Biology subjects

Schwartz, T. W.

Publications and source records attributed to Schwartz, T. W..

4 recordsLinked to original sources

bioRxiv2025

Constitutive activation and allosteric mechanisms underlying Gi/Gq signaling bias in SUCNR1 revealed by AlphaFold-based modeling and enhanced sampling simulations

Succinate Receptor 1 (SUCNR1) is a key metabolite sensing GPCR activated by high local succinate concentrations through Gi and Gq signaling pathways. By combining AlphaFold-based modeling, Gaussian accelerated molecular dynamics simulations (72s in total), receptor mutagenesis, and signaling assays, we here investigate the spontaneous and G-protein binding-associated activation mechanism of SUCNR1. Our molecular simulations predict that, in the absence of G protein or ligand, human SUCNR1 (hSUCNR1), in contrast to murine SUCNR1 (mSUCNR1), adopts canonical active- like conformations in its intracellular domains and undergoes critical conformational changes in its extracellular domain. This finding is consistent with our signaling-assay results showing that hSUCNR1, unlike mSUCNR1, displays a high degree of constitutive signaling. When complexed with either Gi or Gq, both h- and mSUCNR1 adopt highly energetically favorable conformations in the extracellular domain, characterized by the unlocking of extracellular loop 2b (ECL2b) and an expanded ligand entry pathway. Interestingly, the simulations reveal that helix 5 of Gq binds less firmly to the TM3/6 cleft of mSUCNR1, resulting in an unstable extracellular domain of the Gq- mSUCNR1 complex, which however can be stabilized by agonist binding. This result is supported by our BRET assays, which show that, in contrast to hSUCNR1, mSUCNR1 fails to recruit mini-Gq to the cell surface in the absence of an agonist. Furthermore, the signaling effects of key receptor residues at G-protein binding site and within ECL2b, as predicted by our simulations, were confirmed by mutagenesis assays. Overall, our integrative approach demonstrates that hSUCNR1 can spontaneously adopt all key conformations associated with receptor activation, and that G protein binding further primes the extracellular receptor domain for agonist binding, which in turn stabilizes the active complex.

biophysics↗

bioRxiv2023

A new paradigm of intracrine free fatty acid receptor 4 signaling at lipid droplets

G protein-coupled receptors (GPCRs), once thought to be active exclusively at the plasma membrane, have been shown to signal from multiple intracellular membrane compartments, including endosomes and the Golgi. However, the potential occurrence and functional relevance of intracellular signaling for the emerging family of metabolite-sensing GPCRs is largely unknown. Here, we used live-cell imaging, bioluminescence resonance energy transfer (BRET) measurements, and functional readouts to investigate signal compartmentalization of the free fatty acid receptor 4 (FFA4), a prototypical metabolite-sensing GPCR that is activated by medium- and long-chain free fatty acids (FFAs). Unexpectedly, we show that FFA4 largely resides on intracellular membranes that are intimately associated with lipid droplets in adipocytes. Upon lipolysis induction, the released FFAs rapidly bind to and activate this intracellular pool of FFA4, leading to local Gi/o coupling and inhibition of cAMP production in the vicinity of lipid droplets. This provides a spatiotemporally confined negative feedback mechanism allowing individual lipid droplets to rapidly adjust their lipolysis rate. Our results reveal a novel intracrine signaling modality by a prototypical metabolite-sensing GPCR and identify a new lipid-droplet-associated signaling hub implicated in the rapid regulation of lipid metabolism, with important implications for adipocyte physiology and pharmacology.

pharmacology and toxicology↗

bioRxiv2022

Systems analysis of metabolic responses to a mixed meal test in an obese cohort reveals links between tissue metabolism and the gut microbiota

Individuals with prediabetes and type 2 diabetes mellitus (T2DM) have poor ability to adapt to diet-triggered perturbations. We investigated global metabolic responses to a mixed meal test (MMT) in morbidly obese individuals with different diabetic status by performing plasma metabolomic profiling. Abnormal metabolism of carbohydrates, (branched-chain) amino acids, fatty acids and acylcholines in individuals with (pre)diabetes was observed. Moreover, differences in metabolic responses were associated with altered fecal metagenomics and transcriptomes of liver, jejunum and adipose tissues, which revealed a modified gut microbiome and multi-tissue metabolism in individuals having insulin resistance. Finally, using integrative machine learning models, we built a predictive model based on metabolomics data after 2h MMT, and identified possible new biomarkers for glycemic control including N-acetylaspartate and phenylalanine-derived metabolites that may be useful for diagnosis, intervention and prevention of T2DM.

systems biology↗

bioRxiv2019

Rfx6 promotes the differentiation of peptide-secreting enteroendocrine cells while repressing genetic programs controlling serotonin production

ObjectiveEnteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell-Riley syndrome characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function, however, the role of Rfx6 in EECs differentiation remained to be elucidated.\n\nMethodsWe examined the molecular, cellular and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes.\n\nResultsRfx6 is expressed in the gut endoderm; later it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, whose differentiation programs are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1- or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine.\n\nConclusionThese studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell-Riley syndrome.

developmental biology↗