Nanomolar, Noncovalent Antagonism of Hedgehog Cholesterolysis: Exception to the "Irreversibility Rule" for Protein Autoprocessing Inhibition.
Nanomolar, Noncovalent Antagonism of Hedgehog Cholesterolysis: Exception to the "Irreversibility Rule" for Protein Autoprocessing Inhibition.
复制标题
DOI：
10.1021/acs.biochem.1c00697
复制
发表时间：
2022-06-07
期刊：
影响因子：
2.9
通讯作者：
Callahan, Brian P.
中科院分区：
文献类型：
--
作者：
Wagner, Andrew G.;Stagnitta, Robert T.;Xu, Zihan;Pezzullo, John L.;Kandel, Nabin;Giner, Jose-Luis;Covey, Douglas F.;Wang, Chunyu;Callahan, Brian P.
Hedgehog（Hh）信号配体通过专门的自动加工（称为胆固醇裂解）进行羧基末端立体化。Sterylation是通过一个相邻的酶结构域（称为HhC）在分子内进行的，该酶结构域仅存在于前体Hh蛋白中。以前尝试鉴定HhC分子内活性的拮抗剂已经产生了通过共价机制不可逆地结合HhC的抑制剂，这对于蛋白质自动加工抑制剂是常见的。在这里，我们报告一个例外的“不可逆性规则”的自动加工抑制。使用基于荧光共振能量转移的HhC活性测定，我们筛选了非共价抑制剂的甾醇类似物的集中库，并鉴定和验证了四种结构相关的分子，然后将其用于结构活性关系研究。最有效的衍生物tBT-HBT以300 nM的IC 50非共价方式抑制HhC。一个变构结合位点tBT-HBT，包括残基从HhC的两个亚结构域，建议通过动力学分析，诱变研究，和光亲和标记。这里描述的抑制剂类似于我们先前描述的HhC对位催化的非共价变构诱导剂家族。抑制和诱导似乎是由HhC上的一个共享的变构位点介导的。
Hedgehog (Hh) signaling ligands undergo carboxy terminal sterylation through specialized autoprocessing, called cholesterolysis. Sterylation is brought about intramolecularly in a single turnover by an adjacent enzymatic domain, called HhC, which is found in precursor Hh proteins only. Previous attempts to identify antagonists of the intramolecular activity of HhC have yielded inhibitors that bind HhC irreversibly through covalent mechanisms, as is common for protein autoprocessing inhibitors. Here, we report an exception to the “irreversibility rule” for autoprocessing inhibition. Using a fluorescence resonance energy transfer-based activity assay for HhC, we screened a focused library of sterol-like analogues for noncovalent inhibitors and identified and validated four structurally related molecules, which were then used for structure−activity relationship studies. The most effective derivative, tBT-HBT, inhibits HhC noncovalently with an IC50 of 300 nM. An allosteric binding site for tBT-HBT, encompassing residues from the two subdomains of HhC, is suggested by kinetic analysis, mutagenesis studies, and photoaffinity labeling. The inhibitors described here resemble a family of noncovalent, allosteric inducers of HhC paracatalysis which we have described previously. The inhibition and the induction appear to be mediated by a shared allosteric site on HhC.
登录
查看更多内容
影响因子：
3.7
作者：
Buglino JA;Resh MD
通讯作者：
Resh MD
影响因子：
15
作者：
Kirby, AJ;Dutta-Roy, N;Nome, F
通讯作者：
Nome, F
影响因子：
4.8
作者：
Chan, Hon;Pearson, C. Seth;Belfort, Marlene
通讯作者：
Belfort, Marlene
影响因子：
64.5
作者：
Hall, TMT;Porter, JA;Leahy, DJ
通讯作者：
Leahy, DJ
DOI：
10.1083/jcb.201008090
发表时间：
2011-03-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Chen X;Tukachinsky H;Huang CH;Jao C;Chu YR;Tang HY;Mueller B;Schulman S;Rapoport TA;Salic A
通讯作者：
Salic A