Die Transporter SLC22A7 (OAT2) und SLC22A13: Substratsuche, Lokalisation, Entdeckung eines Orotsäure-Transporters

Die Solute Carrier SLC22A7 (OAT2, "Organic Anion Transporter 2") und SLC22A13 liegen in mehreren Mammalia-Spezies konserviert vor. OAT2 wird im Mensch primär in der Leber exprimiert, wohingegen SLC22A13 hauptsächlich in den Nieren vorzufinden ist. Ihre physiologische Bedeutung ist jedoch unklar. Da Transporter im Wesentlichen durch ihre Substrate definiert werden, wurden Substrate für diese beiden verwandten Proteine, mittels LC-MS- (Liquid Chromatography - Mass Spectrometry) Differenz Abtönung, aufgespürt. Über diese Methode konnten Guanidiniumsuccinat (GSA) für SLC22A13h (human) und Trigonellin (Trig) für OAT2h als Substrate identifiziert werden. Darüber hinaus konnten die organischen Anionen a-Ketoglutarat, Glutarat, Glycocholat, Guanidiniumglutarat (GGA), Nikotinat, para-Aminohippurat, Pantothenat (PA), Orotat und Urat sowie die Zwitterionen Glycylprolin (GP), Prolylglycin (PG) und Kreatin als Substrate für SLC22A13h gefunden werden. Die höchste Transporteffizienz (TE) von 15 ul min-1 mg Protein-1 wurde hierbei mit dem Nikotinat erzielt. Auf Grund eines verminderten intrazellulären Gehalts von GSA, GGA, GP, PG und PA bei Expression von SLC22A13h in HEK293-Zellen sowie trans-Stimulations Experimente, ließ die Schlussfolgerung zu, dass SLC22A13h als Austaucher operiert. Neben dem Aufspüren von Substraten wurde die Lokalisation von SLC22A13 durch eine Antikörperfärbung untersucht. So konnte erstmalig gezeigt werden, dass der Transporter in der basolateralen Membran von Schaltzellen im Sammelrohr der Rattenniere lokalisiert ist. Aufgrund dieser Tatsache, wurde die Hypothese aufgestellt, dass SLC22A13 für die Exkretion und/oder Resorption von organischen Anionen, im Zusammenspiel mit dem in Schaltzellen apikal lokalisierten SLC22A9 eine Rolle spielt. Für OAT2 konnte festgestellt werden, dass Trig ein spezifisches Substrat für den humanen und den orthologen Rattentransporter (OAT2r) (TE = 4 ul min-1 mg Protein-1, Km = 409 uM) ist. Die Glutaminsäure an Position 441 im humanen Protein ist am Trig-Transport involviert. Mit Trig als Leitstruktur wurden strukturähnliche Verbindungen auf einen Transport untersucht. Nikotinat-Ribosid wurde hierbei als ein weiteres Substrat nachgewiesen (TE für OAT2r 7 ul min-1 mg Protein1). Des Weiteren wurde erstmalig aufgedeckt, dass OAT2r das Pyrimidin-Derivat Orotsäure bzw. das Salz Orotat effizient transportiert (TE = 74-99 ul min-1 mg Protein-1, Km = 234 uM), wohingegen OAT2h Orotat nicht als Substrat akzeptierte. Aufgrund der hier gewonnenen Erkenntnisse, kann die anatomische und physiologische Veränderung der Rattenleber (Steatosis hepatis (Fettleber)) durch vermehrte Orotsäure-Aufnahme mit der Nahrung auf OAT2r zurückgeführt werden. Mit OAT2r wurde zum ersten mal ein Orotsäure-Transporter in Eukaryonten identifiziert

Substrate discrimination by ergothioneine transporter SLC22A4 and carnitine transporter SLC22A5: Gain-of-function by interchange of selected amino acids

AbstractETT (originally designated as OCTN1; human gene symbol SLC22A4) and CTT (OCTN2; SLC22A5) are highly specific transporters of ergothioneine and carnitine, respectively. Despite a high degree of sequence homology, both carriers discriminate precisely between substrates: ETT does not transport carnitine, and CTT does not transport ergothioneine. Our aim was to turn ETT into a transporter for carnitine and CTT into a transporter for ergothioneine by a limited number of point mutations. From a multiple alignment of several mammalian amino acid sequences, those positions were selected for conversion that were momentously different between ETT and CTT from human but conserved among all orthologues. Mutants were expressed in 293 cells and assayed for transport of ergothioneine and carnitine. Several ETT mutants clearly catalyzed transport of carnitine, up to 35% relative to wild-type CTT. Amazingly, complementary substitutions in CTT did not provoke transport activity for ergothioneine. In similar contrast, carnitine transport by CTT mutants was abolished by very few substitutions, whereas ergothioneine transport by ETT mutants was maintained even with the construct most active in carnitine transport. To explain these results, we propose that ETT and CTT use dissimilar pathways for conformational change, in addition to incongruent substrate binding sites. In other words, carnitine is excluded from ETT by binding, and ergothioneine is excluded from CTT by turnover movement. Our data indicate amino acids critical for substrate discrimination not only in transmembrane segments 5, 7, 8, and 10, but also in segments 9 and 12 which were hitherto considered as unimportant

