Biology subjects

Hofer, P.

Publications and source records attributed to Hofer, P..

6 recordsLinked to original sources

bioRxiv2025

Live-cell quantitative monitoring reveals distinct, high-affinity Gβγ regulations of GIRK2 and GIRK1/2 channels

Gi/o protein-coupled receptors (GPCRs) inhibit cardiac and neuronal excitability via G protein-activated K+ channels (GIRK), assembled by combinations of GIRK1 - GIRK4 subunits. GIRKs are activated by direct binding of the G{beta}{gamma} dimer of inhibitory Gi/o proteins. However, key aspects of this textbook signaling pathway remain debated. Recent studies suggested no Gi/o-GIRK pre-coupling and low (>250 {micro}M) G{beta}{gamma}-GIRK interaction affinity, contradicting earlier sub-{micro}M estimates and implying low signaling efficiency. We show that G{gamma} prenylation, which mediates G{beta}{gamma} membrane attachment required for GIRK activation, also contributes to the G{beta}{gamma}-GIRK interaction, explaining the poor affinity obtained with non-prenylated G{beta}{gamma}. Using quantitative protein titration and electrophysiology in live Xenopus oocytes, G{beta}{gamma} affinity for homotetrameric GIRK2 ranged from 4-30 {micro}M. Heterotetrameric GIRK1/2 showed a higher G{beta}{gamma} apparent affinity due to unique G{beta}{gamma}-docking site (anchor) in GIRK1, which enriches G{beta}{gamma} at the channel. Biochemical approaches and molecular dynamic simulations revealed that the G{beta}{gamma} anchor is formed by interacting N-terminal and distal C-terminal domains of the GIRK1 subunits, distinct from the G{beta}{gamma}-binding "activation" site(s) underlying channel opening. Thus, the affinity of G{beta}{gamma}-GIRK interaction is within the expected physiological range, while dynamic pre-coupling of G{beta}{gamma} to GIRK1-containing channels through high-affinity interactions further enhances the GPCR-Gi/o-GIRK signaling efficiency.

physiology↗

bioRxiv2025

Time-resolved immune dynamics in rheumatoid arthritis under Methotrexate therapy

Rheumatoid arthritis (RA) is characterized by immune dysregulation, including alterations in peripheral blood mononuclear cell (PBMC) populations and aberrant cytokine signaling. Methotrexate (MTX) is the preferred first-line treatment for RA, yet its precise mechanisms of action remain incompletely understood. This study employed a multi-omics strategy--combining single-cell RNA sequencing (scRNA-seq) and immunophenotyping--to identify key effector peripheral immune cells and their cellular responses in RA patients over 12 weeks of MTX treatment. In our study, MTX was associated with significant immune modulation, including the restoration of naive T and B cells and reductions in T cell memory subsets with these effects detectable as early as three weeks post-treatment. Plasmablast levels also emerged as a potential biomarker for early therapeutic response, reflecting MTXs impact on immune homeostasis. Transcriptional analysis revealed modulation of key pathways, including TNF- signaling, B cell receptor signaling, and T cell receptor-mediated apoptosis. Network analysis identified critical regulatory hubs, such as EGR1, JAK2, and SOCS1, in monocytes and CD4 memory T cells, highlighting these cell types as key mediators of MTXs effects. In conclusion, these findings advance our understanding of MTXs effects on immune cell dynamics at different stages of treatment, showing for the first time the early cellular changes leading to immune modulation in RA. Altogether, our results provide the foundation for further mechanistic investigations into MTX.

genomics↗

bioRxiv2024

Non-shivering thermogenesis is intact upon brown-adipocyte specific loss of ATGL and HSL due to white adipose tissue browning

