Der Protonentransport über den humanen Monocarboxylattransporter 1 wird durch das anionische Substrat vermittelt

Es ist bekannt, dass Laktat einen universellen Nährstoff für Zellen darstellt. Der Austausch des Anions über Zellmembranen hinweg wird im Menschen zu einem großen Anteil von Vertretern der Monocarboxylattransporter- Familie (MCT-Familie) vermittelt. Daher gelten MCT als Arzneistoff-Target. Ein umfassendes Verständnis der mechanistischen Zusammenhänge innerhalb des Transporters könnte die Entwicklung hoch wirksamer Inhibitoren erleichtern und so den Fortschritt in der Behandlung von Krebserkrankungen beschleunigen. In dieser Arbeit wurden MCT1 und verschiedene Punktmutanten des Transporters heterolog in Saccharomyces cerevisiae exprimiert. In verschiedenen Funktionsassays wurden zum einen Substrate und Nicht-Substrate des Transporters identifiziert, zum anderen wurden Substrataffinität, die pH-Abhängigkeit des Transportes und dessen kinetische Parameter bestimmt. Das gewählte Expressionssystem zeichnet sich durch eine hohe Stabilität gegenüber extrazellulären pH-Werten aus und lässt bekanntermaßen die Expression von MCT1 auch ohne das Begleitprotein Basigin zu. Dies erlaubte in der vorliegenden Arbeit die Charakterisierung des Einflusses von Basigin auf den Transport und die funktionelle Charakterisierung des Transporters ohne diese Interferenzen. Die in dieser Arbeit präsentierten Messwerte resultieren in einem postulierten Transportmechanismus. Dabei wird das Proton vom Lysin 38 über das gebundene Substrat hinweg bis zum Aspartat 309 transportiert. Der präsentierte Mechanismus ist mit dem aktuellen Wissensstand zu MCT1 vereinbar und könnte auch für die Zwitterionen-Transporter der Familie gelten. Damit legt diese Arbeit die Grundlage, um die Familie der MCT vollständig zu charakterisieren und zu verstehen

Genome-Wide Association Study Identifies Two Novel Regions at 11p15.5-p13 and 1p31 with Major Impact on Acute-Phase Serum Amyloid A

Elevated levels of acute-phase serum amyloid A (A-SAA) cause amyloidosis and are a risk factor for atherosclerosis and its clinical complications, type 2 diabetes, as well as various malignancies. To investigate the genetic basis of A-SAA levels, we conducted the first genome-wide association study on baseline A-SAA concentrations in three population-based studies (KORA, TwinsUK, Sorbs) and one prospective case cohort study (LURIC), including a total of 4,212 participants of European descent, and identified two novel genetic susceptibility regions at 11p15.5-p13 and 1p31. The region at 11p15.5-p13 (rs4150642; p = 3.20×10−111) contains serum amyloid A1 (SAA1) and the adjacent general transcription factor 2 H1 (GTF2H1), Hermansky-Pudlak Syndrome 5 (HPS5), lactate dehydrogenase A (LDHA), and lactate dehydrogenase C (LDHC). This region explains 10.84% of the total variation of A-SAA levels in our data, which makes up 18.37% of the total estimated heritability. The second region encloses the leptin receptor (LEPR) gene at 1p31 (rs12753193; p = 1.22×10−11) and has been found to be associated with CRP and fibrinogen in previous studies. Our findings demonstrate a key role of the 11p15.5-p13 region in the regulation of baseline A-SAA levels and provide confirmative evidence of the importance of the 1p31 region for inflammatory processes and the close interplay between A-SAA, leptin, and other acute-phase proteins

Lactic Acid Permeability of Aquaporin-9 Enables Cytoplasmic Lactate Accumulation via an Ion Trap

(1) Background: Human aquaporin-9 (AQP9) conducts several small uncharged metabolites, such as glycerol, urea, and lactic acid. Certain brain tumors were shown to upregulate AQP9 expression, and the putative increase in lactic acid permeability was assigned to severity. (2) Methods: We expressed AQP9 and human monocarboxylate transporter 1 (MCT1) in yeast to determine the uptake rates and accumulation of radiolabeled l-lactate/l-lactic acid in different external pH conditions. (3) Results: The AQP9-mediated uptake of l-lactic acid was slow compared to MCT1 at neutral and slightly acidic pH, due to low concentrations of the neutral substrate species. At a pH corresponding to the pKa of l-lactic acid, uptake via AQP9 was faster than via MCT1. Substrate accumulation was fundamentally different between AQP9 and MCT1. With MCT1, an equilibrium was reached, at which the intracellular and extracellular l-lactate/H+ concentrations were balanced. Uptake via AQP9 was linear, theoretically yielding orders of magnitude of higher substrate accumulation than MCT1. (4) Conclusions: The selectivity of AQP9 for neutral l-lactic acid establishes an ion trap for l-lactate after dissociation. This may be physiologically relevant if the transmembrane proton gradient is steep, and AQP9 acts as the sole uptake path on at least one side of a polarized cell

Basigin drives intracellular accumulation of l-lactate by harvesting protons and substrate anions.

Transmembrane transport of l-lactate by members of the monocarboxylate transporter family, MCT, is vital in human physiology and a malignancy factor in cancer. Interaction with an accessory protein, typically basigin, is required to deliver the MCT to the plasma membrane. It is unknown whether basigin additionally exerts direct effects on the transmembrane l-lactate transport of MCT1. Here, we show that the presence of basigin leads to an intracellular accumulation of l-lactate 4.5-fold above the substrate/proton concentrations provided by the external buffer. Using basigin truncations we localized the effect to arise from the extracellular Ig-I domain. Identification of surface patches of condensed opposite electrostatic potential, and experimental analysis of charge-affecting Ig-I mutants indicated a bivalent harvesting antenna functionality for both, protons and substrate anions. From these data, and determinations of the cytosolic pH with a fluorescent probe, we conclude that the basigin Ig-I domain drives lactate uptake by locally increasing the proton and substrate concentration at the extracellular MCT entry site. The biophysical properties are physiologically relevant as cell growth on lactate media was strongly promoted in the presence of the Ig-I domain. Lack of the domain due to shedding, or misfolding due to breakage of a stabilizing disulfide bridge reversed the effect. Tumor progression according to classical or reverse Warburg effects depends on the transmembrane l-lactate distribution, and this study shows that the basigin Ig-I domain is a pivotal determinant

Predissociation spectroscopy of cold CN − H 2 and CN − D 2

International audienc