Activation of Rac-1 and RhoA contributes to podocyte injury in chronic kidney disease

Rho-family GTPases like RhoA and Rac-1 are potent regulators of cellular signaling that control gene expression, migration and inflammation. Activation of Rho-GTPases has been linked to podocyte dysfunction, a feature of chronic kidney diseases (CKD). We investigated the effect of Rac-1 and Rho kinase (ROCK) inhibition on progressive renal failure in mice and studied the underlying mechanisms in podocytes. SV129 mice were subjected to 5/6-nephrectomy which resulted in arterial hypertension and albuminuria. Subgroups of animals were treated with the Rac-1 inhibitor EHT1846, the ROCK inhibitor SAR407899 and the ACE inhibitor Ramipril. Only Ramipril reduced hypertension. In contrast, all inhibitors markedly attenuated albumin excretion as well as glomerular and tubulo-interstitial damage. The combination of SAR407899 and Ramipril was more effective in preventing albuminuria than Ramipril alone. To study the involved mechanisms, podocytes were cultured from SV129 mice and exposed to static stretch in the Flexcell device. This activated RhoA and Rac-1 and led via TGFβ to apoptosis and a switch of the cells into a more mesenchymal phenotype, as evident from loss of WT-1 and nephrin and induction of α-SMA and fibronectin expression. Rac-1 and ROCK inhibition as well as blockade of TGFβ dramatically attenuated all these responses. This suggests that Rac-1 and RhoA are mediators of podocyte dysfunction in CKD. Inhibition of Rho-GTPases may be a novel approach for the treatment of CKD

Simultaneous fitting of real-time PCR data with efficiency of amplification modeled as Gaussian function of target fluorescence

<p>Abstract</p> <p>Background</p> <p>In real-time PCR, it is necessary to consider the efficiency of amplification (EA) of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough.</p> <p>Results</p> <p>Here we introduce simultaneous non-linear fitting to determine – without standard curves – an optimal common EA for all samples of a group. In order to adjust EA as a function of target fluorescence, and still to describe fluorescence as a function of cycle number, we use an iterative algorithm that increases fluorescence cycle by cycle and thus simulates the PCR process. A Gauss peak function is used to model the decrease of EA with increasing amplicon accumulation. Our approach was validated experimentally with hydrolysis probe or SYBR green detection with dilution series of 5 different targets. It performed distinctly better in terms of accuracy than standard curve, DART-PCR, and LinRegPCR approaches. Based on reliable EAs, it was possible to detect that for some amplicons, extraordinary fluorescence (EA > 2.00) was generated with locked nucleic acid hydrolysis probes, but not with SYBR green.</p> <p>Conclusion</p> <p>In comparison to previously reported approaches that are based on the separate analysis of each curve and on modelling EA as a function of cycle number, our approach yields more accurate and precise estimates of relative initial target levels.</p

Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants.

In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-β (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions

SLC22A13 catalyses unidirectional efflux of aspartate and glutamate at the basolateral membrane of type A intercalated cells in the renal collecting duct

In vertebrates, SLC22A13 is an evolutionarily conserved transport protein of the plasma membrane. In humans and rat, it is principally expressed in the kidney. The precise localization and physiological function are unknown. In the present study, immunohistochemistry revealed that expression of SLC22A13 is confined to the basolateral membrane of type A intercalated cells in rat kidney. Double-staining confirmed that SLC22A13 co-localizes with anion exchanger 1. LC-MS difference shading showed that heterologous expression of human and rat SLC22A13 in HEK (human embryonic kidney)-293 cells stimulates efflux of guanidinosuccinate, aspartate, glutamate and taurine. Time courses of uptake of [H-3]aspartate and [H-3]glutamate revealed that SLC22A13 counteracted endogenous uptake. By contrast, OAT2 (organic anion transporter 2), a bidirectional glutamate transporter, increased accumulation of [H-3]glutamate. Thus SLC22A13 catalyses unidirectional efflux. Velocity of efflux of standard amino acids was measured by LC-MS/MS. Expression of SLC22A13 strongly stimulated efflux of aspartate, taurine and glutamate. When the intracellular concentrations of aspartate and taurine were increased by pre-incubation, velocities of efflux increased linearly. We propose that in type A intercalated cells, SLC22A13 compensates luminal exit of protons by mediating the basolateral expulsion of the anions aspartate and glutamate. In this context, unidirectional efflux is essential to avoid anion reentering. Loss of SLC22A13 function could cause distal tubular acidosis

Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300

Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene