The Ileal Lipid Binding Protein Is Required for Efficient Absorption and Transport of Bile Acids in the Distal Portion of the Murine Small Intestine

<div><p>The ileal lipid binding protein (ilbp) is a cytoplasmic protein that binds bile acids with high affinity. However evidence demonstrating the role of this protein in bile acid transport and homeostasis is missing. We created a mouse strain lacking ilbp (<em>Fabp6^−/−</em> mice) and assessed the impact of ilbp deficiency on bile acid homeostasis and transport in vivo. Elimination of ilbp increased fecal bile acid excretion (54.2%, <em>P</em><0.05) in female but not male <em>Fabp6^−/−</em> mice. The activity of cholesterol 7α-hydroxylase (cyp7a1), the rate-controlling enzyme of the classical bile acid biosynthetic pathway, was significantly increased in female (63.5%, <em>P</em><0.05) but not in male <em>Fabp6^−/−</em> mice. The amount of [³H]taurocholic acid (TCA) excreted by 24 h after oral administration was 102% (<em>P</em><0.025) higher for female <em>Fabp6^−/−</em> mice whereas it was 57.3% (<em>P</em><0.01) lower for male <em>Fabp6^−/−</em> mice, compared to wild-type mice. The retained fraction of the [³H]TCA localized in the small and large intestines was increased by 22% (<em>P</em><0.02) and decreased by 62.7% (<em>P</em><0.01), respectively, in male <em>Fabp6^−/−</em> mice relative wild-type mice, whereas no changes were seen in female <em>Fabp6^−/−</em> mice. Mucosal to serosal bile acid transport using everted distal gut sacs was decreased by 74% (<em>P</em><0.03) in both sexes of <em>Fabp6^−/−</em> mice as compared to wild-type mice. The results demonstrate that ilbp is involved in the apical to basolateral transport of bile acids in ileal enterocytes, and is vital for the maintenance of bile acid homeostasis in the enterohepatic circulation (EHC) in mice.</p> </div

Survey of gene expression in the small intestine.

<p>(A) qPCR analysis of RNA from small intestines of <i>Fabp6</i>^+/+ (black bars) and <i>Fabp6^−/−</i> (white bars) were done in duplicates. The normalized abundance (mean±SEM) of target mRNAs is expressed relative to male <i>Fabp6</i>^+/+ mice. <i>P</i><0.05, vs. wild-type of the same sex. (B) Protein blots of small intestine homogenates of <i>Fabp6</i>^+/+ and <i>Fabp6^−/−</i> mice were probed with antisera to ilbp, asbt and L-FABP.</p

Plasma chemistry.

*<p>P<0.01,</p>**<p>P<0.05, vs. wild-type, n = 10 per group. Values for ALT and AST are mean ± SEM.</p

Hepatic cholesterol concentration and enzyme activities of <i>Fabp6</i>^+/+ (black bars) and <i>Fabp6^−/−</i> (white bars) mice.

<p>(A) Unesterified cholesterol concentration and (B) Cholesteryl ester concentration, (n = 3). (C) Relative cyp7a1 activity (n = 5). (D) Relative HMGR activity (n = 5). Groups are compared relative to male <i>Fabp6</i>^+/+ mice.</p

Survey of gene expression in the liver.

<p>qPCR analysis of liver RNA from <i>Fabp6</i>^+/+ (black bars) and <i>Fabp6^−/−</i> (white bars) mice were done in duplicates. The abundance (mean ± SEM) of target mRNAs was normalized to internal standards and expressed relative male wild-type mice. *<i>P</i><0.05, vs. wild-type.</p

Transport of bile acids in everted gut sacs.

<p>(A) Amount of TCA accumulated in the serosal fluid after a 30 min incubation of gut sacs from the proximal one-third and distal one-third portions of the small intestine of male <i>Fabp6</i>^+/+ (n = 4), female <i>Fabp6</i>^+/+ (n = 3–4) mice (black bars), male <i>Fabp6^−/−</i> (n = 4) and female <i>Fabp6^−/−</i> (n = 5) mice (white bars). (B) Amount of TCA accumulated in the gut sac tissue at the end of the assay incubation period. *<i>P</i><0.025, **<i>P</i><0.01, vs. wild-type of the same sex.</p

Targeted disruption of the murine <i>Fabp6</i> gene.

<p>(A) Structure of wild-type (wt) and disrupted <i>Fabp6</i> alleles. The targeting vector was designed to replace the region of the <i>Fabp6</i> gene encompassing exons 2 and 3 with the neo resistance (neo^r) gene cassette. X, Xba I site. (B) RNA blot of small intestine RNA from <i>Fabp6</i>^+/+ and <i>Fabp6^−/−</i> mice probed with [³²P]-labeled ilbp cDNA. (C) Protein blot of small intestine homogenates from <i>Fabp6</i>^+/+ and <i>Fabp6^−/−</i> mice probed with antiserum to murine ilbp.</p

Fyn and TOM1L1 are recruited to clathrin-coated pits and regulate Akt signaling

The epidermal growth factor (EGF) receptor (EGFR) controls many aspects of cell physiology. EGF binding to EGFR elicits the membrane recruitment and activation of phosphatidylinositol-3-kinase, leading to Akt phosphorylation and activation. Concomitantly, EGFR is recruited to clathrin-coated pits (CCPs), eventually leading to receptor endocytosis. Previous work uncovered that clathrin, but not receptor endocytosis, is required for EGF-stimulated Akt activation, and that some EGFR signals are enriched in CCPs. Here, we examine how CCPs control EGFR signaling. The signaling adaptor TOM1L1 and the Src-family kinase Fyn are enriched within a subset of CCPs with unique lifetimes and protein composition. Perturbation of TOM1L1 or Fyn impairs EGF-stimulated phosphorylation of Akt2 but not Akt1. EGF stimulation also triggered the TOM1L1- and Fyn-dependent recruitment of the phosphoinositide 5-phosphatase SHIP2 to CCPs. Thus, the recruitment of TOM1L1 and Fyn to a subset of CCPs underlies a role for these structures in the support of EGFR signaling leading to Akt activation.</p

Multiscale interactome analysis coupled with off‑target drug predictions reveals drug repurposing candidates for human coronavirus disease

  The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.  </p