5-gene differential expression predicts stability of human intestinal allografts.

In intestinal allografts, endoscopy and histology detect the injury once changes in the bowel wall architecture have occurred. We aimed to identify a molecular signature that could predict early deterioration, within histologically indistinguishable biopsies with "minimal changes" (MC) pathology. Sixty biopsies from 12 adult recipients were longitudinally taken during 8years post-transplant. They were classified as either stable (STA) or non-stable (NSTA) according to the prospectively recorded number, frequency and severity of rejection events of the allograft. In a discovery set of MC samples analyzed by RNA-Seq, 816 genes were differentially expressed in STA vs NSTA biopsies. A group of 5 genes (ADH1C, SLC39A4, CYP4F2, OPTN and PDZK1) correctly classified all NSTA biopsies in the discovery set and all STA biopsies from an independent set. These results were validated by qPCR in a new group of MC biopsies. Based on a logistic regression model, a cutoff of 0.28 predicted the probability of being a NSTA biopsy with 85% sensitivity and 69% specificity. In conclusion, by analyzing MC samples early after transplantation, the expression of a 5-gene set may predict the evolution of the bowel allograft. This prognostic biomarker may be of help to personalize care of the intestinal transplant recipient.

Plasma membrane-associated superstructure: Have we overlooked a new type of organelle in eukaryotic cells?

A variety of intriguing plasma membrane-associated regions, including focal adhesions, adherens junctions, tight junctions, immunological synapses, neuromuscular junctions and the primary cilia, among many others, have been described in eukaryotic cells. Emphasizing their importance, alteration in their molecular structures induces or correlates with different pathologies. These regions display surface proteins connected to intracellular molecules, including cytoskeletal component, which maintain their cytoarchitecture, and signalling proteins, which regulate their organization and functions. Based on the molecular similarities and other common features observed, we suggest that, despite differences in external appearances, all these regions are just the same superstructure that appears in different locations and cells. We hypothesize that this superstructure represents an overlooked new type of organelle that we call plasma membrane-associated superstructure (PMAS). Therefore, we suggest that eukaryotic cells include classical organelles (e.g. mitochondria, Golgi and others) and also PMAS. We speculate that this new type of organelle might be an innovation associated to the emergence of eukaryotes. Finally we discuss the implications of the hypothesis proposed.

SPO-Seq: An Accessible Method for Efficient Evaluation of Spo11 Catalytic Activity and Profiling Meiotic DSB Hotspots in Saccharomyces cerevisiae.

Meiotic recombination is a key process facilitating the formation of crossovers and the exchange of genetic material between homologous chromosomes in early meiosis. This involves controlled double-strand breaks (DSBs) formation catalyzed by Spo11. DSBs exhibit a preferential location in specific genomic regions referred to as hotspots, and their variability is tied to varying Spo11 activity levels. We have refined a ChIP-Seq technique, called SPO-Seq, to map Spo11-specific DSB formation in Saccharomyces cerevisiae. The chapter describes our streamlined approach and the developed bioinformatic tools for processing data and comparing with existing DSB hotspot maps. Through this combined experimental and computational approach, we aim to enhance our understanding of meiotic recombination and genetic exchange processes in budding yeast, with the potential to expand this methodology to other organisms by applying a few modifications.

Comparative analysis of transcriptomic and hormonal responses to compatible and incompatible plant-virus interactions that lead to cell death.

