Daniël A. Lionarons

as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target.

The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia, as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the postmetamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic, with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (∼10 μM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free-swimming adults revealed that ∼35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72-hour period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts, and BSP, were predominantly excreted by way of the kidney, with minor amounts also detected in feces. Gene expression analysis detected marked up-regulation of orthologs of known organic anion and bile salt transporters in the kidney, with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis. (HEPATOLOGY 2013;57:2418-2426).

Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma.

Cholestasis impairs liver regeneration following partial liver resection (PHx). Bile acid receptor farnesoid X-receptor (FXR) is a key mediator of liver regeneration. The effects of FXR agonist obeticholic acid (OCA) on liver (re)growth were therefore studied in cholestatic rats. Animals underwent sham surgery or reversible bile duct ligation (rBDL). PHx with concurrent internal biliary drainage was performed 7 days after rBDL. Animals were untreated or received OCA (10 mg/kg/day) per oral gavage from rBDL until sacrifice. After 7 days of OCA treatment, dry liver weight increased in the rBDL + OCA group, indicating OCA-mediated liver growth. Enhanced proliferation in the rBDL + OCA group prior to PHx concurred with a rise in Ki67-positive hepatocytes, elevated hepatic Ccnd1 and Cdc25b expression, and an induction of intestinal fibroblast growth factor 15 expression. Liver regrowth after PHx was initially stagnant in the rBDL + OCA group, possibly due to hepatomegaly prior to PHx. OCA increased hepatobiliary injury markers during BDL, which was accompanied by upregulation of the bile salt export pump. There were no differences in histological liver injury. In conclusion, OCA induces liver growth in cholestatic rats prior to PHx but exacerbates biliary injury during cholestasis, likely by forced pumping of bile acids into an obstructed biliary tree.

The apical Na+-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution. The apical Na+-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution. apical sodium-dependent bile salt transporter farnesoid X receptor human ASBT lamprey Asbt skate Asbt taurocholic acid untranslated region Bile alcohols and bile acids are the end products of cholesterol metabolism. Most bile alcohols and bile acids are conjugated with sulfate, taurine, or glycine at the terminal carbon of the side chain and are isolated as bile salts (1Hofmann A.F. Hagey L.R. Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51: 226-246Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Bile salts play critical physiological roles in vertebrates. They facilitate lipid absorption, inhibit microbe growth in the biliary tract and intestine, and function as signaling molecules that regulate energy expenditure and carbohydrate and lipid metabolism (2Trauner M. Claudel T. Fickert P. Moustafa T. Wagner M. Bile acids as regulators of hepatic lipid and glucose metabolism.Dig. Dis. 2010; 28: 220-224Crossref PubMed Scopus (235) Google Scholar). The bile salt pool is maintained in an enterohepatic circulation by bile salt transporters in the distal ileum and the liver (3Dawson P.A. Lan T. Rao A. Bile acid transporters.J. Lipid Res. 2009; 50: 2340-2357Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Central to this process is the apical sodium (Na+)-dependent bile salt transporter (ASBT/SLC10A2) located on the luminal membrane in the distal ileum and proximal tubule of the kidney in humans and rodents (4Dawson P.A. Role of the intestinal bile acid transporters in bile acid and drug disposition.Handb. Exp. Pharmacol. 2011; 201: 169-203Crossref PubMed Scopus (155) Google Scholar). ASBT maintains the enterohepatic and renal-hepatic circulation of bile salts by facilitating their reabsorption from the intestinal lumen and renal tubules. Dysfunction of ASBT/Asbt interrupts the bile salt enterohepatic circulation, reduces the bile salt pool size by 80% in mice, and leads to bile salt malabsorption, diarrhea, and steatorrhea in humans, where reduced plasma levels of cholesterol are also observed (5Dawson P.A. Haywood J. Craddock A.L. Wilson M. Tietjen M. Kluckman K. Maeda N. Parks J.S. Targeted deletion of the ileal bile acid transporter eliminates enterohepatic cycling of bile acids in mice.J. Biol. Chem. 2003; 278: 33920-33927Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 6Oelkers P. Kirby L.C. Heubi J.E. Dawson P.A. Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2).J. Clin. Invest. 1997; 99: 1880-1887Crossref PubMed Scopus (307) Google Scholar). Thus, there is considerable pharmaceutical interest in ASBT inhibition as a potential target for drug discovery for the treatment of hypercholesterolemia and diabetes mellitus type 2 (7Lewis M.C. Brieaddy L.E. Root C. Effects of 2164U90 on ileal bile acid absorption and serum cholesterol in rats and mice.J. Lipid Res. 1995; 36: 1098-1105Abstract Full Text PDF PubMed Google Scholar, 8Bhat B.G. Rapp S.R. Beaudry J.A. Napawan N. Butteiger D.N. Hall K.A. Null C.L. Luo Y. Keller B.T. Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE2/2 mice by SC-435.J. Lipid Res. 2003; 44: 1614-1621Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Furthermore, an ASBT inhibitor has been demonstrated to be beneficial for patients with chronic idiopathic constipation (9Chey W.D. Camilleri M. Chang L. Rikner L. Graffner H. A randomized placebo-controlled phase IIb trial of a3309, a bile acid transporter inhibitor, for chronic idiopathic constipation.Am. J. Gastroenterol. 2011; 106: 1803-1812Crossref PubMed Scopus (138) Google Scholar, 10Simren M. Bajor A. Gillberg P.G. Rudling M. Abrahamsson H. Randomised clinical trial: the ileal bile acid transporter inhibitor A3309 vs. placebo in patients with chronic idiopathic constipation–a double-blind study.Aliment. Pharmacol. Ther. 2011; 34: 41-50Crossref PubMed Scopus (96) Google Scholar). In contrast to what is known for mammalian species, very little is known about the presence or function of Asbt in other vertebrates. Bile salts demonstrate considerable structural variation across vertebrate classes (1Hofmann A.F. Hagey L.R. Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51: 226-246Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 11Haslewood G.A. Bile salt evolution.J. Lipid Res. 1967; 8: 535-550Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). The enzymatic pathway that converts cholesterol into bile salts is complex and requires a minimum of five enzymes in primitive vertebrates and up to 16 enzymes in humans (12Russell D.W. Fifty years of advances in bile acid synthesis and metabolism.J. Lipid Res. 2009; 50 (Suppl): S120-S125Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar–14Hagey L.R. Moller P.R. Hofmann A.F. Krasowski M.D. Diversity of bile salts in fish and amphibians: evolution of a complex biochemical pathway.Physiol. Biochem. Zool. 2010; 83: 308-321Crossref PubMed Scopus (102) Google Scholar). The most primitive vertebrates (agnathans or jawless fish) use early evolving C27 sulfated bile alcohols with a C-5 hydrogen at α configuration (i.e., 5α), which is an overall planar structure to the four conjoined steroid rings, one that is similar to that of cholesterol (1Hofmann A.F. Hagey L.R. Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51: 226-246Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 11Haslewood G.A. Bile salt evolution.J. Lipid Res. 1967; 8: 535-550Abstract Full Text PDF PubMed Google Scholar). Evolutionarily “intermediate” 5β-C27 sulfated bile alcohols in which the four rings of the steroid possess a tilted structure are mostly detected in jawed cartilaginous fishes (early gnathostomes). More complex vertebrates including mammals primarily use later evolving bile salts, which are 5β-C24 bile acid conjugates with a bent ring juncture and a shortened side chain containing a carboxylic acid. Because of these structural changes, bile salt composition has been proposed as a complementing biochemical trait to characterize evolutionary relationships among species (1Hofmann A.F. Hagey L.R. Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51: 226-246Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). The major structural variation of bile salts seen over the course of vertebrate evolution raises the question as to how the various transporters that move bile salts into and out of cells located in the intestine and liver have adapted to these changes. Specifically, do nonmammalian Slc10a2’s in more primitive species function as Na+-dependent bile salt transporters and how have they adapted structurally and functionally with the changing shapes of bile salts? Insight into the structure/function relationship of SLC10A/Slc10a members has been advanced by solving the crystal structure of a distant ASBT homolog from the bacterium Neisseria meningitidis (15Hu N.J. Iwata S. Cameron A.D. Drew D. Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT.Nature. 