The Nutraceutical Alliin From Garlic Is a Novel Substrate of the Essential Amino Acid Transporter LAT1 (SLC7A5).

The plasma membrane transporter LAT1 (SLC7A5) is a crucial player for cell homeostasis because it is responsible for providing cells with essential amino acids and hormones. LAT1 forms a functional heterodimer with the cell surface antigen heavy chain CD98 (also known as 4F2hc and SLC3A2), a type II membrane glycoprotein, which is essential for LAT1 stability and localization to the plasma membrane. The relevance of LAT1 for human metabolism is also related to its altered expression in human diseases, such as cancer and diabetes. These features boosted research toward molecules that are able to interact with LAT1; in this respect, the recent resolution of the LAT1-CD98 3D structure by Cryo-EM has opened important perspectives in the study of the interaction with different molecules in order to identify new drugs to be used in therapy or new substrates of natural origin to be employed as adjuvants and food supplements. In this work, the interaction of LAT1 with alliin, a garlic derivative, has been investigated by using a combined approach of bioinformatics and in vitro transport assays. Alliin is a nutraceutical that has several beneficial effects on human health, such as antidiabetic, anticarcinogenic, antioxidant, and anti-inflammatory properties. The computational analysis suggested that alliin interacts with the substrate binding site of LAT1, to which alliin was docked. These data were then confirmed by the competitive type inhibition measured in proteoliposomes. Interestingly, in the same experimental model, alliin was also revealed to be a substrate of LAT1.

Mitochondria of Cedrus atlantica and allied species: A new chapter in the horizontal gene transfer history.

The extraordinary incidence of Horizontal Gene Transfer (HGT) mostly in mitochondrial genomes of flowering plants is well known. Here, we report another episode of HGT affecting a large mitochondrial gene region in the evergreen conifer Atlas cedar (Cedrus atlantica). Mitochondria of this Pinaceae species possess an rps3 gene that harbours two introns and shares the same genomic context with a downstream overlapping rpl16 gene, like in the major groups of gymnosperms and angiosperms analyzed so far. Interestingly, C. atlantica contains additional copies of the rps3 and rpl16 sequences that are more closely related to angiosperm counterparts than to those from gymnosperms, as also confirmed by phylogenetic analyses. This suggests that a lateral transfer from a flowering plant donor is the most likely mechanism for the origin of the Atlas cedar extra sequences. Quantitative PCR and reverse-transcription (RT)-PCR analyses demonstrate, respectively, mitochondrial location and lack of expression for the rps3 and rpl16 additional sequences in C. atlantica. Furthermore, our study provides evidence that a similar HGT event takes place in two other Cedrus species, which occurr in Cyprus and North Africa. Only the West Himalayan C. deodara lacks the transferred genes. The potential donor and the molecular mechanism underlying this lateral DNA transfer remain still unclear.

Bacterial production and reconstitution in proteoliposomes of Solanum lycopersicum CAT2: a transporter of basic amino acids and organic cations.

KEY MESSAGE: The vacuolar SlCAT2 was cloned, over-produced in E. coli and reconstituted in proteoliposomes. Arg, Ornithine and Lys were identified as substrates. Unexpectedly, also the organic cations Tetraethylammonium and Acetylcholine were transported indicating involvement of SlCAT2 in signaling. In land plants several transporters are involved in ion and metabolite flux across membranes of cells or intracellular organelles. The vacuolar amino acid transporter CAT2 from Solanum lycopersicum was investigated in this work. SlCAT2 was cloned from tomato flower cDNA, over-produced in Escherichia coli and purified by Nichel-chelating chromatography. For functional studies, the transporter was reconstituted in proteoliposomes. Competence of SlCAT2 for Arg transport was demonstrated measuring uptake of [3H]Arg in proteoliposomes which was trans-stimulated by internal Arg or ornithine. Uptake of [3H]Ornithine and [3H]Lys was also detected at lower efficiency with respect to [3H]Arg. Transport was activated by the presence of intraliposomal ATP suggesting regulation by the nucleotide. The prototype for organic cations tetraethylammonium (TEA) was also transported by SlCAT2. However, scarce reciprocal inhibition between TEA and Arg was found, while the biguanide metformin was able to strongly inhibit uptake of both substrates. These findings suggest that amino acids and organic cations may interact with the transporter through different functional groups some of which are common for the two types of substrates. Interestingly, reconstituted SlCAT2 showed competence for acetylcholine transport, which was also inhibited by metformin. Kinetics of Arg and Ach transport were performed from which Km values of 0.29 and 0.79 mM were derived, respectively.

The evolutionary conservation of rps3 introns and rps19-rps3-rpl16 gene cluster in Adiantum capillus-veneris mitochondria.

Ferns are a large and evolutionarily critical group of vascular land plants for which quite limited mitochondrial gene content and genome organization data are, currently, available. This study reports that the gene for the ribosomal protein S3 (rps3) is preserved and physically clustered to an upstream rps19 and a downstream overlapping rpl16 locus in the mitochondrial DNA of the true fern Adiantum capillus-veneris L. Sequence analysis also revealed that the rps3 gene is interrupted by two cis-splicing group II introns, like the counterpart in lycopod and gymnosperm representatives. A preliminary polymerase chain reaction (PCR) survey confirmed a scattered distribution pattern of both the rps3 introns also in other fern lineages. Northern blot and reverse transcription (RT)-PCR analyses demonstrated that the three ribosomal protein genes are co-transcribed as a polycistronic mRNA and modified by RNA editing. Particularly, the U-to-C type editing amends numerous genomic stop codons in the A. capillus-veneris rps19, rps3 and rpl16 sequences, thus, assuring the synthesis of complete and functional polypeptides. Collectively, the findings from this study further expand our knowledge of the mitochondrial rps3 architecture and evolution, also, bridging the significant molecular data gaps across the so far underrepresented ferns and all land plants.

REDIdb: the RNA editing database.

The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at http://biologia.unical.it/py_script/search.html.