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Bile acid-CoA:amino acid N-acyltransferase (BACAT) catalyzes the conjugation of bile acids to glycine and taurine for excretion into bile. By use of site-directed mutagenesis and sequence comparisons, we have identified Cys-235, Asp-328, and His-362 as constituting a catalytic triad in human BACAT (hBACAT) and identifying BACAT as a member of the type I acyl-CoA thioesterase gene family. We therefore hypothesized that hBA-CAT may also hydrolyze fatty acyl-CoAs and/or conjugate fatty acids to glycine. We show here that recombinant hBACAT also can hydrolyze long-and very long-chain saturated acyl-CoAs (mainly C16:0 -C26:0) and by mass spectrometry verified that hBACAT also conjugates fatty acids to glycine. Tissue expression studies showed strong expression of BACAT in liver, gallbladder, and the proximal and distal intestine. However, BACAT is also expressed in a variety of tissues unrelated to bile acid formation and transport, suggesting important functions also in the regulation of intracellular levels of very long-chain fatty acids. Green fluorescent protein localization experiments in human skin fibroblasts showed that the hBACAT enzyme is mainly cytosolic. Therefore, the cytosolic BACAT enzyme may play important roles in protection against toxicity by accumulation of unconjugated bile acids and non-esterified very long-chain fatty acids.

Human filamins are 280-kDa proteins containing an N-terminal actin-binding domain followed by 24 characteristic repeats. They also interact with a number of other cellular proteins. All of those identified to date, with the exception of actin, bind to the C-terminal third of a filamin. In a yeast two-hybrid search of a human placental library, using as bait repeats 10 -18 of filamin B, we isolated a cDNA coding for a novel 374 amino acid protein containing a proline-rich domain near its N terminus and two LIM domains at its C terminus. We term this protein filamin-binding LIM protein-1, FBLP-1. Yeast two-hybrid studies with deletion mutants localized the areas of interaction in FBLP-1 to its N-terminal domain and in filamin B to repeats 10 -13. FBLP-1 mRNA was detected in a variety of tissues and cells including platelets and endothelial cells. We also have identified two FBLP-1 variants. Both contain three C-terminal LIM domains, but one lacks the N-terminal proline-rich domain. Transfection of FBLP-1 into 293A cells promoted stress fiber formation, and both FBLP-1 and filamin B localized to stress fibers in the transfected cells. The association between filamin B and FBLP-1 may play a hitherto unknown role in cytoskeletal function, cell adhesion, and cell motility.The human filamin (FLN) 1 family consists of three ϳ280-kDa actin-binding paralogs (FLNs A, B, and C), each containing an N-terminal actin-binding domain followed by 24 characteristic ϳ96 residue ␤-pleated sheet homologous repeats (1-3). In addition, each FLN contains two intercalated 24 -36 residue "hinge" regions, the first between repeats 15 and 16 and the second between repeats 23 and 24. FLNs self-associate to form homodimers, an interaction that requires the presence of repeat 24.Current data suggest that FLNs may have multiple functions. 1) They are involved in the organization of the cytoskeleton (4, 5) and appear to be necessary for normal cell adhesion and motility (6). 2) They bind to the cytoplasmic tails of a number of membrane proteins including several ␤-integrins (7, 8), GpIb␣ (9), and tissue factor (10), suggesting that they play a role in cytoskeletal-membrane interactions. 3) They associate with several cytoplasmic components of signaling pathways including Rho GTPases (11), TRAF2 (12), Smads (13), and SEK-1 (14) and thus may serve as a scaffolding or point of convergence for signals between the cell membrane and the cell interior. Nevertheless, a clear mechanistic explanation for their importance is still lacking. Most interactions with FLNs have been identified as a result of yeast two-hybrid studies using as bait a variety of membrane or cytoplasmic proteins. In this manner, one or another filamin has been identified as an interactant with some 25 proteins (2,(15)(16)(17)(18)(19). In every case where localization studies have been performed, the interaction site on the filamin has been in the C-terminal third of the molecule. Because the libraries that were screened tend to be relatively enriched in 3Ј-sequences, it seemed pos...