Using substrate specificity of antiplasmin-cleaving enzyme for fibroblast activation protein inhibitor design.
Using substrate specificity of antiplasmin-cleaving enzyme for fibroblast activation protein inhibitor design.
复制标题
DOI：
10.1021/bi900257m
复制
发表时间：
2009-06-16
期刊：
影响因子：
2.9
通讯作者：
McKee, Patrick A.
中科院分区：
文献类型：
--
作者：
Lee, Kyung N.;Jackson, Kenneth W.;Terzyan, Simon;Christiansen, Victoria J.;McKee, Patrick A.
循环抗纤溶蛋白切割酶（APCE）是一种脯氨酸特异性丝氨酸蛋白酶，本质上与细胞膜插入成纤维细胞激活蛋白（FAP）相同，FAP在上皮源性癌症生长过程中短暂表达。人前体α2-抗纤溶酶（Met-α2AP）是APCE唯一已知的生理底物，它在n端裂解为Asn-α2AP，该蛋白与纤维蛋白迅速交联，并保护其不被纤溶酶消化。鉴定APCE/FAP特异性抑制剂的研究仍在继续。重组FAP对可剪切键- pro12 - asn13 -周围的短氨基酸序列的肽库进行了切割，表明需要P2 Gly和P1 Pro，正如我们在APCE中发现的那样。我们检测了P4-P4 '肽的切割，在每个位置上使用19个氨基酸取代，并选择了P8-P5中的氨基酸取代。对肽底物的Km值测定表明，P7 Arg对APCE的亲和力最高。在P6中，Arg增加了多肽的切割速率，而在P5或天然P7中则没有。在P4或P8中放置精氨酸显著降低了解理速率。在可剪切键之前或之后对延伸肽序列的底物进行切割表明，APCE的内肽酶活性优于二肽酶活性。底物类似物抑制剂phe1 - arg -（8-氨基-3,6-二草辛酸）- gly -[r]-氟吡咯烷醚抑制APCE的Ki值为54 μM，但在2 mM处对二肽基肽酶IV没有抑制作用。该抑制剂还阻断Met-α 2ap的裂解，IC50值为91 μM。在与氟吡咯烷素的距离与P7相同的位置用Gly取代Arg，使其对APCE的抑制作用降低了10倍。结果表明，距离P1的P5、P6或P7位点的Arg增强了底物或抑制剂对APCE或FAP的亲和力和效率。
Circulating antiplasmin-cleaving enzyme (APCE), a prolyl-specific serine proteinase, is essentially identical to membrane-inserted fibroblast activation protein (FAP) that is transiently expressed during epithelial-derived cancer growth. Human precursive α2-antiplasmin (Met-α2AP), the only known physiologic substrate for APCE, is cleaved N-terminally to Asn-α2AP that is rapidly cross-linked to fibrin and protects it from digestion by plasmin. Identifying a specific inhibitor of APCE/FAP continues to be intensely pursued. Recombinant FAP cleavage of peptide libraries of short amino acid sequences surrounding the scissile bond, -Pro12-Asn13-, indicated that P2 Gly and P1 Pro are required, just as we found for APCE. We examined cleavage of P4–P4′ peptides, using 19 amino acid substitutions at each position and selected ones in P8–P5. Km values determined for peptide substrates showed that P7 Arg has the highest affinity for APCE. Peptide cleavage rate increased with Arg in P6 rather than P5 or native P7. Placing Arg in P4 or P8 reduced cleavage rates dramatically. Cleavage of substrates with extended peptide sequences before or after the scissile bond showed endopeptidase to be superior to dipeptidase activity for APCE. A substrate analogue inhibitor, Phe-Arg-(8-amino-3,6-dioxaoctanoic acid)-Gly-[r]-fluoropyrrolidide, inhibited APCE with a Ki of 54 μM but not dipeptidyl peptidase IV even at 2 mM. The inhibitor also blocked cleavage of Met-α2AP with an IC50 of 91 μM. Replacing Arg with Gly at the same distance from fluoropyrrolidide as P7 Arg is from P1 Pro reduced its inhibition of APCE ∼10-fold. Results indicate that Arg at P5, P6, or P7 distances from P1 enhances affinity and efficiency of substrates or inhibitors toward APCE or FAP.
登录
查看更多内容
影响因子：
2.9
作者：
Myles, T;Le Bonniec, BF;Stone, SR
通讯作者：
Stone, SR
影响因子：
11.2
作者：
Chen, Donghai;Kennedy, Alanna;Chen, Wen-Tien
通讯作者：
Chen, Wen-Tien
影响因子：
2.9
作者：
Baggio, R;Shi, YQ;Abeles, RH
通讯作者：
Abeles, RH
影响因子：
6.1
作者：
Cunningham, Charles Casey
通讯作者：
Cunningham, Charles Casey
影响因子：
3.6
作者：
Narra, Kalyani;Mullins, Stefanie R.;Cheng, Jonathan D.
通讯作者：
Cheng, Jonathan D.