Functional properties of a newly cloned fish ortholog of the neutral amino acid transporter B0AT1 (SLC6A19)

The functional properties of an ortholog of the B0AT1 (SLC6A19) amino acid transporter, cloned from the intestine of the sea bass Dicentrachus labrax, were investigated. The two-electrode voltage-clamp technique was applied to Xenopus laevis oocytes heterologously expressing the transporter in order to measure the currents associated with the transport process in different conditions. In particular the substrate specificity, the ionic requirements, and possible effects of pH were examined. Among the organic substrates, leucine, glycine, serine and valine generated the largest transport currents with apparent affinities in the lower millimolar range. The importance of Na+ as the driver ion in the transport process is confirmed, although Li+ is also capable to sustain transport, while K+ is not. No evidence of a relevant role of Cl- in the transport activity was found. Concerning the other two kinds of currents commonly found in electrogenic transporters, very fast presteady-state currents were detected in the absence of organic substrate, while lithium-specific leak currents were not observed. The comparison of these properties with those of the mammalian and insect orthologs may give interesting indication for future structure-function studies in this transporter subfamily

Identification and characterization of the Atlantic salmon peptide transporter 1a

Peptide transporter 1 (PepT1) mediates the uptake of dietary di-/tripeptides in vertebrates. However, in teleost fish gut, more than one PepT1-type transporter might operate, because of teleost-specific whole gen(om)e duplication event(s) that occurred during evolution. Here, we describe a novel teleost di-/tripeptide transporter, i.e., the Atlantic salmon (Salmo salar) peptide transporter 1a [PepT1a; or solute carrier family 15 member 1a (Slc15a1a)], which is a paralog (77% similarity and 64% identity at the amino acid level) of the well-described Atlantic salmon peptide transporter 1b [PepT1b, alias PepT1; or solute carrier family 15 member 1b (Slc15a1b)]. Comparative analysis and evolutionary relationships of gene/protein sequences were conducted after ad hoc database mining. Tissue mRNA expression analysis was performed by quantitative real-time PCR, whereas transport function analysis was accomplished by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp measurements. Atlantic salmon pept1a is highly expressed in the proximal intestine (pyloric ceca ≈ anterior midgut > midgut >> posterior midgut), in the same gut regions as pept1b but notably ~5-fold less abundant. Like PepT1b, Atlantic salmon PepT1a is a low‐affinity/high‐capacity system. Functional analysis showed electrogenic, Na+-independent/pH-dependent transport and apparent substrate affinity (K0.5) values for Gly-Gln of 1.593 mmol/L at pH 7.6 and 0.076 mmol/L at pH 6.5. In summary, we show that a piscine PepT1a-type transporter is functional. Defining the role of Atlantic salmon PepT1a in the gut will help to understand the evolutionary and functional relationships among peptide transporters. Its functional characterization will contribute to elucidate the relevance of peptide transporters in Atlantic salmon nutritional physiology.acceptedVersio

The peptide transporter 1a of the zebrafish Danio rerio, an emerging model in nutrigenomics and nutrition research: Molecular characterization, functional properties, and expression analysis

Background: Peptide transporter 1 (PepT1, alias Slc15a1) mediates the uptake of dietary di/tripeptides in all vertebrates. However, in teleost fish, more than one PepT1-type transporter might function, due to specific whole genome duplication event(s) that occurred during their evolution leading to a more complex paralogue gene repertoire than in higher vertebrates (tetrapods). Results: Here, we describe a novel di/tripeptide transporter in the zebrafish (Danio rerio), i.e., the zebrafish peptide transporter 1a (PepT1a; also known as Solute carrier family 15 member a1, Slc15a1a), which is a paralogue (78% similarity, 62% identity at the amino acid level) of the previously described zebrafish peptide transporter 1b (PepT1b, alias PepT1; also known as Solute carrier family 15 member 1b, Slc15a1b). Also, we report a basic analysis of the pept1a (slc15a1a) mRNA expression levels in zebrafish adult tissues/organs and embryonic/early larval developmental stages. As assessed by expression in Xenopus laevis oocytes and two-electrode voltage clamp measurements, zebrafish PepT1a, as PepT1b, is electrogenic, Na+-independent, and pH-dependent and functions as a low-affinity system, with K0.5 values for Gly-Gln at − 60 mV of 6.92 mmol/L at pH 7.6 and 0.24 mmol/L at pH 6.5 and at − 120 mV of 3.61 mmol/L at pH 7.6 and 0.45 mmol/L at pH 6.5. Zebrafish pept1a mRNA is highly expressed in the intestine and ovary of the adult fish, while its expression in early development undergoes a complex trend over time, with pept1a mRNA being detected 1 and 2 days post-fertilization (dpf), possibly due to its occurrence in the RNA maternal pool, decreasing at 3 dpf (~ 0.5-fold) and increasing above the 1–2 dpf levels at 4 to 7 dpf, with a peak (~ 7-fold) at 6 dpf. Conclusions: We show that the zebrafish PepT1a-type transporter is functional and co-expressed with pept1b (slc15a1b) in the adult fish intestine. Its expression is also confirmed during the early phases of development when the yolk syncytial layer is present and yolk protein resorption processes are active. While completing the missing information on PepT1-type transporters function in the zebrafish, these results open to future investigations on the similar/differential role(s) of PepT1a/PepT1b in zebrafish and teleost fish physiology.publishedVersio

Proline Dehydrogenase (PRODH) Is Expressed in Lung Adenocarcinoma and Modulates Cell Survival and 3D Growth by Inducing Cellular Senescence