2011; 478: 408-411Crossref PubMed Scopus (192) Google Scholar). Still, the structural determinants of ASBT/Asbt for its bile salt substrates remain unclear because of low homology in the substrate binding pocket between ASBT/Asbt and this bacterial homolog. In particular, it is not known which residues directly bind bile salts when ASBT is configured in an outward direction to accept substrates for uptake. In this report, we have characterized two ASBT orthologs at the molecular and functional level that represent early stages in the vertebrate lineage. First, we identified an ASBT ortholog in the sea lamprey (Petromyzon marinus), representing agnathans, the most primitive vertebrate class that diverged from a more complex lineage ∼500 million years ago and whose bile consists of some of the earliest 5α-C27 bile alcohols. Second, we identified an ASBT ortholog in the little skate (Leucoraja erinacea), an early gnathostome that diverged ∼300 million years ago, whose bile salts are 5β-C27 bile alcohols (“intermediate” bile salts). Finally, we compared our findings with the structure and substrate specificity of ASBT from humans, whose bile contains the later evolving 5β-C24 bile acids. Our phylogenetic and experimental findings support the concept that ASBT emerged at the very beginning of vertebrate evolution with a limited ability to transport bile salts. As vertebrate evolution progressed, the substrate specificity of ASBT/Asbt for bile salts expanded while at the same time retaining its ability to transport the earlier evolved forms. These findings also indicate that the enterohepatic circulation of bile salts is a conserved function throughout vertebrate evolution. Unless otherwise stated, all chemicals were from Sigma (St. Louis, MO). 3H-taurocholic acid (TCA, activity 5.0 Ci/mmol) and 3H-estrone-3-sulfate (activity 57.3 Ci/mmol) were purchased from PerkinElmer (Waltham, MA). 5α-petromyzonol sulfate (PZS) was from Toronto Research Chemicals. 5α-cyprinol sulfate was kindly provided by Dr. Lee Hagey (University of California at San Diego, San Diego, CA). Taurodehydrocholic acid was from Calbiochem (San Diego, CA) and bilirubin-ditaurate was from Frontier Scientific (Logan, UT). Oligonucleotides and DNA sequencing were provided by the Keck Biotechnology Resource Laboratory at Yale University. Fluorescence dye labeled DNA probes were made by Integrated DNA Technologies (Coralville, IA). All animal were at the Laboratory in were by the and and in with the on and of Laboratory were from were while in the in and were in at were in from the of and maintained in at All were with The intestine was at the liver or bile and at the of the and distal gut sacs were as by and L. In absorption of bile salts by intestine of rats and J. PubMed Scopus Google Scholar). sacs were four in lamprey to or as of a bile salt in skate J. Google Scholar). all sacs were in their with 50 with and at for or with sacs were four in and in was at for and for of and was to the using a was in a and was to was isolated using CA) and using a CA). of from was into using a from was on an CA). The and probes are in Because the of the gene in the of that were we to of were in where the were to a lamprey Asbt we from the lamprey by ortholog using human ASBT as We and a from lamprey intestine using DNA sequencing and phylogenetic that this a of A was by and using a from skate Asbt we a DNA by using that two conserved of The was also by we directly the region from cells and it into a and with functionally characterize and both were also into both and at were and one with to the lamprey or products was for In we made and in to these two at the for of and The are in was with the the P.A. H. A. and X PubMed Scopus Google Scholar). We with using phylogenetic 2003; PubMed Scopus Google Scholar). The was the for acid an with four and for with one chain and three were by and the as The phylogenetic was using as an and with cells were maintained at low in growth with 50 and 50 all from cells 80% they were with or using or cells were to the as P. Craddock A.L. Dawson P.A. cloning and of the ileal sodium-dependent bile acid Biol. Chem. Full Text PDF PubMed Google Scholar). activity was to are as and were by of to the using A gene was to the ability of conjugated bile salts to be transported into cells with cells were maintained in growth cells 80% the was to with and with 50 or and or 50 or 50 with 50 and in using for in cells were with bile salts for an in was to and was detected in a were to the the bile salt transport function of cells were with lipid isolated from lamprey was with was to between two and was to between more two by for was to be by our demonstrated that bile salts are from the intestine of the little that an transport is for bile salts J. circulation of sulfate in an the little skate J. 1997; Google Scholar). However, it was not known