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Diaz A.,Barcelona Institute for Research in Biomedicine | Diaz A.,CIBER ISCIII | Diaz-Lobo M.,Barcelona Institute for Research in Biomedicine | Diaz-Lobo M.,CIBER ISCIII | And 8 more authors.

Despite the biological relevance of glycosyltrasferases (GTs) and the many efforts devoted to this subject, the catalytic mechanism through which a subclass of this large family of enzymes, namely those that operate with net retention of the anomeric configuration, has not been fully established. Here, we show that in the absence of an acceptor, an archetypal retaining GT such as Pyrococcus abyssi glycogen synthase (PaGS) reacts with its glucosyl donor substrate, uridine 5'-diphosphoglucose (UDP-Glc), to produce the scission of the covalent bond between the terminal phosphate oxygen of UDP and the sugar ring. X-ray diffraction analysis of the PaGS/UDP-Glc complex shows no electronic density attributable to the UDP moiety, but establishes the presence in the active site of the enzyme of a glucose-like derivative that lacks the exocyclic oxygen attached to the anomeric carbon. Chemical derivatization followed by gas chromatography/mass spectrometry of the isolated glucose-like species allowed us to identify the molecule found in the catalytic site of PaGS as 1,5-anhydro-D-arabino-hex-1-enitol (AA) or its tautomeric form, 1,5-anhydro-D-fructose. These findings are consistent with a stepwise S Ni-like mechanism as the modus operandi of retaining GTs, a mechanism that involves the discrete existence of an oxocarbenium intermediate. Even in the absence of a glucosyl acceptor, glycogen synthase (GS) promotes the formation of the cationic intermediate, which, by eliminating the proton of the adjacent C2 carbon atom, yields AA. Alternatively, these observations could be interpreted assuming that AA is a true intermediate in the reaction pathway of GS and that this enzyme operates through an elimination/addition mechanism. Copyright © 2012 Wiley Periodicals, Inc. Source

Morata J.,Institute Of Biologia Molecular Of Barcelona Ibmb | Bejar S.,Institute Of Biologia Molecular Of Barcelona Ibmb | Talavera D.,University of Manchester | Riera C.,Vall Dhebron Institute Of Research Vhir | And 5 more authors.

At present we know that phenotypic differences between organisms arise from a variety of sources, like protein sequence divergence, regulatory sequence divergence, alternative splicing, etc. However, we do not have yet a complete view of how these sources are related. Here we address this problem, studying the relationship between protein divergence and the ability of genes to express multiple isoforms. We used three genome-wide datasets of human-mouse orthologs to study the relationship between isoform multiplicity co-occurrence between orthologs (the fact that two orthologs have more than one isoform) and protein divergence. In all cases our results showed that there was a monotonic dependence between these two properties. We could explain this relationship in terms of a more fundamental one, between exon number of the largest isoform and protein divergence. We found that this last relationship was present, although with variations, in other species (chimpanzee, cow, rat, chicken, zebrafish and fruit fly). In summary, we have identified a relationship between protein divergence and isoform multiplicity co-occurrence and explained its origin in terms of a simple gene-level property. Finally, we discuss the biological implications of these findings for our understanding of inter-species phenotypic differences. © 2013 Morata et al. Source

Cuenca-Bono B.,Research Center Principe Felipe | Garcia-Molinero V.,Research Center Principe Felipe | Pascual-Garcia P.,Research Center Principe Felipe | Pascual-Garcia P.,Salk Institute for Biological Studies | And 5 more authors.
Nucleic Acids Research

Efficient coupling between mRNA synthesis and export is essential for gene expression. Sus1/ENY2, a component of the SAGA and TREX-2 complexes, is involved in both transcription and mRNA export. While most yeast genes lack introns, we previously reported that yeast SUS1 bears two. Here we show that this feature is evolutionarily conserved and critical for Sus1 function. We determine that while SUS1 splicing is inefficient, it responds to cellular conditions, and intronic mutations either promoting or blocking splicing lead to defects in mRNA export and cell growth. Consistent with this, we find that an intron-less SUS1 only partially rescues sus1Δ phenotypes. Remarkably, splicing of each SUS1 intron is also affected by the presence of the other and by SUS1 exonic sequences. Moreover, by following SUS1 RNA and protein levels we establish that nonsense-mediated decay (NMD) pathway and the splicing factor Mud2 both play a role in SUS1 expression. Our data (and those of the accompanying work by Hossain et al.) provide evidence of the involvement of splicing, translation, and decay in the regulation of early events in mRNP biogenesis; and imply the additional requirement for a balance in splicing isoforms from a single gene. © The Author(s) 2011. Published by Oxford University Press. Source

Janowski R.,Institute Of Biologia Molecular Of Barcelona Ibmb | Janowski R.,Barcelona Institute for Research in Biomedicine | Mortuza G.,National Research Center Oncologica | Bertero M.G.,Center for Genomic Regulation | And 21 more authors.

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. © 2013 Dos Santos et al. Source

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