A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a beta-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

The Spanish corporate structure through interlocking directorates

[EN] The aim of this paper is to determine to what extent globalization pressures are changing interlocking directorate networks modeled on continental capitalism into Anglo-Saxon models. For this purpose we analyse the Spanish network of interlocks, comparing the present structure (2012) with that of 1993 and 2006. We show how, although Spanish corporative structure continues to display characteristics of the continental economies, some major banks are significantly reducing industrial activity. Nevertheless, the financial organizations continue to maintain a close relationship with sectors such as construction and services. The analysis of the network of directorates shows a retreat in activity of industrial banking in Spain. Two large Spanish financial institutions, BSCH and La Caixa, continue to undertake activities of industrial banking in 2006, but this activity is significantly reduced in 2012. According to the theories on the role of the interlocking directorates, the companies in these sectors assure their access to banking credit by incorporating advisors from financial organizations to their board of directors. We cannot conclude that the structure of the Spanish corporate network has become a new case of Anglo-Saxon structure, but we got indications that are becoming less hierarchic as banks seem to slowly abandon centrality positions. This is especially salient if we compare the networks of 2006 and 2012, which show a continuing decrease of the role of banks and insurance companies in the network.

Two Nuclear Localization Signals in USP1 Mediate Nuclear Import of the USP1/UAF1 Complex

9 p. : il.

Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

11 p.

Analysis of SUMOylated proteins using SUMO-traps

6 p. [+ 7 p. Supplementary Information]

Mechanisms of E2F2-mediated transcriptional repression

In this work we wanted to study the mechanism of E2F2-mediated repression. Our hypothesis is that E2F2 activates the expression of one or more E2F members of the “repressor” subset of the family through the E2F motifs present in their promoters, and those repressor E2F(s) would subsequently repress the target promoters. To address this hypothesis, we focused on E2F7. E2F7 is a repressor that lacks the Rb binding domain, and associates with DNA through E2F binding sites (de Bruin et al., 2003). Furthermore, E2F7 itself is also regulated by E2F motifs on its own promoter, and it has been shown to repress DNA metabolism and replication genes in late S-phase (de Bruin et al., 2003; Westendorp et al., 2012). E2F7, together with E2F8 has been found to form heterodimers, being critical on cell proliferation and development, and both seem to have similar functions (Li et al., 2008). Preliminary results from Zubiaga’s group have indicated that E2F2 activates E2F7 transcription in U2OS cells, suggesting that E2F2’s repressor function could be mediated by E2F7. For this purpose, we focused on studying E2F7’s role on the target genes previously known to be repressed by E2F2: Chk1 and Mcm5. The specific aims for this work were the following: - Confirm that E2F2 induces E2F7 in HEK-293T cells - Assess whether E2F7 acts as a transcriptional repressor on E2F sites - Evaluate the role of E2F7 on E2F2-mediated transcriptional repression of Chk1 and Mcm5.

Assembly of viral genomes from metagenomes

Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

Structure-function analysis of USP1: insights into the role of Ser313 phosphorylation site and the effect of cancer-associated mutations on autocleavage

Background: In complex with its cofactor UAF1, the USP1 deubiquitinase plays an important role in cellular processes related to cancer, including the response to DNA damage. The USP1/UAF1 complex is emerging as a novel target in cancer therapy, but several aspects of its function and regulation remain to be further clarified. These include the role of the serine 313 phosphorylation site, the relative contribution of different USP1 sequence motifs to UAF1 binding, and the potential effect of cancer-associated mutations on USP1 regulation by autocleavage. Methods: We have generated a large set of USP1 structural variants, including a catalytically inactive form (C90S), non-phosphorylatable (S313A) and phosphomimetic (S313D) mutants, deletion mutants lacking potential UAF1 binding sites, a mutant (GG/AA) unable to undergo autocleavage at the well-characterized G670/G671 diglycine motif, and four USP1 mutants identified in tumor samples that cluster around this cleavage site (G667A, L669P, K673T and A676T). Using cell-based assays, we have determined the ability of these mutants to bind UAF1, to reverse DNA damage-induced monoubiquitination of PCNA, and to undergo autocleavage. Results: A non-phosphorylatable S313A mutant of USP1 retained the ability to bind UAF1 and to reverse PCNA ubiquitination in cell-based assays. Regardless of the presence of a phosphomimetic S313D mutation, deletion of USP1 fragment 420-520 disrupted UAF1 binding, as determined using a nuclear relocation assay. The UAF1 binding site in a second UAF1-interacting DUB, USP46, was mapped to a region homologous to USP1(420-520). Regarding USP1 autocleavage, co-expression of the C90S and GG/AA mutants did not result in cleavage, while the cancer-associated mutation L669P was found to reduce cleavage efficiency. Conclusions: USP1 phosphorylation at S313 is not critical for PCNA deubiquitination, neither for binding to UAF1 in a cellular environment. In this context, USP1 amino acid motif 420-520 is necessary and sufficient for UAF1 binding. This motif, and a homologous amino acid segment that mediates USP46 binding to UAF1, map to the Fingers sub-domain of these DUBs. On the other hand, our results support the view that USP1 autocleavage may occur in cis, and can be altered by a cancer-associated mutation.