In Vitro Evolution of Ligands to the Membrane Protein Caveolin
In Vitro Evolution of Ligands to the Membrane Protein Caveolin
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DOI：
10.1021/ja201792q
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发表时间：
2011-06-29
影响因子：
15
通讯作者：
Weiss, Gregory A.
中科院分区：
文献类型：
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作者：
Majumdar, Sudipta;Hajduczki, Agnes;Weiss, Gregory A.
膜蛋白占人类基因组的三分之一，但对反向化学遗传学来说，这是一个具有挑战性的目标。例如，尽管膜蛋白caveolin-1与包括多发性骨髓瘤在内的许多疾病有关，但由于其大部分未知的结构和不溶性，它似乎为发现合成配体提供了一个很差的靶标。为了打破这一僵局并确定控制先导化合物的新类小泡，我们应用基于噬菌体的反向化学遗传学方法发现了来自抗hiv治疗药物T20的小泡蛋白-1配体。将同源残基替换到T20序列中使用了类似于药物化学的过程，以使结合小窝蛋白的亲和力成熟。与野生型T20相比，与>结合的溶洞蛋白-1配体的亲和力提高了1000倍。两种类型的elisa和等温滴定量热法（ITC）测量表明，T20变体与溶洞olin具有高亲和力结合，K(d)值在150 nM范围内。使用亲和性最高的小窝蛋白配体进行显微镜实验，证实了这些配体与内源性小窝蛋白在NIH 3T3细胞中的共定位。该结果为靶向小泡蛋白及小泡在活细胞中的形成奠定了基础。
Membrane proteins comprise a third of the human genome, yet present challenging targets for reverse chemical genetics. For example, although implicated in numerous diseases including multiple myeloma, the membrane protein caveolin-1 appears to offer a poor target for the discovery of synthetic ligands due to its largely unknown structure and insolubility. To break this impasse and identify new classes of caveolae controlling lead compounds, we applied phage-based, reverse chemical genetics for the discovery of caveolin-1 ligands derived from the anti-HIV therapeutic T20. Substitution of homologous residues into the T20 sequence used a process analogous to medicinal chemistry for the affinity maturation to bind caveolin. The resultant caveolin-1 ligands bound with >1000-fold higher affinity than wild-type T20. Two types of ELISAs and isothermal titration calorimetry (ITC) measurements demonstrated high affinity binding to cave olin by the T20 variants with K(d) values in the 150 nM range. Microscopy experiments with the highest affinity caveolin ligands confirmed colocalization of the ligands with endogenous caveolin in NIH 3T3 cells. The results establish the foundation for targeting caveolin and caveolae formation in living cells.