Hypersensitive response-related programmed cell death (PCD) has been extensively analyzed in various plant-virus interactions. However, little is known about the changes in gene expression and phytohormone levels associated with cell death caused by compatible viruses. The synergistic interaction of Potato virus X (PVX) with a number of Potyvirus spp. results in increased symptoms that lead to systemic necrosis (SN) in Nicotiana benthamiana. Here, we show that SN induced by a PVX recombinant virus expressing a potyviral helper component-proteinase (HC-Pro) gene is associated with PCD. We have also compared transcriptomic and hormonal changes that occur in response to a compatible synergistic virus interaction that leads to SN, a systemic incompatible interaction conferred by the Tobacco mosaic virus-resistance gene N, and a PCD response conditioned by depletion of proteasome function. Our analysis indicates that the SN response clusters with the incompatible response by the similarity of their overall gene expression profiles. However, the expression profiles of both defense-related genes and hormone-responsive genes, and also the relative accumulation of several hormones in response to SN, relate more closely to the response to depletion of proteasome function than to that elicited by the incompatible interaction. This suggests a potential contribution of proteasome dysfunction to the increased pathogenicity observed in PVX-Potyvirus mixed infections. Furthermore, silencing of coronatine insensitive 1, a gene involved in jasmonate perception, in N. benthamiana accelerated cell death induced by PVX expressing HC-Pro.

Identification of the mitochondrial carrier that provides Yarrowia lipolytica with a fatty acid-induced and nucleotide-sensitive uncoupling protein-like activity.

Uncoupling proteins (UCPs) are mitochondrial carriers distributed throughout the eukaryotic kingdoms. While genes coding for UCPs have been identified in plants and animals, evidences for the presence of UCPs in fungi and protozoa are only functional. Here, it is reported that in the yeast Yarrowia lipolytica there is a fatty acid-promoted and GDP-sensitive uncoupling activity indicating the presence of a UCP. The uncoupling activity is higher in the stationary phase than in the mid-log growth phase. The in silico search on the Y. lipolytica genome led to the selection of two genes with the highest homology to the UCP family, XM_503525 and XM_500457. By phylogenetic analysis, XP_503525 was predicted to be an oxaloacetate carrier while XP_500457 would be a dicarboxylate carrier. Each of these two genes was cloned and heterologously expressed in Saccharomyces cerevisiae and the resulting phenotype was analyzed. The transport activity of the two gene products confirmed the phylogenetic predictions. In addition, only mitochondria isolated from yeasts expressing XP_503525 showed bioenergetic properties characteristic of a UCP: the proton conductance was increased by linoleic acid and inhibited by GDP. It is concluded that the XM_503525 gene from Y. lipolytica encodes for an oxaloacetate carrier although, remarkably, it also displays an uncoupling activity stimulated by fatty acids and inhibited by nucleotides.

Characterization of a pathogenesis-related protein 4 (PR-4) induced in Capsicum chinense L3 plants with dual RNase and DNase activities.

Resistance conferred by the L(3) gene is active against most of the tobamoviruses, including the Spanish strain (PMMoV-S), a P(1),(2) pathotype, but not against certain strains of pepper mild mottle virus (PMMoV), termed as P(1),(2),(3) pathotype, such as the Italian strain (PMMoV-I). PMMoV-S induces a hypersensitive reaction (HR) in C. chinense PI159236 plant leaves with the formation of necrotic local lesions and restriction of the virus at the primary infection sites. In this paper, a C. chinense PR-4 protein induced during both the compatible and the incompatible interactions has been identified. It was strongly associated with HR induction and to a lesser extent with the compatible interaction, but only in the later stages of infection. Moreover, it was found to accumulate during the necrogenic reaction induced by Potato virus X. The C. chinense PR-4 protein belongs to the PR-4 protein subgroup II, based on the absence of a hevein domain. Furthermore, it is shown that the purified protein does not have chitinase activity, as previously proposed for PR-4 proteins. Instead, it has both RNase and DNase activity, although its contribution to the bulk activity of nucleases in infected plants is very low.

Features of the plasmid pMV158-encoded MobM, a protein involved in its mobilization.

