Anna De Grassi

Evolution, structure and function of mitochondrial carriers: a review with new insights

The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying different substrate specificities, patterns of gene expression and even non-mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans-membrane domain proteins. The number in other sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomyces cerevisiae and comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of specific symmetry-related amino acid triplets. We assessed the predictive power of these triplets by comparing predictions with experimentally determined data for Arabidopsis MCs, and applied these predictions to the not yet functionally characterized mitochondrial carriers of the grass, Brachypodium distachyon, and the alga, Ostreococcus lucimarinus. We additionally studied evolutionary aspects of the plant MCF by comparing sequence data of the Arabidopsis MCF with those of Saccharomyces cerevisiae and Homo sapiens, then with those of Brachypodium distachyon and Ostreococcus lucimarinus, employing intra- and inter-genome comparisons. Finally, we discussed the importance of the approaches of global gene expression analysis and in vivo characterizations in order to address the relevance of these vital carrier proteins.

p53FamTaG: a database resource of human p53, p63 and p73 direct target genes combining in silico prediction and microarray data

BackgroundThe p53 gene family consists of the three genes p53, p63 and p73, which have polyhedral non-overlapping functions in pivotal cellular processes such as DNA synthesis and repair, growth arrest, apoptosis, genome stability, angiogenesis, development and differentiation. These genes encode sequence-specific nuclear transcription factors that recognise the same responsive element (RE) in their target genes. Their inactivation or aberrant expression may determine tumour progression or developmental disease. The discovery of several protein isoforms with antagonistic roles, which are produced by the expression of different promoters and alternative splicing, widened the complexity of the scenario of the transcriptional network of the p53 family members. Therefore, the identification of the genes transactivated by p53 family members is crucial to understand the specific role for each gene in cell cycle regulation. We have combined a genome-wide computational search of p53 family REs and microarray analysis to identify new direct target genes. The huge amount of biological data produced has generated a critical need for bioinformatic tools able to manage and integrate such data and facilitate their retrieval and analysis.DescriptionWe have developed the p53FamTaG database (p53 FAMily TArget Genes), a modular relational database, which contains p53 family direct target genes selected in the human genome searching for the presence of the REs and the expression profile of these target genes obtained by microarray experiments. p53FamTaG database also contains annotations of publicly available databases and links to other experimental data.The genome-wide computational search of the REs was performed using PatSearch, a pattern-matching program implemented in the DNAfan tool. These data were integrated with the microarray results we produced from the overexpression of different isoforms of p53, p63 and p73 stably transfected in isogenic cell lines, allowing the comparative study of the transcriptional activity of all the proteins in the same cellular background.p53FamTaG database is available free at http://www2.ba.itb.cnr.it/p53FamTaG/Conclusionp53FamTaG represents a unique integrated resource of human direct p53 family target genes that is extensively annotated and provides the users with an efficient query/retrieval system which displays the results of our microarray experiments and allows the export of RE sequences. The database was developed for supporting and integrating high-throughput in silico and experimental analyses and represents an important reference source of knowledge for research groups involved in the field of oncogenesis, apoptosis and cell cycle regulation.

Ultradeep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability

Ultradeep sequencing of genomes permits the detection of very low-level genomic instability in non-neoplastic tissues of patients with the most common form of inherited colorectal cancer.

Single-nucleotide evolution quantifies the importance of each site along the structure of mitochondrial carriers

Mitochondrial carriers are membrane-embedded proteins consisting of a tripartite structure, a three-fold pseudo-symmetry, related sequences, and similar folding whose main function is to catalyze the transport of various metabolites, nucleotides, and coenzymes across the inner mitochondrial membrane. In this study, the evolutionary rate in vertebrates was screened at each of the approximately 50,000 nucleotides corresponding to the amino acids of the 53 human mitochondrial carriers. Using this information as a starting point, a scoring system was developed to quantify the evolutionary pressure acting on each site of the common mitochondrial carrier structure and estimate its functional or structural relevance. The degree of evolutionary selection varied greatly among all sites, but it was highly similar among the three symmetric positions in the tripartite structure, known as symmetry-related sites or triplets, suggesting that each triplet constitutes an evolutionary unit. Based on evolutionary selection, 111 structural sites (37 triplets) were found to be important. These sites play a key role in structure/function of mitochondrial carriers and are involved in either conformational changes (sites of the gates, proline–glycine levels, and aromatic belts) or in binding and specificity of the transported substrates (sites of the substrate-binding area in between the two gates). Furthermore, the evolutionary pressure analysis revealed that the matrix short helix sites underwent different degrees of selection with high inter-paralog variability. Evidence is presented that these sites form a new sequence motif in a subset of mitochondrial carriers, including the ADP/ATP translocator, and play a regulatory function by interacting with ligands and/or proteins of the mitochondrial matrix.

