Domenico Raimondo

The implications of alternative splicing in the ENCODE protein complement.

Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.

Protein function annotation by homology-based inference.

With many genomes now sequenced, computational annotation methods to characterize genes and proteins from their sequence are increasingly important. The BioSapiens Network has developed tools to address all stages of this process, and here we review progress in the automated prediction of protein function based on protein sequence and structure.

Evaluating the usefulness of protein structure models for molecular replacement

MOTIVATION We investigate the relationship between the quality of models of protein structure and their usefulness as search models in molecular replacement, a widely used method to experimentally determine protein structures by X-ray crystallography. RESULTS We used the available models submitted to the Critical Assessment of Techniques for Protein Structure Prediction to verify in which cases they can be automatically used as search templates for molecular replacement. Our results show that there is a correlation between the quality of the models and their suitability for molecular replacement but that the traditional method of relying on sequence identity between the model and the template used to build it is not diagnostic for the success of the procedure. AVAILABILITY Additional data are available at http://cassandra.bio.uniroma1.it/mr-results-casp.html

Verrocchio, a Drosophila OB fold-containing protein, is a component of the terminin telomere-capping complex

Drosophila telomeres are elongated by transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the terminal DNA sequence. Drosophila telomeres are protected by terminin, a complex that includes the HOAP (Heterochromatin Protein 1/origin recognition complex-associated protein) and Moi (Modigliani) proteins and shares the properties of human shelterin. Here we show that Verrocchio (Ver), an oligonucleotide/oligosaccharide-binding (OB) fold-containing protein related to Rpa2/Stn1, interacts physically with HOAP and Moi, is enriched only at telomeres, and prevents telomere fusion. These results indicate that Ver is a new terminin component; we speculate that, concomitant with telomerase loss, Drosophila evolved terminin to bind chromosome ends independently of the DNA sequence.

Cancer-Selective Targeting of the Nf-ΚB Survival Pathway With Gadd45Β/Mkk7 Inhibitors

Summary Constitutive NF-κB signaling promotes survival in multiple myeloma (MM) and other cancers; however, current NF-κB-targeting strategies lack cancer cell specificity. Here, we identify the interaction between the NF-κB-regulated antiapoptotic factor GADD45β and the JNK kinase MKK7 as a therapeutic target in MM. Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45β/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells. DTP3 has similar anticancer potency to the clinical standard, bortezomib, but more than 100-fold higher cancer cell specificity in vitro. Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses. Hence, cancer-selective targeting of the NF-κB pathway is possible and, at least for myeloma patients, promises a profound benefit.

Site-directed enzymatic PEGylation of the human granulocyte colony-stimulating factor

Poly(ethylene glycol) (PEG) is a widely used polymer employed to increase the circulating half‐life of proteins in blood and to decrease their immunogenicity and antigenicity. PEG attaches to free amines, typically at lysine residues or at the N‐terminal amino acid. This lack of selectivity can present problems when a PEGylated protein therapeutic is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic modification of proteins is one route to overcome this limitation. Bacterial transglutaminases are enzyme candidates for site‐specific modification, but they also have rather broad specificity. The need arises to be able to predict a priori potential PEGylation sites on the protein of interest and, especially, to be able to design mutants where unique PEGylation sites can be introduced when needed. We investigated the feasibility of a computational approach to the problem, using human granulocyte colony‐stimulating factor as a test case. The selected protein is therapeutically relevant and represents a challenging problem, as it contains 17 potential PEGylation sites. Our results show that a combination of computational methods allows the identification of the specific glutamines that are substrates for enzymatic PEGylation by a microbial transglutaminase, and that it is possible to rationally modify the protein and introduce PEG moieties at desired sites, thus allowing the selection of regions that are unlikely to interfere with the biological activity of a therapeutic protein.

Automatic procedure for using models of proteins in molecular replacement.

