RD12/0036/0051/FEDER to PN. GFM is funded by a Juan de la Cierva contract from MINECO. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). CIBERehd is an initiative from the Instituto de Salud Carlos III. We also thank Dr Francisco X Real for his contribution to knockout mice generation

Protein-protein interaction networks for interactions experimentally observed for human centrosomal proteins.

<p>The color code represents betweeness centrality, a graph theoretic measure of the centrality of a node in a network, red representing the most central node.</p

Empirical energy functions evaluated for each models and for the corresponding region of the template show that the predicted stability decrease is moderate.

<p>Empirical energy functions evaluated for each models and for the corresponding region of the template show that the predicted stability decrease is moderate.</p

Number of occurrences of domains predicted by SMART.

<p>Only domains with more than 3 occurrences are shown.</p

Fraction of protein length that is predicted to be disordered, coiled-coil, or modeled by homology.

<p>The plots represent the distribution of the percentage of protein length that has been (A) predicted to be disordered and coiled-coil at the same time; (B) Predicted to be disordered and not coiled-coil; (C) modeled; (D) Predicted to have regular secondary structure and not to be disordered neither coiled-coil, but not modeled.</p

Number of residues and number of models for each protein.

<p>The plots represent the distribution of the number of residues of the longest isoform of centrosomal genes (A) and the number of structural models obtained for each protein (B).</p