Lattices for ab initio protein structure prediction.

In the study of the protein folding problem with ab initio methods, the protein backbone can be built on some periodic lattices. Any vertex of these lattices can be occupied by a “ball,” which can represent the mass center of an amino acid in a simplified coarse‐grained model of the protein. The backbone, at a coarse‐grained level, can be constituted of a No Reverse Self Avoiding Walk, which cannot intersect itself and cannot go back on itself. There is still much debate between those who use lattices to simplify the study of the protein folding problem and those preferring to work by using an off‐lattice approach. Lattices can help to identify the protein tertiary structure in a computational less‐expensive way, than off‐lattice approaches that have to consider a potentially infinite number of possible structures. However, the use of a lattice, constituted of insufficiently accurate direction vectors, constrains the predictive ability of the model. The aim of this study is to perform a systematic screening of 7 known classic and 11 newly proposed lattices in terms of predictive power. The crystal structures of 42 different proteins (14 mainly alpha helical, 14 mainly beta sheet and 14 mixed structure proteins) were compared to the most accurate simulated models for each lattice. This strategy defines a scale of fitness for all the analyzed lattices and demonstrates that an increase in the coordination number and in the degrees of freedom is necessary but not sufficient to reach the best result. Instead, the introduction of a good set of direction vectors, as developed and tested in this study, strongly increases the lattice performance. Proteins 2008.

Mutations in the Mitochondrial Citrate Carrier SLC25A1 are Associated with Impaired Neuromuscular Transmission.

Background and Objective Congenital myasthenic syndromes are rare inherited disorders characterized by fatigable weakness caused by malfunction of the neuromuscular junction. We performed whole exome sequencing to unravel the genetic aetiology in an English sib pair with clinical features suggestive of congenital myasthenia. Methods We used homozygosity mapping and whole exome sequencing to identify the candidate gene variants. Mutant protein expression and function were assessed in vitro and a knockdown zebrafish model was generated to assess neuromuscular junction development. Results We identified a novel homozygous missense mutation in the SLC25A1 gene, encoding the mitochondrial citrate carrier. Mutant SLC25A1 showed abnormal carrier function. SLC25A1 has recently been linked to a severe, often lethal clinical phenotype. Our patients had a milder phenotype presenting primarily as a neuromuscular (NMJ) junction defect. Of note, a previously reported patient with different compound heterozygous missense mutations of SLC25A1 has since been shown to suffer from a neuromuscular transmission defect. Using knockdown of SLC25A1 expression in zebrafish, we were able to mirror the human disease in terms of variable brain, eye and cardiac involvement. Importantly, we show clear abnormalities in the neuromuscular junction, regardless of the severity of the phenotype. Conclusions Based on the axonal outgrowth defects seen in SLC25A1 knockdown zebrafish, we hypothesize that the neuromuscular junction impairment may be related to pre-synaptic nerve terminal abnormalities. Our findings highlight the complex machinery required to ensure efficient neuromuscular function, beyond the proteomes exclusive to the neuromuscular synapse.

Tandem repeats modify the structure of human genes hosted in segmental duplications.