In a crystallography experiment, a crystal is irradiated with X‐rays whose diffracted waves are collected and measured. The reconstruction of the structure of the molecule in the crystal requires knowledge of the phase of the diffracted waves, information that is lost in the passage from the three‐dimensional structure of the molecule to its diffraction pattern. It can be recovered using experimental methods such as heavy‐atom isomorphous replacement and anomalous scattering or by molecular replacement, which relies on the availability of an atomic model of the target structure. This can be the structure of the target protein itself, if a previous structure determination is available, or a computational model or, in some cases, the structure of a homologous protein. It is not straightforward to predict beforehand whether or not a computational model will work in a molecular replacement experiment, although some rules of thumb exist. The consensus is that even minor differences in the quality of the model, which are rather difficult to estimate a priori, can have a significant effect on the outcome of the procedure. We describe here a method for quickly assessing whether a protein structure can be solved by molecular replacement. The procedure consists in submitting the sequence of the target protein to a selected list of freely available structure prediction servers, cluster the resulting models, select the representative structures of each cluster and use them as search models in an automatic phasing procedure. We tested the procedure using the structure factors of newly released proteins of known structure downloaded from the Protein Data Bank as soon as they were made available. Using our automatic procedure we were able to obtain an interpretable electron density map in more than half the cases. Proteins 2007.

Coding potential of the products of alternative splicing in human

BackgroundAnalysis of the human genome has revealed that as much as an order of magnitude more of the genomic sequence is transcribed than accounted for by the predicted and characterized genes. A number of these transcripts are alternatively spliced forms of known protein coding genes; however, it is becoming clear that many of them do not necessarily correspond to a functional protein.ResultsIn this study we analyze alternative splicing isoforms of human gene products that are unambiguously identified by mass spectrometry and compare their properties with those of isoforms of the same genes for which no peptide was found in publicly available mass spectrometry datasets. We analyze them in detail for the presence of uninterrupted functional domains, active sites as well as the plausibility of their predicted structure. We report how well each of these strategies and their combination can correctly identify translated isoforms and derive a lower limit for their specificity, that is, their ability to correctly identify non-translated products.ConclusionsThe most effective strategy for correctly identifying translated products relies on the conservation of active sites, but it can only be applied to a small fraction of isoforms, while a reasonably high coverage, sensitivity and specificity can be achieved by analyzing the presence of non-truncated functional domains. Combining the latter with an assessment of the plausibility of the modeled structure of the isoform increases both coverage and specificity with a moderate cost in terms of sensitivity.

Multistep Current Signal in Protein Translocation through Graphene Nanopores

In nanopore sensing experiments, the properties of molecules are probed by the variation of ionic currents flowing through the nanopore. In this context, the electronic properties and the single-layer thickness of graphene constitute a major advantage for molecule characterization. Here we analyze the translocation pathway of the thioredoxin protein across a graphene nanopore, and the related ionic currents, by integrating two nonequilibrium molecular dynamics methods with a bioinformatic structural analysis. To obtain a qualitative picture of the translocation process and to identify salient features we performed unsupervised structural clustering on translocation conformations. This allowed us to identify some specific and robust translocation intermediates, characterized by significantly different ionic current flows. We found that the ion current strictly anticorrelates with the amount of pore occupancy by thioredoxin residues, providing a putative explanation of the multilevel current scenario observed in recently published translocation experiments.

MAISTAS: a tool for automatic structural evaluation of alternative splicing products

Motivation: Analysis of the human genome revealed that the amount of transcribed sequence is an order of magnitude greater than the number of predicted and well-characterized genes. A sizeable fraction of these transcripts is related to alternatively spliced forms of known protein coding genes. Inspection of the alternatively spliced transcripts identified in the pilot phase of the ENCODE project has clearly shown that often their structure might substantially differ from that of other isoforms of the same gene, and therefore that they might perform unrelated functions, or that they might even not correspond to a functional protein. Identifying these cases is obviously relevant for the functional assignment of gene products and for the interpretation of the effect of variations in the corresponding proteins. Results: Here we describe a publicly available tool that, given a gene or a protein, retrieves and analyses all its annotated isoforms, provides users with three-dimensional models of the isoform(s) of his/her interest whenever possible and automatically assesses whether homology derived structural models correspond to plausible structures. This information is clearly relevant. When the homology model of some isoforms of a gene does not seem structurally plausible, the implications are that either they assume a structure unrelated to that of the other isoforms of the same gene with presumably significant functional differences, or do not correspond to functional products. We provide indications that the second hypothesis is likely to be true for a substantial fraction of the cases. Availability: http://maistas.bioinformatica.crs4.it/. Contact: anna.tramontano@uniromal.it