A DFT investigation of a bulky biomimetic model catalyzing the 5′-outer ring deiodination of thyroxine

This paper illustrates the outcomes of a density functional theory investigation aimed at unraveling mechanistic aspects of the 5′-outer ring deiodination process of thyroxine (T4) assisted by the sterically protected organoselenol compound BpqSeH. BpqSeH, which was previously synthesized and tested for its deiodinase activity, is able to afford the active hormone 3,5,3′-tetraiodothyronine (T3) by selective outer-ring deiodination of T4, and to protect the SeH moiety inside the nano-sized molecular cavity from further reactivity, allowing its isolation and characterization. Calculations were also performed including an imidazole ring that, mimicking a His residue in the active site of the original enzyme, plays an crucial role in deprotonating the selenol moiety. Both the suggested enol/keto tautomerization and the previously proven formation of an intermediate whose main characteristic is the presence of a Se?I?C halogen bond, were examined along the pathway leading to 5′-outer ring deiodination. The calculated potential energy surface showed that neither the pathway encompassing enol/keto tautomerism nor the formation of a halogen bond paving the way to C–I bond breaking and chalcogen-I bond forming is viable. The exergonic formation of the final selenenyl iodide product confirms the stabilization effect of the molecular cavity.

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Graphical Abstract Computed free energy profile describing the 5′-outer deiodination of thyroxine assisted by the steric hindered organoselenol BpqSH compound. The molecular electrostatic potential map reoported for the INT1 intermediate shows the non-covalent Se-I interaction, due to the attraction between charges of opposite sign, that weakens the C-I bond and prepares the formation of the new Se-I bond.

     

Quantitative assessment of chemical artefacts produced by propionylation of histones prior to mass spectrometry analysis

Histone PTMs play a crucial role in regulating chromatin structure and function, with impact on gene expression. MS is nowadays widely applied to study histone PTMs systematically. Because histones are rich in arginine and lysine, classical shot‐gun approaches based on trypsin digestion are typically not employed for histone modifications mapping. Instead, different protocols of chemical derivatization of lysines in combination with trypsin have been implemented to obtain “Arg‐C like” digestion products that are more suitable for LC‐MS/MS analysis. Although widespread, these strategies have been recently described to cause various side reactions that result in chemical modifications prone to be misinterpreted as native histone marks. These artefacts can also interfere with the quantification process, causing errors in histone PTMs profiling. The work of Paternoster V. et al. 1 is a quantitative assessment of methyl‐esterification and other side reactions occurring on histones after chemical derivatization of lysines with propionic anhydride [Proteomics 2016, 16, 2059–2063]. The authors estimate the effect of different solvents, incubation times, and pH on the extent of these side reactions. The results collected indicate that the replacement of methanol with isopropanol or ACN not only blocks methyl‐esterification, but also significantly reduces other undesired unspecific reactions. Carefully titrating the pH after propionic anhydride addition is another way to keep methyl‐esterification under control. Overall, the authors describe a set of experimental conditions that allow reducing the generation of various artefacts during histone propionylation.     

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.     

Protective effect of orally administered carnosine on bleomycin-induced lung injury

Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and L-histidine. It acts as a free radical scavenger and possesses antioxidant properties. Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), IL-1, and TNF-alpha in different experimental settings. In the present study, we investigated the efficacy of carnosine on the animal model of bleomycin-induced lung injury. Mice were subjected to intratracheal administration of bleomycin and were assigned to receive carnosine daily by an oral bolus of 150 mg/kg. One week after fibrosis induction, bronchoalveolar lavage (BAL) cell counts and TGF-beta levels, lung histology, and immunohistochemical analyses for myeloperoxidase, TGF-beta, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase were performed. Finally, apoptosis was quantified by terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay. After bleomycin administration, carnosine-treated mice exhibited a reduced degree of lung damage and inflammation compared with wild-type mice, as shown by the reduction of 1) body weight, 2) mortality rate, 3) lung infiltration by neutrophils (myeloperoxidase activity and BAL total and differential cell counts), 4) lung edema, 5) histological evidence of lung injury and collagen deposition, 6) lung myeloperoxidase, TGF-beta, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase immunostaining, 7) BAL TGF-beta levels, and 8) apoptosis. Our results indicate that orally administered carnosine is able to prevent bleomycin-induced lung injury likely through its direct antioxidant properties. Carnosine is already available for human use. It might prove useful as an add-on therapy for the treatment of fibrotic disorders of the lung where oxidative stress plays a role, such as for idiopathic pulmonary fibrosis, a disease that still represents a major challenge to medical treatment.     

