Zhang Xizi, Cattoglio Claudia, Zoltek Madeline, Vetralla Carlo, Mozumdar Deepto, Schepartz Alanna
Department of Chemistry, University of California, Berkeley, California 94720, United States.
Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720, United States.
ACS Cent Sci. 2023 Feb 3;9(2):277-288. doi: 10.1021/acscentsci.2c01226. eCollection 2023 Feb 22.
The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic acids fail to efficiently cross cell or endosomal membranes. The impact of biologic-based therapeutics would expand exponentially if proteins and nucleic acids could reliably evade endosomal degradation, escape endosomal vesicles, and remain functional. Using the cell-permeant mini-protein ZF5.3, here we report the efficient nuclear delivery of functional Methyl-CpG-binding-protein 2 (MeCP2), a transcriptional regulator whose mutation causes Rett syndrome (RTT). We report that ZF-MeCP2, a conjugate of ZF5.3 and MeCP2(Δaa13-71, 313-484), binds DNA in a methylation-dependent manner , and reaches the nucleus of model cell lines intact to achieve an average concentration of 700 nM. When delivered to live cells, ZF-MeCP2 engages the NCoR/SMRT corepressor complex, selectively represses transcription from methylated promoters, and colocalizes with heterochromatin in mouse primary cortical neurons. We also report that efficient nuclear delivery of ZF-MeCP2 relies on an endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-MeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of biologic-based therapeutics.
绝大多数基于生物制剂的疗法在血清中、细胞表面或内吞小泡内发挥作用，这在很大程度上是因为蛋白质和核酸无法有效穿过细胞膜或内体膜。如果蛋白质和核酸能够可靠地避免内体降解、逃离内体小泡并保持功能，那么基于生物制剂的疗法的影响将呈指数级扩大。利用细胞穿透性微型蛋白ZF5.3，我们在此报告了功能性甲基化CpG结合蛋白2（MeCP2）的有效核递送，MeCP2是一种转录调节因子，其突变会导致雷特综合征（RTT）。我们报告称，ZF-MeCP2是ZF5.3与MeCP2（Δaa13 - 71，313 - 484）的缀合物，以甲基化依赖的方式结合DNA，并完整地到达模型细胞系的细胞核，平均浓度达到700 nM。当递送至活细胞时，ZF-MeCP2与NCoR/SMRT共抑制复合物结合，选择性抑制甲基化启动子的转录，并与小鼠原代皮质神经元中的异染色质共定位。我们还报告称，ZF-MeCP2的有效核递送依赖于由HOPS依赖的内体融合提供的内体逃逸通道。为作比较而评估的MeCP2的Tat缀合物（Tat-MeCP2）在细胞核内被降解，对甲基化启动子没有选择性，并且以不依赖HOPS的方式运输。这些结果支持了利用细胞穿透性微型蛋白ZF5.3通过HOPS依赖通道将功能性大分子递送至细胞内部的可行性。这样的策略可能会扩大多个基于生物制剂的治疗家族的影响。
