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1.
Cell Mol Gastroenterol Hepatol ; : 101392, 2024 Aug 22.
Article in English | MEDLINE | ID: mdl-39179177

ABSTRACT

BACKGROUNDS & AIMS: Bile acids (BAs) are core gastrointestinal metabolites with dual functions in lipid absorption and cell signaling. BAs circulate between the liver and distal small intestine (ie, ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with host intestinal cells in vivo remain poorly understood. Because ileal absorption is rate-limiting in determining which BAs in the intestinal lumen gain access to host intestinal cells and receptors, and at what concentrations, we hypothesized that defining the rates and routes of ileal BA absorption in vivo would yield novel insights into the physiological forms and functions of mouse enterohepatic BA pools. METHODS: Using ex vivo mass spectrometry, we quantified 88 BA species and metabolites in the intestinal lumen and superior mesenteric vein of individual wild-type mice, and cage-mates lacking the ileal BA transporter, Asbt/Slc10a2. RESULTS: Using these data, we calculated that the pool of BAs circulating through ileal tissue (ie, the ileal BA pool) in fasting C57BL/6J female mice is ∼0.3 mmol/g. Asbt-mediated transport accounted for ∼80% of this pool and amplified size. Passive permeability explained the remaining ∼20% and generated diversity. Compared with wild-type mice, the ileal BA pool in Asbt-deficient mice was ∼5-fold smaller, enriched in secondary BA species and metabolites normally found in the colon, and elicited unique transcriptional responses on addition to ex vivo-cultured ileal explants. CONCLUSIONS: This study defines quantitative traits of the mouse enterohepatic BA pool and reveals how aberrant BA metabolism can impinge directly on host intestinal physiology.

2.
bioRxiv ; 2024 Feb 18.
Article in English | MEDLINE | ID: mdl-38405928

ABSTRACT

Bile acids (BAs) are gastrointestinal metabolites that serve dual functions in lipid absorption and cell signaling. BAs circulate actively between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with intestinal cells in vivo remain ill-defined. Through multi-site sampling of nearly 100 BA species in individual wild type mice, as well as mice lacking the ileal BA transporter, Asbt/Slc10a2, we calculate the ileal BA pool in fasting C57BL/6J mice to be ~0.3 µmoles/g. Asbt-mediated transport accounts for ~80% of this pool and amplifies size, whereas passive absorption explains the remaining ~20%, and generates diversity. Accordingly, ileal BA pools in mice lacking Asbt are ~5-fold smaller than in wild type controls, enriched in secondary BA species normally found in the colon, and elicit unique transcriptional responses in cultured ileal explants. This work quantitatively defines ileal BA pools in mice and reveals how BA dysmetabolism can impinge on intestinal physiology.

3.
Nucleic Acids Res ; 48(8): 4507-4520, 2020 05 07.
Article in English | MEDLINE | ID: mdl-32170306

ABSTRACT

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , RNA, Bacterial/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Escherichia coli Proteins/genetics , Genetic Techniques , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Models, Molecular , Protein Binding , Protein Domains
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