BackgroundRecently duplicated genes are often subject to genomic rearrangements that can lead to the development of novel gene structures. Here we specifically investigated the effect of variations in internal tandem repeats (ITRs) on the gene structure of human paralogs located in segmental duplications.ResultsWe found that around 7% of the primate-specific genes located within duplicated regions of the genome contain variable tandem repeats. These genes are members of large groups of recently duplicated paralogs that are often polymorphic in the human population. Half of the identified ITRs occur within coding exons and may be either kept or spliced out from the mature transcript. When ITRs reside within exons, they encode variable amino acid repeats. When located at exon-intron boundaries, ITRs can generate alternative splicing patterns through the formation of novel introns.ConclusionsOur study shows that variation in the number of ITRs impacts on recently duplicated genes by modifying their coding sequence, splicing pattern, and tissue expression. The resulting effect is the production of a variety of primate-specific proteins, which mostly differ in number and sequence of amino acid repeats.

Molecular modeling of antibodies for the treatment of TNFα-related immunological diseases.

Therapeutic monoclonal antibodies (mAbs) have high efficacy in treating TNFα‐related immunological diseases. Other than neutralizing TNFα, these IgG1 antibodies exert Fc receptor‐mediated effector functions such as the complement‐dependent cytotoxicity (CDC) and antibody‐dependent cell cytotoxicity (ADCC). The crystallizable fragment (Fc) of these IgG1 contains a single glycosylation site at Asn 297/300 that is essential for the CDC and ADCC. Glycosylated antibodies lacking core fucosylation showed an improved ADCC. However, no structural data are available concerning the ligand‐binding interaction of these mAbs used in TNFα‐related diseases and the role of the fucosylation. We therefore used comparative modeling for generating complete 3D mAb models that include the antigen‐binding fragment (Fab) portions of infliximab, complexed with TNFα (4G3Y.pdb), the Fc region of the human IGHG1 fucosylated (3SGJ) and afucosylated (3SGK) complexed with the Fc receptor subtype FcγRIIIA, and the Fc region of a murine immunoglobulin (1IGT). After few thousand steps of energy minimization on the resulting 3D mAb models, minimized final models were used to quantify interactions occurring between FcγRIIIA and the fucosylated/afucosylated Fc fragments. While fucosylation does not affect Fab‐TNFα interactions, we found that in the absence of fucosylation the Fc–mAb domain and FcγRIIIA are closer and new strong interactions are established between G129 of the receptor and S301 of the Chimera 2 Fc mAb; new polar interactions are also established between the Chimera 2 Fc residues Y299, N300, and S301 and the FcγRIIIA residues K128, G129, R130, and R155. These data help to explain the reduced ADCC observed in the fucosylated mAbs suggesting the specific AA residues involved in binding interactions.

Prediction of high- and low-affinity quinol-analogue-binding sites in the aa3 and bo3 terminal oxidases from Bacillus subtilis and Escherichia coli1.

Haem-copper oxidases are the terminal enzymes in both prokaryotic and eukaryotic respiratory chains. They catalyse the reduction of dioxygen to water and convert redox energy into a transmembrane electrochemical proton gradient during their catalytic activity. Haem-copper oxidases show substantial structure similarity, but spectroscopic and biochemical analyses indicate that these enzymes contain diverse prosthetic groups and use different substrates (i.e. cytochrome c or quinol). Owing to difficulties in membrane protein crystallization, there are no definitive structural data about the quinol oxidase physiological substrate-binding site(s). In the present paper, we propose an atomic structure model for the menaquinol:O2 oxidoreductase of Bacillus subtilis (QOx.aa3). Furthermore, a multistep computational approach is used to predict residues involved in the menaquinol/menaquinone binding within B. subtilis QOx.aa3 as well as those involved in quinol/quinone binding within Escherichia coli QOx.bo3. Two specific sequence motifs, R70GGXDX4RXQX3PX3FX[D/N/E/Q]X2HYNE97 and G159GSPX2GWX2Y169 (B. subtilis numbering), were highlighted within QOx from Bacillales. Specific residues within the first and the second sequence motif participate in the high- and low-affinity substrate-binding sites respectively. Using comparative analysis, two analogous motifs, R71GFXDX4RXQX8[Y/F]XPPHHYDQ101 and G163EFX3GWX2Y173 (E. coli numbering) were proposed to be involved in Enterobacteriales/Rhodobacterales/Rhodospirillales QOx high- and low-affinity quinol-derivative-binding sites. Results and models are discussed in the context of the literature.

Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster.

CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.