this transport is as is human and this was in gut sacs isolated from the little skate L. In absorption of bile salts by intestine of rats and J. PubMed Scopus Google Scholar). As demonstrated in a of was observed in the distal but not proximal was by in the of was that a Na+-dependent bile salt transporter was in the distal intestine of A similar transport was out in and in contrast to skate intestine, the proximal distal intestine of the lamprey showed Na+-dependent of (Fig. that an ASBT ortholog has not evolved in lamprey or its substrate specificity is limited and not the modern bile salt TCA. A of the lamprey DNA with the potential to of an ASBT and identified a which acids with a region and we using by We a in for acids with at the and at the and and acid to and as the most primitive of known orthologs (Fig. of of the sea an that is to the with vertebrates, 16 that be members of the However, phylogenetic not a potential ortholog of (Fig. and The that was most was on the of Slc10a2 and on these we that a emerged from an gene the beginning of vertebrate from a gene between and lamprey at a time that with the of the of bile salts, the of a biliary and the ability to of of ASBT/Asbt's that the proposed are including the and the substrate binding pocket on the crystal structure of a distant ASBT homolog in N. meningitidis (15Hu N.J. Iwata S. Cameron A.D. Drew D. Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT.Nature. 2011; 478: 408-411Crossref PubMed Scopus (192) Google Scholar) and In contrast, the of the and while a in is conserved A. Chang C. and structure of the binding of the apical sodium-dependent bile acid transporter PubMed Scopus Google an at in the of and we As in was primarily in the kidney and in and and was in and A similar was in lamprey was in the intestine with levels in the and it was from other (Fig. In we of intestine, which demonstrated that both and are most in the distal intestine (Fig. was in skate with a in the distal compared with the proximal These are similar in human and rodents and with the known bile salt function of functionally characterize and and to their substrate specificity to we these three into a and into cells for As in and demonstrated Na+-dependent of that over In contrast, not transport activity for with or in the by and that the was on the plasma membrane of cells These results are with the results of transport in the gut (Fig. and suggest that is not a functional bile salt transporter or it is functionally for the modern 5β-C24 bile salt TCA. of transport that has a of with a of whereas has a of with a of 2 (Fig. the was from to in not to transporter which between transport and of However, is of transport and be compared to in The findings of of affinity for and low affinity for for suggest that structural changes in Asbt have evolved that of this bile acid structure between the of and early whereas affinity was between early gnathostome and mammalian evolution. bile salts, we a Because conjugated bile salts a transporter to the membrane and they are for human of the ability of ASBT to transport a bile salt into the In this of or were with into The cells were with sulfate, and TCA, the three major bile salts in and transport this bile salt (Fig. and also demonstrated transport activity for 5β-C27 sulfate was showed activity at or whereas and demonstrated activity with (Fig. cells were with 5β-C24 TCA, not transport showed low whereas showed high activity (Fig. that and have for bile salts, we early and evolving bile salts with the As by transport lamprey bile salts from its liver lipid and 5α-cyprinol sulfate 5α-C27 bile but not modern 5β-C24 bile salts, including acid acid TCA, and acid. acid is an and was transported by transported sulfate in to all the but demonstrated low affinity for most modern bile salts (Fig. In contrast, all bile salts were transported by these findings indicate that affinity for bile salts is to the 5α-C27 early evolving bile salts, transport both the and “intermediate” bile salts whereas bile salts with the of substrate we an of bile salts, steroid and in assays with of In more bile salt more with (Fig. Specifically, the early evolved bile salts 5α-cyprinol “intermediate” bile salts and modern 5β-C24 bile acids and acid reduced transport activity in both and reduced transport activity in but not in with results from the Taurodehydrocholic acid on transport activity with or We the affinity of for 5β-C27 sulfate, the major bile salt in in a for uptake. sulfate has a of whereas the for was as In we or not steroid inhibit and transport of These molecules are structurally similar to bile salts and are transported by J. K. N. H. and functional of human sodium-dependent