Release LTP_12_2020, featuring a new ARB alignment and improved 16S rRNA tree for prokaryotic type strains

The new release of the All-Species Living Tree Project (LTP) represents an important step forward in the reconstruction of 16S rRNA gene phylogenies, since we not only provide an updated set of type strain sequences until December 2020, but also a series of improvements that increase the quality of the database. An improved universal alignment has been introduced that is implemented in the ARB format. In addition, all low-quality sequences present in the previous releases have been substituted by new entries with higher quality, many of them as a result of whole genome sequencing. Altogether, the improvements in the dataset and 16S rRNA sequence alignment allowed us to reconstruct robust phylogenies. The trees made available through this current LTP release feature the best topologies currently achievable. The given nomenclature and taxonomic hierarchy reflect all the changes available up to December 2020. The aim is to regularly update the validly published nomenclatural classification changes and new taxa proposals. The new release can be found at the following URL: https://imedea.uib-csic.es/mmg/ltp/.     

Amino acids activate mammalian target of rapamycin complex 2 (mTORC2) via PI3K/Akt signaling

The activity of mammalian target of rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation, or survival. Nutrients such as amino acids are important regulators of mTOR complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis, and autophagy. Here, we show that amino acids may also activate mTOR complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr-308 and Ser-473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser-473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.     

Modification of hormonal balance in larvae of the corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae) due to sublethal Bacillus thuringiensis protein ingestion

Bacillus thuringiensis (Bt) corn, Zea mays L., is highly efficient against the corn borer Sesamia nonagrioides (Lefèbvre) (Lepidoptera: Noctuidae) when the larvae feed only on the transgenic plants. However, when they feed on Bt leaves during only part of their development, thus ingesting sublethal amounts of Bt toxins, some larvae survive. A previous study reported a prolonged development and precocious diapause induction in larvae fed on a diet with sublethal amounts of Cry1Ab protein. To determine whether these effects were accompanied by a modification of the hormonal balance, S. nonagrioides larvae were fed on sublethal amounts of Bt protein provided in Bt leaves or in the diet. The larvae that survived had higher levels of juvenile hormone (JH), whereas their level of ecdysteroids did not increase sufficiently to allow pupation, leading to a longer larval development and more larval molts. This response may be considered a defense mechanism that allows some larvae to survive toxin ingestion; it is similar the response to insecticidal toxins or viruses observed in other larvae. Changes in the hormone levels in diapausing larvae were undetectable, probably because these changes were masked by the higher level of JH in the hemolymph of diapausing larvae and because of lack of ecdysteroid titer increase, a phenomenon that is usually observed a few days before pupation in nondiapausing larvae. These results should be taken into account in the establishment of non-Bt refuges to prevent development of Bt-resistance in S. non-agrioides populations.     

Salt effects on the conformational stability of the visual G-protein-coupled receptor rhodopsin

Membrane protein stability is a key parameter with important physiological and practical implications. Inorganic salts affect protein stability, but the mechanisms of their interactions with membrane proteins are not completely understood. We have undertaken the study of a prototypical G-protein-coupled receptor, the α-helical membrane protein rhodopsin from vertebrate retina, and explored the effects of inorganic salts on the thermal decay properties of both its inactive and photoactivated states. Under high salt concentrations, rhodopsin significantly increased its activation enthalpy change for thermal bleaching, whereas acid denaturation affected the formation of a denatured loose-bundle state for both the active and inactive conformations. This behavior seems to correlate with changes in protonated Schiff-base hydrolysis. However, chromophore regeneration with the 11-cis-retinal chromophore and MetarhodopsinII decay kinetics were slower only in the presence of sodium chloride, suggesting that in this case, the underlying phenomenon may be linked to the activation of rhodopsin and the retinal release processes. Furthermore, the melting temperature, determined by means of circular dichroism and differential scanning calorimetry measurements, was increased in the presence of high salt concentrations. The observed effects on rhodopsin could indicate that salts favor electrostatic interactions in the retinal binding pocket and indirectly favor hydrophobic interactions at the membrane protein receptor core. These effects can be exploited in applications where the stability of membrane proteins in solution is highly desirable.     

