Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

Genome Size: A Novel Genomic Signature in Support of Afrotheria

Molecular phylogenetic analyses suggest an emerging phylogeny for the extant Placentalia (eutherian) that radically departs from morphologically based constructions of the past. Placental mammals are partitioned into four supraordinal clades: Afrotheria, Xenarthra, Laurasiatheria, and Euarchontoglires. Afrotheria form an endemic African clade that includes elephant shrews, golden moles, tenrecs, aardvarks, hyraxes, elephants, dugongs, and manatees. Datamining databases of genome size (GS) shows that till today just one afrotherian GS has been evaluated, that of the aardvark Orycteropus afer. We show that the GSs of six selected representatives across the Afrotheria supraordinal group are among the highest for the extant Placentalia, providing a novel genomic signature of this enigmatic group. The mean GS value of Afrotheria, 5.3 ± 0.7 pg, is the highest reported for the extant Placentalia. This should assist in planning new genome sequencing initiatives. [Reviewing Editor: Dmitri Petrov]     

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.     

Molecular Genetics of Cystinuria: Identification of Four New Mutations and Seven Polymorphisms, and Evidence for Genetic Heterogeneity

A cystinuria disease gene (rBAT) has been recently identified, and some mutations causing the disease have been described. The frequency of these mutations has been investigated in a large sample of 51 Italian and Spanish cystinuric patients. In addition, to identify new mutated alleles, genomic DNA has been analyzed by an accurate and sensitive method able to detect nucleotide changes. Because of the lack of information available on the genomic structure of rBAT gene, the study was carried out using the sequence data so far obtained by us. More than 70% of the entire coding sequence and 8 intron-exon boundaries have been analyzed. Four new mutations and seven intragenic polymorphisms have been detected. All mutations so far identified in rBAT belong only to cystinuria type I alleles, accounting for ~44% of all type I cystinuric chromosomes. Mutation M467T is the most common mutated allele in the Italian and Spanish populations. After analysis of 70% of the rBAT coding region, we have detected normal sequences in cystinuria type II and type III chromosomes. The presence of rBAT mutated alleles only in type I chromosomes of homozygous (type I/I) and heterozygous (type I/III) patients provides evidence for genetic heterogeneity where rBAT would be responsible only for type I cystinuria and suggests a complementation mechanism to explain the intermediate type I/type III phenotype.     

Trypanosoma rangeli sialidase: cloning, expression and similarity to T.cruzi trans-sialidase

Sialidases are hydrolytic enzymes present from virus to highereukaryotes, catalyzing the removal of sialic acid from glycoconjugates.Some protozoa Trypanosomatidae secrete high levels of sialidaseinto the medium. We have now purified the secreted sialidasefrom Trypanosoma rangeli Its N-terminal sequence reveals 100%identity with the corresponding region of the trans-sialidasefrom T.cruzi Trans-sialidase, although homologous to viral andbacterial sialidases, displays a novel sialyltransferase activityand is involved in host cell invasion. Several homologous trans-sialidase-likegenes were cloned from genomic DNA of T.rangeli, and groupedin three subfamilies. Active siali-dase-encoding genes werefound in one of them. The re-combinant sialidase shows similarproperties to those of the native enzyme, including undetectabletrans-sialidase activity. Nevertheless, it has an overall identityof 68.9% with the catalytic domain of T.cruzi trans-sialidase,increasing to 86.7% admitting conservative substitutions. Onlythree other eukaryotic sialidases have been previously cloned,none of them showing significant homology to trans-sialidase.The isolation of a highly similar sialidase is relevant to furtheridentify the molecular determinants allowing trans-sialidaseactivity. As a first approach, chimeric constructs between sialidaseand trans-sialidase were generated, one of them rendering asialidase with three times lower K_m than the natural enzyme. eukaryotic sialidase gene family glycosidase parasite sialic acid     

2-D DIGE analysis of UV-C radiation-responsive proteins in globe artichoke leaves

Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a ～144,000 customized Compositae protein database, which included about 19,000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress.     

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.     

DNA repair-deficient premature aging models display accelerated epigenetic age

Several premature aging mouse models have been developed to study aging and identify interventions that can delay age-related diseases. Yet, it is still unclear whether these models truly recapitulate natural aging. Here, we analyzed DNA methylation in multiple tissues of four previously reported mouse models of premature aging (Ercc1, LAKI, Polg, and Xpg). We estimated DNA methylation (DNAm) age of these samples using the Horvath clock. The most pronounced increase in DNAm age could be observed in Ercc1 mice, a strain which exhibits a deficit in DNA nucleotide excision repair. Similarly, we detected an increase in epigenetic age in fibroblasts isolated from patients with progeroid syndromes associated with mutations in DNA excision repair genes. These findings highlight that mouse models with deficiencies in DNA repair, unlike other premature aging models, display accelerated epigenetic age, suggesting a strong connection between DNA damage and epigenetic dysregulation during aging.