Mapping Interactions of Microbial Metabolites with Human G-Protein-Coupled Receptors.

Despite evidence linking the human microbiome to health and disease, how the microbiota affects human physiology remains largely unknown. Microbiota-encoded metabolites are expected to play an integral role in human health. Therefore, assigning function to these metabolites is critical to understanding these complex interactions and developing microbiota-inspired therapies. Here, we use large-scale functional screening of molecules produced by individual members of a simplified human microbiota to identify bacterial metabolites that agonize G-protein-coupled receptors (GPCRs). Multiple metabolites, including phenylpropanoic acid, cadaverine, 9-10-methylenehexadecanoic acid, and 12-methyltetradecanoic acid, were found to interact with GPCRs associated with diverse functions within the nervous and immune systems, among others. Collectively, these metabolite-receptor pairs indicate that diverse aspects of human health are potentially modulated by structurally simple metabolites arising from primary bacterial metabolism.

Transient Ischemic Dilatation during Stress Echocardiography: An Additional Marker of Significant Myocardial Ischemia.

AIM: Left ventricular (LV) transient ischemic dilatation (TID) is not clear how it relates to inducible myocardial ischemia during stress echocardiography (SE). METHODS AND RESULTS: Eighty-eight SEs were examined from the site certification phase of the ISCHEMIA Trial. LV end-diastolic volume (EDV) and end-systolic volume (ESV) were measured at rest and peak stages and the percent change calculated. Moderate or greater ischemia was defined as ≥3 segments with stress-induced severe hypokinesis or akinesis. Optimum cut points in stress-induced percent EDV and ESV change that identified moderate or greater myocardial ischemia were analyzed. Analysis from percentage distribution identified a > 13% LV volume increase in EDV or a > 9% LV volume increase in ESV as the optimum cutoff points for moderate or greater ischemia. Using these definitions for TID, there were 27 (31%) with TIDESV and 12 (14%) with TIDEDV . By logistic regression analysis and receiver operating characteristic curves, the percent change in ESV had a stronger association with moderate or greater myocardial ischemia than that of EDV change. Compared to those without TIDESV , cases with TIDESV had larger extent of inducible wall-motion abnormalities, lower peak stress LVEF, and higher likelihood of moderate or grater ischemia. For moderate or greater myocardial ischemia detection, TIDESV had a sensitivity of 46%, specificity of 83%, positive predictive value of 70%, and negative predictive value of 64%. CONCLUSION: Transient ischemic dilatation by SE is a marker of extensive myocardial ischemia and can be used as an additional marker of higher risk.

Unexpected Allosteric Network Contributes to LRH-1 Co-regulator Selectivity.

Phospholipids (PLs) are unusual signaling hormones sensed by the nuclear receptor liver receptor homolog-1 (LRH-1), which has evolved a novel allosteric pathway to support appropriate interaction with co-regulators depending on ligand status. LRH-1 plays an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of metabolic and neoplastic diseases. Although the prospect of modulating LRH-1 via small molecules is exciting, the molecular mechanism linking PL structure to transcriptional co-regulator preference is unknown. Previous studies showed that binding to an activating PL ligand, such as dilauroylphosphatidylcholine, favors LRH-1's interaction with transcriptional co-activators to up-regulate gene expression. Both crystallographic and solution-based structural studies showed that dilauroylphosphatidylcholine binding drives unanticipated structural fluctuations outside of the canonical activation surface in an alternate activation function (AF) region, encompassing the ß-sheet-H6 region of the protein. However, the mechanism by which dynamics in the alternate AF influences co-regulator selectivity remains elusive. Here, we pair x-ray crystallography with molecular modeling to identify an unexpected allosteric network that traverses the protein ligand binding pocket and links these two elements to dictate selectivity. We show that communication between the alternate AF region and classical AF2 is correlated with the strength of the co-regulator interaction. This work offers the first glimpse into the conformational dynamics that drive this unusual PL-mediated nuclear hormone receptor activation.

Phospholipid--driven gene regulation.

Phospholipids (PLs), well known for their fundamental role in cellular structure, play critical signaling roles via their derivatives and cleavage products acting as second messengers in signaling cascades. Recent work has shown that intact PLs act as signaling molecules in their own right by modulating the activity of nuclear hormone transcription factors responsible for tuning genes involved in metabolism, lipid flux, steroid synthesis and inflammation. As such, PLs have been classified as novel hormones. This review highlights recent work in PL-driven gene regulation with a focus on the unique structural features of phospholipid-sensing transcription factors and what sets them apart from well known soluble phospholipid transporters.

Deciphering modern glucocorticoid cross-pharmacology using ancestral corticosteroid receptors.

Steroid receptors (SRs) are the largest family of metazoan transcription factors and control genes involved in development, endocrine signaling, reproduction, immunity, and cancer. The entire hormone receptor system is driven by a molecular switch triggered by the binding of small lipophilic ligands. This makes the SRs ideal pharmaceutical targets, yet even the best clinically approved synthetic steroidal agonists are prone to cross-reactivity and off-target pharmacology. The mechanism underlying this promiscuity is derived from the fact that SRs share common structural features derived from their evolutionary relationship. More often than not, rational attempts to probe SR drug selectivity via mutagenesis fail even when high quality structural and functional data are available due to the fact that important mutations often result in nonfunctional receptors. This highlights the fact that SRs suffer from instability, preventing in-depth mutational analysis and hampering crystallization of key receptor-ligand complexes. We have taken a unique approach to address this problem by using a resurrected ancestral protein to determine the structure of a previously intractable complex and identified the structural mechanisms that confer activation and selectivity for a widely used glucocorticoid, mometasone furoate. Moreover, we have identified a single residue located outside of the ligand-binding pocket that controls mometasone furoate antagonism versus agonism in the human mineralocorticoid receptor.

Development of photo-crosslinking reagents for protein kinase-substrate interactions.

The identification of relevant protein kinase-protein substrate partners remains a serious challenge on a genome-wide scale. The design and synthesis of a photo-activatable nucleotide reagent to crosslink protein kinases with their substrates is described in which an azido group is appended to the gamma-phosphoryl and purine moieties of ATP. In the absence of UV, compounds of this class were shown to act as competitive inhibitors versus ATP and non-competitive inhibitors versus peptide substrate for the protein tyrosine kinase Csk, suggesting that they can form a ternary complex with kinase and protein substrate. In vitro experiments with protein kinases indicate the bifunctional reagent can induce covalent protein-protein crosslinking that is dependent on UV irradiation. That significant kinase-substrate crosslinking occurs is suggested by the fact that this crosslinking is competitively inhibited by ATP. The crosslinked adducts can be readily cleaved by phosphodiesterase which supports the model for crosslinking and provides a simple method to deconvolute the linked protein partners.