Biology subjects

Kleinau, G.

Publications and source records attributed to Kleinau, G..

6 recordsLinked to original sources

bioRxiv2025

Murine Toll-like receptor 8 is a nucleic acid multi-sensor detecting 2',3'-cyclic monophosphate guanosine as well as combinations of ribo-, deoxy-, cyclic nucleotides, and nucleosides

Toll-like receptor 8 (TLR8) in humans senses RNA degradation products and elicits an inflammatory immune response. In contrast, the ligand specificity and function of its murine counterpart mTLR8, long considered non-functional, remain poorly defined. Here, we established an agonist combination model of poly-deoxythymidine (poly-dT) DNA and TLR7/8 binding site 1 agonists such as uridine or the benzazepine compound TL8-506, which activates mTLR8, while suppressing mTLR7 signaling. Extensive agonist analysis based on this model revealed that 2,3-cyclic guanosine monophosphate (2,3-cGMP) serves as a natural ligand for mTLR8, suggesting functionality of its binding site 1 without engagement of site 2. In addition, 2,3-cyclic uridine monophosphate, bacterial single-stranded (ss) DNA, double-stranded (ds) DNA fragments, microRNAs, ssRNA derived from HIV1, SARS-CoV-2, or bacterial sources all potentiate mTLR8 sensing of site 1 agonists. All these stimuli induce distinct inflammatory responses from murine macrophages and microglia via TLR8. In vivo, intrathecal administration of TL8-506 and poly-dT led to microglial accumulation and neuronal injury in the murine cerebral cortex through TLR8, highlighting the potential neuropathological consequences of mTLR8 activation. Taken together, our study defines mTLR8 as a nucleic acid sensor detecting 2,3-cGMP as well as combinations of ssDNA, dsDNA, ssRNA fragments, 2,3-cyclic nucleotide monophosphates, and nucleosides, with implications for host defense and neuroinflammation.

immunology↗

bioRxiv2024

MRAP2 modifies signaling and oligomerization state of the melanocortin-4 receptor

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) and a key regulator of appetite and metabolism. It can interact with the melanocortin-receptor accessory protein 2 (MRAP2), a single transmembrane helix protein known to interact with several different GPCRs. However, the consequences of this interaction are not completely understood. Here we report that co-expression of MRAP2 has multiple effects on the MC4R: it enhances G protein-mediated signaling and simultaneously impairs {beta}-arrestin2 recruitment and, consequently, internalization. In addition, co-expression of MRAP2 leads to an increased number of monomers of MC4R by disrupting receptor oligomers. A structural homology model of the active state MC4R-MRAP2-Gs complex suggests interaction sites between MRAP2 and MC4R that are relevant for receptor activation. Taken together, our data indicate that MRAP2 is an accessory protein that interacts with and influences MC4R structure, biasing its signaling towards G protein-mediated effects.

pharmacology and toxicology↗

bioRxiv2023

Is the neuropeptide PEN a ligand of GPR83?

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second-messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.

pharmacology and toxicology↗

bioRxiv2021

Functional modulation of PTH1R activation and signalling by RAMP2

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs) including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signalling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signalling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signalling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered {beta}-arrestin2 recruitment to PTH1R. Employing homology modelling we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signalling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

pharmacology and toxicology↗

bioRxiv2021

Structures of active melanocortin-4 receptor--Gs-protein complexes with NDP-α-MSH and setmelanotide

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a G-protein coupled receptor and a prime target for the treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R- Gs-protein complexes with two recently FDA-approved drugs, the peptide agonists NDP--MSH and setmelanotide, with 2.9 [A] and 2.6 [A] resolution. Together with signaling data, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6 regulated receptor activation. In both structures, different ligand binding modes of NDP--MSH, a high-affinity variant of the endogenous agonist, and setmelanotide, an anti-obesity drug with biased signaling, underline the key role of TM3 for ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-TM3 interplay subsequently impacts the receptor- Gs-protein interfaces, mainly at intracellular loop 2. These structures reveal mechanistic details of MC4R activation or inhibition and provide important insights into receptor selectivity that will facilitate the development of tailored anti-obesity drugs.

biochemistry↗

bioRxiv2019

Human endogenous retrovirus HERV-K(HML-2) RNA causes neurodegeneration through Toll-like receptors

Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs have been suggested to be involved in neurological disorders, little is known about their biological function and pathophysiological relevance. HERV-K(HML-2) comprises evolutionarily young proviruses transcribed in the brain. We report that RNA derived from an HERV-K(HML-2) env gene region binds to the human RNA-sensing Toll-like receptor (TLR) 8, activates human TLR8, as well as murine Tlr7, and causes neurodegeneration through TLR8 and Tlr7 in neurons and microglia. HERV-K(HML-2) RNA introduced extracellularly into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimers disease (AD), resulted in neurodegeneration. Tlr7-deficient mice were protected against neurodegenerative effects, but were re-sensitized towards HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Accordingly, transcriptome datasets of human brain samples from AD patients revealed a specific correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from AD individuals compared to controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for human TLR8 and murine Tlr7 and imply a functional contribution of specific human endogenous retroviral transcripts to neurodegenerative processes such as AD.

neuroscience↗