The identification of markers for early diagnosis, prognosis, and improvement of therapeutic options represents an unmet clinical need to increase survival in Non-Small Cell Lung Cancer (NSCLC), a neoplasm still characterized by very high incidence and mortality. Here, we investigated whether proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, played a role in NSCLC tumorigenesis. PRODH expression was investigated by immunohistochemistry; digital PCR, quantitative PCR, immunoblotting, measurement of reactive oxygen species (ROS), and functional cellular assays were carried out. PRODH expression was found in the majority of lung adenocarcinomas (ADCs). Patients with PRODH-positive tumors had better cancer-free specific and overall survival compared to those with negative tumors. Ectopic modulation of PRODH expression in NCI-H1299 and the other tested lung ADC cell lines decreased cell survival. Moreover, cell proliferation curves showed delayed growth in NCI-H1299, Calu-6 and A549 cell lines when PRODH-expressing clones were compared to control clones. The 3D growth in soft agar was also impaired in the presence of PRODH. PRODH increased reactive oxygen species production and induced cellular senescence in the NCI-H1299 cell line. This study supports a role of PRODH in decreasing survival and growth of lung ADC cells by inducing cellular senescence

The “www” of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research

After 50 years, the heterologous expression of proteins in Xenopus laevis oocytes is still essential in many research fields. New approaches and revised protocols, but also classical methods, such as the two-electrode voltage clamp, are applied in studying membrane transporters. New and old methods for investigating the activity and the expression of Solute Carriers (SLC) are reviewed, and the kinds of experiment that are still useful to perform with this kind of cell are reported. Xenopus laevis oocytes at the full-grown stage have a highly efficient biosynthetic apparatus that correctly targets functional proteins at the defined compartment. This small protein factory can produce, fold, and localize almost any kind of wild-type or recombinant protein; some tricks are required to obtain high expression and to verify the functionality. The methodologies examined here are mainly related to research in the field of membrane transporters. This work is certainly not exhaustive; it has been carried out to be helpful to researchers who want to quickly find suggestions and detailed indications when investigating the functionality and expression of the different members of the solute carrier families

The D-amino acid transport by the invertebrate SLC6 transporters KAAT1 and CAATCH1 from Manduca sexta

The ability of the SLC6 family members, the insect neutral amino acid cotransporter KAAT1(K(+)-coupled amino acid transporter 1) and its homologous CAATCH1(cation anion activated amino acid transporter/channel), to transport D-amino acids has been investigated through heterologous expression in Xenopus laevis oocytes and electrophysiological techniques. In the presence of D-isomers of leucine, serine, and proline, the msKAAT1 generates inward, transport-associated, currents with variable relative potencies, depending on the driving ion Na(+) or K(+). Higher concentrations of D-leucine ( 651\ua0mmol/L) give rise to an anomalous response that suggests the existence of a second binding site with inhibitory action on the transport process. msCAATCH1 is also able to transport the D-amino acids tested, including D-leucine, whereas L-leucine acts as a blocker. A similar behavior is exhibited by the KAAT1 mutant S308T, confirming the relevance of the residue in this position in L-leucine binding and the different interaction of D-leucine with residues involved in transport mechanism. D-leucine and D-serine on various vertebrate orthologs B(0)AT1 (SLC6A19) elicited only a very small current and singular behavior was not observed, indicating that it is specific of the insect neutral amino acid transporters. These findings highlight the relevance of D-amino acid absorption in the insect nutrition and metabolism and may provide new evidences in the molecular transport mechanism of SLC6 family

Intracellular ANKRD1 protein levels are regulated by 26S proteasome-mediated degradation

The ANKRD1/CARP gene encodes a muscle-specific protein which has been implicated in transcriptional regulation and myofibrillar assembly. Several features at both the mRNA and protein levels define ANKRD1 as a gene whose expression is tightly regulated, and deregulated expression of this protein has been recently associated to human congenital heart disease. It is therefore crucial to define the intracellular pathways that regulate the ANKRD1 protein's steady-state levels. Here, we show that ANKRD1 is a short-lived protein whose levels are tightly regulated by the 26S proteasome. In addition, a critical role for a putative PEST motif was established, although other degrons within the ANKRD1 protein are likely implicated in the control of its intracellular level

Hyperpolarization-activated cyclic nucleotide-gated channels in peripheral diaphragmatic lymphatics

Diaphragmatic lymphatic function is mainly sustained by pressure changes in the tissue and serosal cavities during cardiorespiratory cycles. The most peripheral diaphragmatic lymphatics are equipped with muscle cells (LMCs) which exhibit spontaneous contraction, whose molecular machinery is still undetermined. Hypothesizing that spontaneous contraction might involve hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in lymphatic LMCs, diaphragmatic specimens including spontaneously contracting lymphatics were excised from 33 anesthetized rats, moved to a perfusion chamber containing HEPES-Tyrode's solution and treated with HCN channels inhibitors Cesium Cloride, ivabradine and ZD-7288. Compared to control, exposure to 10 mM CsCl reduced (- 65%, n=13, p<0.01) the contraction frequency (FL) and increased end-diastolic diameter (DL-d, + 7.3 ± 0.7%, p<0.01) without changes in end-systolic (DL-s) diameter. 300 µM ivabradine abolished contraction and increased DL-d (≈ 14%, n=10, p<0.01) or caused an incomplete inhibition of FL (n=3, p<0.01), leaving DL-d and DL-s unaltered. 200 µM ZD-7288, completely (n=12, p<0.01) abolished FL, while DL-d decreased to 90.9 ± 2.7% of control. HCN gene expression and immunostaining confirmed the presence of HCN1-4 channels isoforms, likely arranged in different configurations, in LMCs. Hence, all together data suggest that HCN channels might play an important role in affecting contraction frequency of LMCs