Synthetic peptides derived from the prosegments of proprotein convertase 1/3 and furin are potent inhibitors of both enzymes
Synthetic peptides derived from the prosegments of proprotein convertase 1/3 and furin are potent inhibitors of both enzymes
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DOI：
10.1042/bj20030120
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发表时间：
2003-07-01
影响因子：
4.1
通讯作者：
Lazure, C
中科院分区：
文献类型：
--
作者：
Basak, A;Lazure, C
前蛋白转化酶（PC）是枯草杆菌蛋白酶/kexin家族的Ca 2+依赖性丝氨酸蛋白酶，已知其特异性地在R-X-（K/R）-R向下箭头的C-末端切割前肽和前蛋白底物以产生相关的生物活性肽。PC最初合成为无酶活性的酶原形式，其中前片段对相应的酶起重要的抑制作用。在这里，我们研究了是否合成肽来源于前区也可以代表特定的和有效的抑制剂。通过序列比对、二级结构分析和亲水性分析，筛选出8 ~ 33个残基的多肽。这些包括包含原小鼠PC 1/3序列中的残基55-62、50-62、39-62、50-83、55-83、64-83和74-83以及原人弗林蛋白酶序列的残基54-62、48-62和39-62的区段。所有肽均通过固相FastMoc化学制备，通过反相HPLC纯化，并通过MS和氨基酸分析进行表征。体外测试这些肽对重组小鼠PC 1/3和人弗林蛋白酶的抑制活性。进展曲线和结束时间动力学分析表明，这些肽，特别是那些含有初级和二级加工位点，显示出强烈的抑制两种酶的抑制常数（Ki）在高纳摩尔范围内。与整个前肽不同，这些小的合成肽抑制剂根据序列表现出真正的竞争性或混合竞争性抑制。我们的数据进一步揭示了前结构域的最后两个碱性氨基酸残基（例如小鼠PC 1/3序列的Lys（82）-Arg（83））在赋予强的抗转化酶活性中的关键作用。该研究还建立了两种转化酶的前片段内所含的某些区域的抑制潜力。
Proprotein convertases (PCs) are Ca2+-dependent serine proteases of the subtilisin/kexin family which are known specifically to cleave propeptide and proprotein substrates at the C-terminal of R-X-(K/R)-Rdown arrow to generate the relevant biologically active peptides. PCs are initially synthesized as enzymically inactive proenzyme forms where the prosegments play an important inhibitory role to the respective enzymes. Here we investigated whether synthetic peptides derived from the pro-region could also represent specific and potent inhibitors. Based upon sequence alignment, secondary structure analysis and hydrophilicity plot, a number of peptides ranging from 8 to 33 residues were selected. These included segments encompassing residues 55-62, 50-62, 39-62, 50-83, 55-83, 64-83 and 74-83 in the pro-mouse PC1/3 sequence and residues 54-62, 48-62 and 39-62 of the pro-human furin sequence. All peptides were prepared by solid-phase FastMoc chemistry, purified by reversed-phase HPLC and characterized by MS and amino acid analysis. These peptides were tested in vitro for inhibitory activity towards recombinant mouse PC1/3 and human furin. Progress-curve and end-time kinetic analysis demonstrated that a number of these peptides, particularly those containing both the primary and the secondary processing sites, displayed strong inhibition of both enzymes with inhibition constants (K-i) in the high nanomolar range. Unlike the whole propeptide, these small synthetic peptide inhibitors exhibited either true competitive or mixed competitive inhibition, depending on the sequence. Our data revealed further the critical role of the last two basic amino acid residues (e.g. Lys(82)-Arg(83) for the mouse PC1/3 sequence) of the prodomain in imparting a strong anti-convertase activity. The study also establishes the inhibitory potential of certain regions contained within the prosegment of the two convertases.