An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine
An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine
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DOI：
10.1074/jbc.c117.800136
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发表时间：
2017-08-18
影响因子：
4.8
通讯作者：
Resh, Marilyn D.
中科院分区：
文献类型：
--
作者：
Asciolla, James J.;Miele, Matthew M.;Resh, Marilyn D.
Wnt蛋白是一个分泌性信号蛋白家族，在调节胚胎和成体组织中的细胞增殖中发挥关键作用。活性Wnt的产生取决于棕榈油酸酯（一种单不饱和脂肪酸）通过酰基转移酶Porcupine（PORCN）与保守丝氨酸的连接。PORCN活性的研究依赖于基于细胞的脂肪酰化和信号传导测定，因为尚未开发直接的酶测定。在这里，我们提出了第一个体外试验，准确地概括PORN介导的脂肪酰化的Wnt底物。关键特征是使用双二硫键结合的Wnt肽，其模拟Wnt酰化位点周围的二维结构。当Wnt肽用DTT处理时，PORCN介导的Wnt酰化被消除，并且当线性（非二硫键）肽或双二硫键结合的Wnt肽含有Ala取代Ser酰化位点时，PORCN介导的Wnt酰化不发生。我们利用这种体外Wnt酰化测定来提供直接证据，即用于管理Wnt驱动的肿瘤的临床试验中的小分子LGK 974是真正的PORCN抑制剂，其体外抑制Wnt脂肪酰化的IC 50与抑制Wnt信号传导的IC 50密切匹配。并排比较PORCN和刺猬酰基转移酶（HHAT），两种将16-碳脂肪酸连接到分泌蛋白质的酶，揭示了两种酶都不会接受另一种的脂肪酰辅酶A或肽底物。这些发现说明了独特的酶底物的选择性所表现出的膜结合的O-酰基转移酶家族的成员。
Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O-acyl transferase family.