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Braun D.,Charite - Medical University of Berlin | Lelios I.,Charite - Medical University of Berlin | Krause G.,Forschungsinstitut For Molekulare Pharmakologie | Schweizer U.,Charite - Medical University of Berlin | Schweizer U.,University of Bonn
Endocrinology | Year: 2013

Mutations in monocarboxylate transporter 8 (MCT8; SLC16A2) cause the Allan-Herndon-Dudley syndrome, a severe X-linked psychomotor retardation syndrome. MCT8 belongs to the major facilitator superfamily of 12 transmembrane-spanning proteins and transports thyroid hormones across the blood-brain barrier and into neurons. How MCT8 distinguishes thyroid hormone substrates from structurally closely related compounds is not known. The goal of this study was to identify critical amino acids along the transport channel cavity, which participate in thyroid hormone recognition. The fact that T 3 is bound between a His-Arg clamp in the crystal structure of the T3 receptor/T3 complex prompted us to investigate whether such a motif might potentially be relevant for T3 recognition in MCT8. We therefore replaced candidate histidines and arginines by site-directed mutagenesis and performed activity assays in MDCK-1 cells and Xenopus oocytes. Histidines were replaced by alanine, phenylalanine, and glutamine to probe for molecular properties like aromatic ring structure and H-bonding properties. It was found that some mutations in His192 and His415 significantly changed substrate transport kinetics. Arg301 at the intracellular end of the substrate channel is at an ideal distance to His415 to participate in a His-Arg clamp and mutation to alanine-abrogated hormone transport. Molecular modeling demonstrates a perfect fit of T3 poised into the substrate channel between His415 and Arg301 and observing the same geometry as in the T3 receptor. Copyright © 2013 by The Endocrine Society.

Khatri Y.,Saarland University | Ringle. M.,Saarland University | Lisurek M.,Forschungsinstitut For Molekulare Pharmakologie | VonKries J.P.,Forschungsinstitut For Molekulare Pharmakologie | And 2 more authors.
ChemBioChem | Year: 2016

Cytochromes P450 catalyze a variety of synthetically useful reactions. However, it is difficult to determine their physiological or artificial functions when a plethora of orphan P450 systems are present in a genome. CYP260A1 from Sorangium cellulosum So ce56 is a new member among the 21 available P450s in the strain. To identify putative substrates for CYP260A1 we used high-throughput screening of a compound library (ca. 17 000 ligands). Structural analogues of the typeI hits were searched for biotechnologically relevant compounds, and this led us to select C-19 steroids as potential substrates. We identified efficient surrogate redox partners for CYP260A1, and an Escherichia coli-based whole-cell biocatalyst system was developed to convert testosterone, androstenedione, and their derivatives methyltestosterone and 11-oxoandrostenedione. A detailed 1H and 13CNMR characterization of the product(s) from C-19 steroids revealed that CYP260A1 is the very first 1α-steroid hydroxylase. The first 1α: CYP260A1 performed predominant 1α-hydroxylation of testosterone, androstenedione, and 11-oxoandrostenedione; however, testosterone acetate was hydroxylated at both the 1α- and the 9α-positions. This hydroxylation offers scope for further chemical modification at the steroidal C-1 position, which is of significant pharmaceutical interest. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Jankowski V.,Charite - Medical University of Berlin | Tolle M.,Charite - Medical University of Berlin | Santos R.A.S.,Federal University of Minas Gerais | Gunthner T.,Charite - Medical University of Berlin | And 11 more authors.
FASEB Journal | Year: 2011

The family of angiotensin peptides has been steadily growing in recent years. Most are fragments of angiotensin II (Ang II) with different affinities to the known angiotensin receptors. Here, we describe a novel endogenous Ang II-like octapeptide in plasma from healthy humans and patients with end-stage renal failure, which acts as a stronger agonist at Mas receptors than Ang 1-7. Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an Ang II-like octapeptide, angioprotectin, with the sequence Pro-Glu-Val-Tyr-Ile- His-Pro-Phe, which differs from Ang II in Pro 1 and Glu 2 instead of Asp 1 and Arg 2. Pro-Glu-Val-Tyr-Ile-His-Pro-Phe in angioprotectin is most likely generated enzymatically from Ang II. Angioprotectin antagonized the contractile actions of Ang II on rat aortic rings. The physiological antagonism of vasoconstrictor actions of Ang II by angioprotectin is mediated by the Mas receptor. Angioprotectin has a stronger affinity to the Mas receptor than Ang-1-7. Plasma concentrations were ∼15% of plasma Ang II concentrations in healthy volunteers and up to 50% in patients with renal failure. A commercially available Ang II antibody did not discriminate between angioprotectin and Ang II; thus, angioprotectin can contribute to Ang II concentrations measured by antibody- based assays. This novel peptide is likely to be a relevant component of the human renin-angiotensin-system. © FASEB.

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