A gamma-glutamyl transpeptidase of Aphidius ervi venom induces apoptosis in the ovaries of host aphids

Parasitism by the endophagous braconid Aphidius ervi (Hymenoptera, Braconidae) has a negative impact on the reproductive activity of its host, Acyrthosiphon pisum (Homoptera, Aphididae). The host castration is induced by the parasitoid venom and is reproduced by the injection of chromatographic fractions highly enriched with two proteins, of 18 (p18) and 36 kDa (p36) in size, respectively. Here we demonstrate that these bioactive proteins trigger apoptosis of the cells in the germaria and ovariole sheath of the host aphid. Both p18 and p36 were internally sequenced and the gathered information was matched against the deduced amino acid sequence of the putative proteins encoded by cDNA clones, randomly selected from a cDNA library, which was raised using mRNA extracted from A. ervi venom glands. The identified cDNA clones contained an insert corresponding to the RNA product of an interrupted gene, made of six exons and five introns, which was found to be transcribed at higher levels in adult females of A. ervi than in males. This gene codes for a putative protein composed of 541 amino acids, with a calculated molecular mass of 56.9 kDa, which contained the amino acid sequences experimentally determined for both p18 and p36. This putative protein showed a significant level of sequence identity with gamma-glutamyl transpeptidases (gamma-GT), and it was named Ae-gamma-GT. The gamma-GTs are enzymes which play a key role in the metabolism of glutathione (GSH) and, as observed in most organisms, they are membrane-bound heterodimers formed by a large and a small subunit, which originate by post-translational processing of a single-chain precursor. The expression in insect cells of Ae-gamma-GT confirmed the occurrence of the expected post-translational processing, and demonstrated that, unlike other gamma-GTs, this protein is secreted in the extracellular environment. A measurable gamma-GT activity was detected in the venom of A. ervi and in the chromatographic fractions containing Ae-gamma-GT. Thus, we suggest that this venom protein may induce apoptosis in the host ovarioles by generating an alteration of the GSH metabolism and a consequent oxidative stress.     

HLA class I and class II polymorphism in three Sicilian populations

Two human leukocyte antigen (HLA) class I loci (HLA-A and HLA-B) and one class II locus (HLA-DR) were typed at the DNA level in the Sicilian population. Study participants were of Sicilian origin (183 for class I loci and 260 for class II loci) and live in three towns, chosen on the basis of geographic position and different historical events. These towns are Sciacca (southwest Sicily, located at sea level, conquered by Arabs in A.D. 814), Piana degli Albanesi (northwest Sicily, 720 m above sea level, has maintained religious, cultural, and linguistic peculiarities traced to Albanian settlement in 1488), and Troina (northeast Sicily, 1120 m above sea level, known as the first settlement of Normans). The assumptions underlying the study of genetic structure, based on HLA allele polymorphism, are that these three towns are located in areas that can be distinguished according to historical criteria and that they are likely to have contributed to cultural and probably genetic differences. As such, the high frequency of some alleles in Sciacca and Troina seems to be correlated with Greek, Phoenician, North African, and Arab influence. In accordance with different human settlements in Sicily, we found that the HLA allele frequencies support the existence of genetic differentiation between the western and eastern sides of Sicily. This separation is attributed to Greek colonization in the east and to Phoenician-Carthaginian-Arab influence in the west. Moreover, the comparisons of all allele frequencies between Mediterranean and AfrIcan populations show the same trend, highlighting in some cases European origin and in other cases non-European origin.     

Anodic microbial community analysis of microbial fuel cells based on enriched inoculum from freshwater sediment

Bioprocess and Biosystems Engineering - The characterization of anodic microbial communities is of great importance in the study of microbial fuel cells (MFCs). These kinds of devices mainly...     

hmSEEKER: Identification of hmSILAC Doublets in MaxQuant Output Data

Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic labeling strategy employed in proteomics to increase the confidence of global identification of methylated peptides by MS. However, to this day, the automatic and robust identification of heavy and light peak doublets from MS‐raw data of hmSILAC experiments is a challenging task, for which the choice of computational methods is very limited. Here, hmSEEKER, a software designed to work downstream of a MaxQuant analysis for in‐depth search of MS peak pairs that correspond to light and heavy methyl‐peptide within MaxQuant‐generated tables is described with good sensitivity and specificity. The software is written in Perl, and its code and user manual are freely available at Bitbucket ( https://bit.ly/2scCT9u ).