Herbert G. Bull

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.

Peptidylarginine deiminases (PADs) play a critical role in generating autoantigens in rheumatoid arthritis (RA), but the mechanisms underlying their dysregulation in this disease remain unknown. Although PADs require supraphysiologic concentrations of calcium for activity in vitro, the enzymes are active in vivo (for example, in RA synovial fluid) where calcium concentrations are much lower. We have discovered a subset of anti-PAD4 autoantibodies (identified by their cross-reactivity with PAD3) that markedly increase the catalytic efficiency of PAD4 by decreasing the enzyme's requirement for calcium into the physiologic range. Patients with these PAD3/PAD4 cross-reactive autoantibodies had higher baseline radiographic damage scores and a higher likelihood of radiographic progression compared to individuals negative for these antibodies. The ability of autoantibodies to activate an enzyme that itself generates citrullinated autoantigens identifies an important feed-forward loop, which may drive the erosive outcome observed in RA patients with these autoantibodies. PAD3 autoantibodies may therefore identify RA patients who would benefit from early aggressive treatment or addition of PAD inhibitor therapy.

UDP-N-acetylglucosamine acyltransferase of Escherichia coli catalyzes the reaction, UDP-GlcNAc + R-3-hydroxymyristoyl-ACP--> UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc + ACP. Using Matrex Gel Green A and heparin-agarose, we have purified the enzyme to near homogeneity from a strain that overproduces it 474-fold. The subunit molecular mass determined by SDS-gel electrophoresis is approximately 30 kDa, consistent with results of previous radiolabeling experiments in mini-cells. The amino-terminal sequence (Met-Ile-Asp-Lys-Ser-Ala-Phe-Val-His-Pro) and the amino acid composition of the purified protein are consistent with DNA sequencing (Coleman, J., and Raetz, C. R. H. (1988) J. Bacteriol. 170, 1268-1274). At saturating concentrations of the second substrate, the apparent Km values for UDP-GlcNAc and R-3-hydroxymyristoyl-ACP are 99 and 1.6 microM, respectively. There is an absolute requirement for the R-3-hydroxy moiety of the fatty acyl-ACP substrate; myristoyl-ACP binds effectively (IC50 = 2 microM) but is inactive (< 0.01%) as an alternate substrate. The most remarkable feature of the reaction is its unfavorable equilibrium constant, Keq approximately equal to 0.01, which is not predicted by model S-->O acyl transfer reactions. Thus, although UDP-GlcNAc acyltransferase catalyzes the first unique step of lipid A biosynthesis, it is the second enzyme (the deacetylase) that commits the substrates to this pathway. The specific activity of the deacetylase is elevated approximately 5-fold when lipid A synthesis is inhibited.