The streptococcal promiscuous plasmid pMV158 can be mobilized between a number of bacterial species by means of three elements: (i) the plasmid-encoded nicking-closing protein MobM, involved in the initiation and termination of the conjugative transfer; (ii) the DNA sequence where the MobM-mediated nick takes place (the oriT(pMV158)); and (iii) the function(s) provided by auxiliary plasmids. MobM belongs to the Pre/Mob family of plasmid-encoded DNA-relaxing proteins (relaxases). Purified MobM protein has been used to assay cleavage conditions on plasmid supercoiled DNA. Some structural features of MobM have been addressed by analytical ultracentrifugation, circular dichroism, thermal denaturation, and fluorescence emission. The protein behaved as a dimer of identical subunits with an ellipsoidal shape. MobM showed a high (about 60%) alpha-helical content and a midpoint denaturation of about 40 degrees C. Cell fractionation assays showed that MobM was associated to the cell membrane. This association was abolished when a great alteration was introduced within a putative coiled-coil located at the C-terminal region of the protein. Emission fluorescence suggested that the three Trp residues of MobM are located within a hydrophobic environment. A molecular model of MobM on the known structure of colicin Ia has been built.

Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate.

Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs.

The yefM-yoeB toxin-antitoxin systems of Escherichia coli and Streptococcus pneumoniae: functional and structural correlation.

Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM(Spn)) and toxin (YoeB(Spn)) products. We showed that overproduction of YoeB(Spn) is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB(Spn)-mediated toxicity could be reversed by the cognate antitoxin, YefM(Spn), but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

The mitochondrial uncoupling proteins.

The uncoupling proteins (UCPs) are transporters, present in the mitochondrial inner membrane, that mediate a regulated discharge of the proton gradient that is generated by the respiratory chain. This energy-dissipatory mechanism can serve functions such as thermogenesis, maintenance of the redox balance, or reduction in the production of reactive oxygen species. Some UCP homologs may not act as true uncouplers, however, and their activity has yet to be defined. The UCPs are integral membrane proteins, each with a molecular mass of 31-34 kDa and a tripartite structure in which a region of around 100 residues is repeated three times; each repeat codes for two transmembrane segments and a long hydrophilic loop. The functional carrier unit is a homodimer. So far, 45 genes encoding members of the UCP family have been described, and they can be grouped into six families. Most of the described genes are from mammals, but UCP genes have also been found in fish, birds and plants, and there is also functional evidence to suggest their presence in fungi and protozoa. UCPs are encoded in their mature form by nuclear genes and, unlike many nuclear-encoded mitochondrial proteins, they lack a cleavable mitochondrial import signal. The information for mitochondrial targeting resides in the first loop that protrudes into the mitochondrial matrix; the second matrix loop is essential for insertion of the protein into the inner mitochondrial membrane. UCPs are regulated at both the transcriptional level and by activation and inhibition in the mitochondrion.

Modeling the transmembrane arrangement of the uncoupling protein UCP1 and topological considerations of the nucleotide-binding site.

The uncoupling protein from brown adipose tissue (UCP1) is a mitochondrial proton transporter whose activity is inhibited by purine nucleotides. UCP1, like the other members of the mitochondrial transporter superfamily, is an homodimer and each subunit contains six transmembrane segments. In an attempt to understand the structural elements that are important for nucleotide binding, a model for the transmembrane arrangement of UCP1 has been built by computational methods. Biochemical and sequence analysis considerations are taken as constraints. The main features of the model include the following: (i) the six transmembrane alpha-helices (TMHs) associate to form an antiparallel helix bundle; (ii) TMHs have an amphiphilic nature and thus the hydrophobic and variable residues face the lipid bilayer; (iii) matrix loops do not penetrate in the core of the bundle; and (iv) the polar core constitutes the translocation pathway. Photoaffinity labeling and mutagenesis studies have identified several UCP1 regions that interact with the nucleotide. We present a model where the nucleotide binds deep inside the bundle core. The purine ring interacts with the matrix loops while the polyphosphate chain is stabilized through interactions with essential Arg residues in the TMH and whose side chains face the core of the helix bundle.