Positive and negative regulation of angiogenesis by soluble vascular endothelial growth factor receptor-1

Vascular endothelial growth factor receptor (VEGFR)-1 exists in different forms, derived from alternative splicing of the same gene. In addition to the transmembrane form, endothelial cells produce a soluble VEGFR-1 (sVEGFR-1) isoform, whereas non-endothelial cells produce both sVEGFR-1 and a different soluble molecule, known as soluble fms-like tyrosine kinase (sFlt)1-14. By binding members of the vascular endothelial growth factor (VEGF) family, the soluble forms reduce the amounts of VEGFs available for the interaction with their transmembrane receptors, thereby negatively regulating VEGFR-mediated signaling. In agreement with this activity, high levels of circulating sVEGFR-1 or sFlt1-14 are associated with different pathological conditions involving vascular dysfunction. Moreover, sVEGFR-1 and sFlt1-14 have an additional role in angiogenesis: they are deposited in the endothelial cell and pericyte extracellular matrix, and interact with cell membrane components. Interaction of sVEGFR-1 with α5β1 integrin on endothelial cell membranes regulates vessel growth, triggering a dynamic, pro-angiogenic phenotype. Interaction of sVEGFR-1/sFlt1-14 with cell membrane glycosphingolipids in lipid rafts controls kidney cell morphology and glomerular barrier functions. These cell-matrix contacts represent attractive novel targets for pharmacological intervention in addition to those addressing interactions between VEGFs and their receptors

The phenotypic expression of mitochondrial tRNA-mutations can be modulated by either mitochondrial leucyl-tRNA synthetase or the C-terminal domain thereof

Mutations in mitochondrial (mt) DNA determine important human diseases. The majority of the known pathogenic mutations are located in transfer RNA (tRNA) genes and are responsible for a wide range of currently untreatable disorders. Experimental evidence both in yeast and in human cells has shown that the detrimental effects of mt-tRNA point mutations can be attenuated by increasing the expression of the cognate mt-aminoacyl-tRNA synthetases (aaRSs). In addition, constitutive high levels of isoleucyl-tRNA syntethase have been shown to reduce the penetrance of a homoplasmic mutation in mt-tRNAIle in a small kindred. More recently, we showed that the isolated carboxy-terminal domain of human mt-leucyl tRNA synthetase (LeuRS-Cterm) localizes to mitochondria and ameliorates the energetic defect in transmitochondrial cybrids carrying mutations either in the cognate mt-tRNALeu(UUR) or in the non cognate mt-tRNAIle gene. Since the mt-LeuRS-Cterm does not possess catalytic activity, its rescuing ability is most likely mediated by a chaperon-like effect, consisting in the stabilization of the tRNA structure altered by the mutation. All together, these observations open potential therapeutic options for mt-tRNA mutations-associated diseas

Insights into the Catalytic Mechanism of Glutathione S-Transferase: The Lesson from Schistosoma haematobium

SummaryGlutathione S-transferases (GSTs) are involved in detoxification of xenobiotic compounds and in the biosynthesis of important metabolites. All GSTs activate glutathione (GSH) to GS−; in many GSTs, this is accomplished by a Tyr at H-bonding distance from the sulfur of GSH. The high-resolution structure of GST from Schistosoma haematobium revealed that the catalytic Tyr occupies two alternative positions, one external, involving a π-cation interaction with the conserved Arg21, and the other inside the GSH binding site. The interaction with Arg21 lowers the pKa of the catalytic Tyr10, as required for catalysis. Examination of several other GST structures revealed the presence of an external pocket that may accommodate the catalytic Tyr, and suggested that the change in conformation and acidic properties of the catalytic Tyr may be shared by other GSTs. Arginine and two other residues of the external pocket constitute a conserved structural motif, clearly identified by sequence comparison

Exploring the cytochrome c folding mechanism: cytochrome c552 from thermus thermophilus folds through an on-pathway intermediate.

Understanding the role of partially folded intermediate states in the folding mechanism of a protein is a crucial yet very difficult problem. We exploited a kinetic approach to demonstrate that a transient intermediate of a thermostable member of the widely studied cytochrome c family (cytochrome c552 from Thermus thermophilus) is indeed on-pathway. This is the first clear indication of an obligatory intermediate in the folding mechanism of a cytochrome c. The fluorescence properties of this intermediate demonstrate that the relative position of the heme and of the only tryptophan residue cannot correspond to their native orientation. Based on an analysis of the three-dimensional structure of cytochrome c552, we propose an interpretation of the data which explains the residual fluorescence of the intermediate and is consistent with the established role played by some conserved interhelical interactions in the folding of other members of this family. A limited set of topologically conserved contacts may guide the folding of evolutionary distant cytochromes c through the same partially structured state, which, however, can play different kinetic roles, acting either as an intermediate or a transition state

Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration

Endoribonucleases participate in almost every step of eukaryotic RNA metabolism, acting either as degradative or biosynthetic enzymes. We previously identified the founding member of the Eukaryotic EndoU ribonuclease family, whose components display unique biochemical features and are flexibly involved in important biological processes, such as ribosome biogenesis, tumorigenesis and viral replication. Here we report the discovery of the CG3303 gene product, which we named DendoU, as a novel family member in Drosophila. Functional characterisation revealed that DendoU is essential for Drosophila viability and nervous system activity. Pan-neuronal silencing of dendoU resulted in fly immature phenotypes, highly reduced lifespan and dramatic motor performance defects. Neuron-subtype selective silencing showed that DendoU is particularly important in cholinergic circuits. At the molecular level, we unveiled that DendoU is a positive regulator of the neurodegeneration-associated protein dTDP-43, whose downregulation recapitulates the ensemble of dendoU-dependent phenotypes. This interdisciplinary work, which comprehends in silico, in vitro and in vivo studies, unveils a relevant role for DendoU in Drosophila nervous system physio-pathology and highlights that DendoU-mediated neurotoxicity is, at least in part, contributed by dTDP-43 loss-of-function

Arabidopsis thaliana sirtuins control proliferation and glutamate dehydrogenase activity

Sirtuins are part of a gene family of NAD-dependent deacylases that act on histone and non-histone proteins and control a variety of activities in all living organisms. Their roles are mainly related to energy metabolism and include lifetime regulation, DNA repair, stress resistance, and proliferation. A large amount of knowledge concerning animal sirtuins is available, but data about their plant counterparts are scarce. Plants possess few sirtuins that have, like in animals, a recognized role in stress defense and metabolism regulation. However, engagement in proliferation control, which has been demonstrated for mammalian sirtuins, has not been reported for plant sirtuins so far. In this work, srt1 and srt2 Arabidopsis mutant seedlings have been used to evaluate in vivo the role of sirtuins in cell proliferation and regulation of glutamate dehydrogenase, an enzyme demonstrated to be involved in the control of cell cycle in SIRT4-defective human cells. Moreover, bioinformatic analyses have been performed to elucidate sequence, structure, and function relationships between Arabidopsis sirtuins and between each of them and the closest mammalian homolog. We found that cell proliferation and GDH activity are higher in mutant seedlings, suggesting that both sirtuins exert a physiological inhibitory role in these processes. In addition, mutant seedlings show plant growth and root system improvement, in line with metabolic data. Our data also indicate that utilization of an easy to manipulate organism, such as Arabidopsis plant, can help to shed light on the molecular mechanisms underlying the function of genes present in interkingdom species

Novel compound mutations in the mitochondrial translation elongation factor (TSFM) gene cause severe cardiomyopathy with myocardial fibro-adipose replacement

Primary mitochondrial dysfunction is an under-appreciated cause of cardiomyopathy, especially when cardiac symptoms are the unique or prevalent manifestation of disease. Here, we report an unusual presentation of mitochondrial cardiomyopathy, with dilated phenotype and pathologic evidence of biventricular fibro-adipose replacement, in a 33-year old woman who underwent cardiac transplant. Whole exome sequencing revealed two novel compound heterozygous variants in the TSFM gene, coding for the mitochondrial translation elongation factor EF-Ts. This protein participates in the elongation step of mitochondrial translation by binding and stabilizing the translation elongation factor Tu (EF-Tu). Bioinformatics analysis predicted a destabilization of the EF-Ts variants complex with EF-Tu, in agreement with the dramatic steady-state level reduction of both proteins in the clinically affected myocardium, which demonstrated a combined respiratory chain enzyme deficiency. In patient fibroblasts, the decrease of EF-Ts was paralleled by up-regulation of EF-Tu and induction of genes involved in mitochondrial biogenesis, along with increased expression of respiratory chain subunits and normal oxygen consumption rate. Our report extends the current picture of morphologic phenotypes associated with mitochondrial cardiomyopathies and confirms the heart as a main target of TSFM dysfunction. The compensatory response detected in patient fibroblasts might explain the tissue-specific expression of TSFM-associated disease

Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

Vide

The isolated carboxy-terminal domain of human mitochondrial leucyl-tRNA synthetase rescues the pathological phenotype of mitochondrial tRNA mutations in human cells.

Mitochondrial (mt) diseases are multisystem disorders due to mutations in nuclear or mtDNA genes. Among the latter, more than 50% are located in transfer RNA (tRNA) genes and are responsible for a wide range of syndromes, for which no effective treatment is available at present. We show that three human mt aminoacyl-tRNA syntethases, namely leucyl-, valyl-, and isoleucyl-tRNA synthetase are able to improve both viability and bioenergetic proficiency of human transmitochondrial cybrid cells carrying pathogenic mutations in the mt-tRNA(Ile) gene. Importantly, we further demonstrate that the carboxy-terminal domain of human mt leucyl-tRNA synthetase is both necessary and sufficient to improve the pathologic phenotype associated either with these "mild" mutations or with the "severe" m.3243A>G mutation in the mt-tRNA(L)(eu(UUR)) gene. Furthermore, we provide evidence that this small, non-catalytic domain is able to directly and specifically interact in vitro with human mt-tRNA(Leu(UUR)) with high affinity and stability and, with lower affinity, with mt-tRNA(Ile). Taken together, our results sustain the hypothesis that the carboxy-terminal domain of human mt leucyl-tRNA synthetase can be used to correct mt dysfunctions caused by mt-tRNA mutations