Intracellular fatty acids (FAs) activate and fuel non-shivering thermogenesis (NST) via uncoupling protein 1 (UCP1). Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) control FA availability. Since mice lacking ATGL in brown adipose tissue (BAT) exhibit intact recruitable adrenergic thermogenesis, we hypothesized that HSL-mediated FA release is sufficient to activate UCP1-dependent NST. We demonstrate that mice with inducible brown adipocyte-specific loss of ATGL and HSL (iBDKO) exhibit normal recruitable adrenergic thermogenesis upon prolonged cold exposure. Mechanistically, we show that BAT thermogenic capacity is impaired in cold-adapted iBDKO mice due to diminished mitochondrial numbers. Increased browning of white adipose tissue (WAT) in iBDKO mice indicates a shift in thermogenesis from BAT to WAT. Consistently, the loss of ATGL and HSL in BAT and WAT disrupts thermogenesis in both depots, resulting in blunted UCP1-dependent NST. Our study highlights the metabolic adaptability of adipose tissue and the critical role of intracellular lipolysis in regulating thermogenesis.

physiology↗

bioRxiv2024

Dietary control of peripheral adipose storage capacity through membrane lipid remodelling

Complex genetic and dietary cues contribute to the development of obesity, but how these are integrated on a molecular level is incompletely understood. Here, we show that PPAR{gamma} supports hypertrophic expansion of adipose tissue via transcriptional control of LPCAT3, a membrane-bound O-acyltransferase that enriches diet-derived omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in the phospholipidome. In high-fat diet-fed mice, lowering membrane n-6 PUFA levels by adipocyte-specific Lpcat3 knockout (Lpcat3AKO) or by dietary lipid manipulation leads to dysfunctional triglyceride (TG) storage, ectopic fat deposition and insulin resistance. Aberrant lipolysis of stored TGs in Lpcat3AKO adipose tissues instigates a non-canonical adaptive response that engages a futile lipid cycle to increase energy expenditure and limit further body weight gain. Mechanistically, we find that adipocyte LPCAT3 activity promotes TG storage by selectively enriching n-6 arachidonoyl-phosphatidylethanolamine at the ER-lipid droplet interface, which in turn favours the budding of large droplets that exhibit greater resistance to ATGL-dependent hydrolysis. Thus, our study highlights the PPAR{gamma}-LPCAT3 pathway as a molecular link between dietary n-6 PUFA intake, adipose expandability and systemic energy balance.

physiology↗

bioRxiv2023

Unmasking Crucial Residues in Adipose Triglyceride Lipase (ATGL) for Co-Activation with Comparative Gene Identification-58 (CGI-58)

Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be co-activated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL stimulation by CGI-58 is incompletely understood. Based on analysis of evolutionary conservation, we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein co-activation on a per-residue level. We identified the region from residues N209-N215 in mouse ATGL as essential for co-activation by mouse CGI-58. ATGL variants with amino-acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 co-activation compared to C-terminally truncated ATGL variants. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the co-activation process. Three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrated that a large surface area is involved in the protein-protein interaction. Mapping important amino acids for co-activation of both proteins, ATGL and CGI-58, onto the 3D model of the complex locates these essential amino acids at the predicted ATGL/CGI-58 interface thus strongly corroborating the significance of these residues in CGI-58 mediated co-activation of ATGL.

biochemistry↗

bioRxiv2022

Adipocyte lipolysis protects the host against Trypanosoma brucei infection

Trypanosoma brucei, an etiological agent of African trypanosomiasis, colonizes the interstitial spaces of the adipose tissue in disproportionately high numbers, inducing a robust local immune response. Loss of body weight, including loss of adipose mass, is a hallmark symptom of African trypanosomiasis. Nevertheless, it is unclear which molecular mechanisms drive this loss of adipose mass and in turn whether it contributes to pathology. Here we show that lipolysis is activated early in infection in adipose tissue of T. brucei-infected mice. This activation is dependent on immune activation, as mice deficient for both B and T lymphocytes failed to upregulated adipocyte lipolysis upon infection and retained higher fat mass. Genetic ablation of the rate limiting adipose triglyceride lipase specifically from adipocytes in mice (AdipoqCre/+-Atglfl/fl) prevented the upregulation of adipocyte lipolysis during infection, leading to reduced loss of fat mass and adipocyte volume. Surprisingly infected AdipoqCre/+-Atglfl/fl mice succumbed earlier to infection and presented a higher parasite burden in the gonadal adipose tissue, indicating that lipolysis limits the growth of the parasite population. Collectively, this work provides molecular mechanistic insight into the loss of fat mass in African trypanosomiasis and identifies adipocyte lipolysis as a host-protective mechanism during a T. brucei infection.

microbiology↗