Biology subjects

Klenk, C.

Publications and source records attributed to Klenk, C..

4 recordsLinked to original sources

bioRxiv2026

Integrated Multiomics Links Metabolic and Inflammatory Remodeling to Arterial Stiffness After the 4,486-km Trans Europe Footrace

RationaleRegular aerobic exercise protects against vascular aging and reshapes the circulating molecular milieu, but the relation between vascular function, circulating molecules, and exercise dose at extreme volumes remains poorly defined. The vascular and molecular consequences of chronic, multi-stage ultra-endurance running are particularly unclear. ObjectiveTo define circulating molecular signatures associated with vascular dysfunction following the 64-stage, 4,486-km Trans Europe Foot Race (TEFR). Methods and ResultsIntegrated multiomics analysis (proteomics, lipidomics, metabolomics) of plasma from 27 finishers revealed a coordinated systemic shift driving an oxidative phenotype. Specifically, we identified altered arginine metabolism and a universal upregulation of lipotoxic ceramides consistent with incomplete fatty acid oxidation. In conjunction, we identified upregulation of innate immune system pathways including the acute phase response and the complement system. Central pulse wave velocity (cPWV) increased significantly after the race, consistent with arterial stiffening. To test whether the post-race circulating milieu could directly influence vascular mechanics, naive murine aortic rings were incubated with participant plasma. Post-race plasma acutely increased aortic elastic modulus, and this effect was attenuated by the superoxide dismutase mimetic TEMPOL, supporting a ROS-dependent component. In human aortic endothelial cells (HAECs), post-race plasma increased reactive oxygen species generation without detectable changes in eNOS phosphorylation, total eNOS abundance, or stimulated nitric oxide production. Endothelial ROS responses were associated with components of the terminal complement pathway. ConclusionsExtreme multi-stage ultra-endurance exercise induces a distinct systemic milieu associated with arterial stiffening through ROS-sensitive mechanisms. This response is characterized by remodeling of arginine-related metabolism, ceramide accumulation, innate immune activation, and oxidative stress, without evidence of reduced measured eNOS abundance or stimulated NO production. These findings identify candidate molecular pathways linking prolonged metabolic stress to vascular dysfunction.

physiology↗

bioRxiv2022

A Vaccinia-based system for directed evolution of GPCRs in mammalian cells

Low stability and poor expression levels are inherent in many G protein-coupled receptors, hindering structural and biophysical analyses. Directed evolution in bacterial or yeast display systems has been successfully used to overcome both limitations in some cases. Yet, some receptors cannot be tackled in microbial systems, due to their complex molecular composition or due to unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality of individual mutants, and a rather uniform gene copy number, both prerequisites for strict genotype-phenotype linkage in mammalian cells, we developed a highly efficient viral transduction system based on Vaccinia virus. Using rational design of synthetic DNA libraries, we first evolved neurotensin receptor 1 for high stability and expression, reaching levels similar or higher to what has been achieved in bacterial systems. Second, using parathyroid hormone 1 receptor we demonstrate that also receptors with complex molecular architectures and large ligands can be readily evolved in mammalian cells. Furthermore, in our system, the physiological signaling environment in mammalian cells can be employed to evolve functional receptor properties. We obtained fully functional receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface, resulting in higher signaling efficacy. Thus, our approach provides new means to readily improve the biophysical properties of receptors in a mammalian cellular environment. Moreover, it opens the possibility to modulate receptor signaling and to gain further insights into the intricate molecular interplay required for GPCR activation.

biochemistry↗

bioRxiv2021

Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone receptor 1

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to Gs and increased activation of Gq compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.

pharmacology and toxicology↗

bioRxiv2020

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

G protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals1,2. Although several structures have been solved for GPCR-G protein complexes3-17, structural studies of the complex in a physiological lipid membrane environment are lacking. Additionally, most previous studies required additional antibodies/nanobodies and/or engineered G proteins for complex stabilization. In the absence of a native complex structure, the underlying mechanism of G protein activation leading to GDP/GTP exchange remains unclear. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gi1{beta}1{gamma}1 protein in two conformational states, resolved to 4.1 and 4.2 [A] resolution. The structures were determined without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling under near native conditions.

molecular biology↗