transporter Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a of ASBT (Fig. activity of and for but not whereas but not However, 3H-estrone-3-sulfate was not transported by or Also, both and not affinity for the whereas both these suggest that as the substrate specificity of these transporters expanded for bile salts, this not to the of steroid In to the evolutionary of and its substrate we functionally characterized this intestinal bile salt transport in the sea lamprey and the little two species that represent early evolving members of vertebrate and compared these with the human Our results demonstrate that primitive orthologs in lamprey and skate have the ability to transport bile salts and mammalian the substrate specificity of and to bile salts, as they not demonstrate affinity for structurally steroid and other (Fig. The of both and was most in the distal intestine and kidney (Fig. with the of transporters to bile salts from the intestinal an essential function for an enterohepatic molecular that the enterohepatic circulation of bile salts was at the earliest of vertebrate evolution. Because an ASBT ortholog was not identified in the sea (Fig. 2 and a we that ASBT/Asbt is a bile salt transporter that evolved at the beginning of vertebrate evolution. We also demonstrated that bile salts in a Na+-dependent this we were not to directly bile salt transport by is Na+-dependent bile salt substrates are not However, that acids directly in for N. meningitidis are conserved in mammalian ASBT/Asbt's and Na+-dependent and (15Hu N.J. Iwata S. Cameron A.D. Drew D. Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT.Nature. 2011; 478: 408-411Crossref PubMed Scopus (192) Google that is to be a Na+-dependent In this we transport activity and substrate specificity with assays in intestinal (Fig. and assays as as in assays using recombinant transporters (Fig. these assays demonstrate that has a bile salt substrate specificity with high affinity for bile salt of and 5α-cyprinol sulfate, two early evolving bile alcohols, whereas the more advanced “intermediate” 5β-C27 bile sulfate was a low affinity In contrast, the modern 5β-C24 bile acids and were not In the of not was 5β-C27 sulfate bile a high affinity substrate but were the 5α-bile alcohols. The later evolving 5β-C24 bile acids as and showed low affinity for all the structural of bile salts were high affinity substrates for the major bile salts in humans are 5β-C24 bile acids. these results suggest that ASBT expanded its substrate specificity a class of bile salts emerged in evolution (1Hofmann A.F. Hagey L.R. Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51: 226-246Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 11Haslewood G.A. Bile salt evolution.J. Lipid Res. 1967; 8: 535-550Abstract Full Text PDF PubMed Google Scholar). substrate affinity for human ASBT is as most orthologs affinity for their earlier substrates when they affinity for substrates during evolution Y. of PubMed Scopus Google and of of enzymes and 2011; PubMed Scopus Google a ASBT/Asbt has affinity for substrates as it affinity for more modern bile salts. We that modern ASBT/Asbt its substrate specificity in to as different species of bile salts as In 16 different enzymatic are to cholesterol to 5β-C24 bile acids. As is not of bile salts be and in to bile J. Bile in and Lipid Res. 2010; 51: Full Text Full Text PDF PubMed Scopus Google Scholar). In to most the bile salt pool these of bile salt are also We that other transporters in the enterohepatic circulation of bile salts also specificity for a of bile salt substrates this has not been Our phylogenetic that the crystal structure of N. meningitidis is not a of the gene (Fig. it TCA, it is not known it transport other bile salts or other In N. meningitidis to and Because the four do not transport TCA, this raises a question as to the specificity of the substrate binding pocket of N. meningitidis for bile salts. Because and and demonstrate for bile salts, it be possible to acids that are for the of mammalian ASBT/Asbt substrate specificity using and to ASBT/Asbt's evolutionary while into the determinants of In the that Asbt is a functional bile salt transporter in the most primitive vertebrates, with the of bile salts and the ability of to that ASBT/Asbt and the enterohepatic circulation of bile salts were at the beginning of vertebrate vertebrates the transport ability essential to regulate both bile salt and lipid Our findings also suggest that as ASBT it substrate specificity to bile salt while retaining affinity for its We Dr. Lee Hagey (University of California at San Diego, San Diego, CA) for 5α-cyprinol sulfate and for critical and of this Dr. and in our have provided Also, we are to (University of for and