Potential implication of aniline derivatives in the Toxic Oil Syndrome (TOS)

The Toxic Oil Syndrome (TOS) was an epidemic disease appeared in central Spain in 1981, causing over 400 deaths and affecting more than 20,000 people, mainly women and children. The disease was linked to the consumption of rapeseed oil denatured with aniline, illegally refined at the ITH oil refinery in Seville, mixed with other oils and sold as edible olive oil. Among the aniline derivatives detected in the oil batches generated by an uncontrolled deodorisation procedure during the refining process, fatty acid anilides were first postulated as the causal agents. Nevertheless, compounds identified as 3-(N-phenylamino)propane-1,2-diol (PAP) and its mono-, di-, and triacyl derivatives (mPAP, dPAP and tPAP, respectively), were subsequently considered better biomarkers of toxic oils and the best candidates for causing the intoxication. In this account, we will discuss the results obtained in recent years by our group concerning: (a) The effect of different variables intervening in the deodorisation process that could influence the formation of PAP derivatives. To this end we decided to take the aniline derivatives linked to oleic acid as compound models since this is the fatty acid present in highest amounts in rapeseed oil. The study was focused on the influence of different parameters on the formation of the diester PAP derivative (OOPAP) the monoester derivative (OPAP) and the corresponding amide (oleanilide, OA), and the interactions between any two of these variables. Of particular interest was the interaction observed between OOPAP and OA, due to its potential relevance to the final composition of the toxic oil model. (b) Xenobiochemical aspects of PAP derivatives, specifically: the stereospecific hydrolysis of OPAP and OOPAP by human pancreatic lipase, the in vitro activation of PAP by human and rat liver microsomes as well as by recombinant 450 enzymes, and the formation and stability of GSH and N-acetylcysteine adducts of a highly reactive iminoquinone intermediate generated in the biotransformation of PAP.     

Hydrophobic amino acids at the cytoplasmic ends of helices 3 and 6 of rhodopsin conjointly modulate transducin activation

Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin – where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues – in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V138^3.53 and the multiple mutant containing V227^5.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V138^3.53, V227^5.62, V250^6.33, V254^6.37 and I255^6.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction.     

Indirect inactivation of tyrosinase in its action on tyrosine

Under aerobic conditions, tyrosinase is inactivated by dopa as a result of suicide inactivation, and, under anaerobic conditions, as a result of irreversible inactivation. However, tyrosine protects the enzyme from being inactivated by dopa under anaerobic conditions. This paper describes how under aerobic conditions the enzyme acting on tyrosine is not directly inactivated but undergoes a process of indirect suicide inactivation provoked by reaction with the o-diphenol originated from the evolution of o-dopaquinone and accumulated in the reaction medium.     

Nestin (+) stem cells independently contribute to neural remodelling of the ischemic heart

Recent studies have revealed the existence of multipotent nestin-immunoreactive cells in the adult mammalian heart. These cells were recruited to infarct site following ischemic injury and differentiated to a vascular lineage leading to de novo blood vessel formation. Here, we show that a sub-population of cardiac resident nestin((+)) cells can further differentiate to a neuronal-like fate in vivo following myocardial infarction. In the ischemically damaged rat heart, neurofilament-M((+)) fibres were detected innervating the peri-infarct/infarct region and the preponderance of these fibres were physically associated with processes emanating from nestin((+)) cells. One week after isogenic heterotopic cardiac transplantation, the beating transplanted rat heart was devoid of neurofilament-M((+)) fibre staining. The superimposition of an ischemic insult to the transplanted heart led to the de novo synthesis of neurofilament-M((+)) fibres by cardiac resident nestin((+)) cells. Nerve growth factor infusion and the exposure of normal rats to intermittent hypoxia significantly increased the density of neurofilament-M((+)) fibres in the heart. However, these newly formed neurofilament-M((+)) fibres were not physically associated with nestin((+)) processes. These data highlight a novel paradigm of reparative fibrosis as a subpopulation of cardiac resident nestin((+)) cells directly contributed to neural remodelling of the peri-infarct/infarct region of the ischemically damaged rat heart via the de novo synthesis of neurofilament-M fibres.     

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction,phosphorylation and ER localization

AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca²⁺-pumping.     

“Candidatus Haloectosymbiotes riaformosensis” (Halobacteriaceae), an archaeal ectosymbiont of the hypersaline ciliate Platynematum salinarum

The novel ciliate Platynematum salinarum (Scuticociliatia) was isolated only recently from a thalassohaline solar saltern pond (12%) in Portugal. Scanning electron microscopy showed numerous bacterial-shaped cells covering the complete surface of the ciliate. The rod-shaped epibionts were identified and characterized following the “Full-Cycle rRNA Approach”. The almost full-length 16S rRNA gene sequence was obtained using archaeal-specific primers and two species-specific probes were designed for fluorescence in situ hybridization. The 16S rRNA gene sequence of the epibiotic cells showed 87% sequence identity with the type strain sequence of the closest characterized species Halolamina pelagica. Phylogenetic reconstructions affiliated the novel organism to the genus Halolamina (Halobacteria, Archaea). Attempts to isolate the epibionts failed and, therefore, growth experiments incorporating the antibiotic anisomycin were conducted in order to investigate the potential symbiotic relationship between P. salinarum and the epibionts. The results suggested an obligate symbiosis between the two organisms and revealed the first symbiotic representative of the Halobacteria. Based on the phylogenetic analyses and growth experiments we propose the classification of this novel organism in a new genus, with the taxon name “Candidatus Haloectosymbiotes riaformosensis”.