Abstract：
Senescence-associated heterochromatin foci (SAHF) is often used as a biological marker for senescent cells, but the regulation of its formation process is unclear. To find a new modulator of SAHF, we screened our chemical small molecules and found 7-amino-2,3,4,5-tetrahedrobenzo[b][1,4] oxazepin-3-ol (ABO) that was identified as an inhibitor of annexin A7 GTPase (ANXA7) dramatically suppressed the aggregation of heterochromatin protein (HP1γ), an indicator of SAHF. To understand its action mechanism, we first observed the changes in the karyoplasmic ratio of ANXA7 because HP1γ mainly located in the nucleus. The results showed that ABO elevated the protein level of ANXA7 in the nucleus. Therefore, we raised a hypothesis that ANXA7 interacted with HP1γ and regulated its phosphorylation, which is closely related to the formation of SAHF. The co-immunoprecipitation and Western blot experiment results showed that ANXA7 had no direct interaction with HP1γ, however, the phosphorylation of HP1γ was increased by ABO, which suggested that ANXA7 indirectly regulated HP1γ phosphorylation. Then, based on our previous discovery of ANXA7 interacting with AMP-activated protein kinase (AMPK), we investigated the effect of the AMPK/mammalian target of rapamycin (mTOR) signaling pathway on ABO-increased phosphorylation of HP1γ. We found that ABO decreased AMPK phosphorylation and increased the phosphorylation level and activity of mTOR. In the presence of an AMPK activator or mTOR inhibitor, ABO could not increase HP1γ phosphorylation. As a result, ABO inhibited the senescence of human dermal fibroblasts (HDFs). In this study, we found that ANXA7 was a new regulator of SAHF, it could regulate the formation of SAHF through the AMPK/mTOR pathway. The data suggested that ABO could be used as a powerful tool to inhibit the replicative senescence of HDFs.
摘要：
衰老相关的异染色质灶（SAHF）通常被用作衰老细胞的生物学标记，但其形成过程的规律尚不清楚。为了找到一种新的SAHF调制器，我们筛选了我们的化学小分子，发现7-氨基-2,3,4,5-四氢苯并[b][1,4]恶氮卓-3-醇（ABO）被鉴定为膜联蛋白A7GTP酶（ANXA7）的抑制剂显着抑制聚集异染色质蛋白（HP1γ），SAHF的指标。为了了解其作用机制，我们首先观察到ANXA7的核质比例的变化，因为HP1γ主要位于细胞核中。结果表明，ABO升高了细胞核中ANXA7的蛋白水平。因此,我们提出了一个假设，即ANXA7与HP1γ相互作用并调节其磷酸化，这与SAHF的形成密切相关。免疫共沉淀和Westernblot实验结果表明,ANXA7与HP1γ无直接相互作用,然而,ABO增加了HP1γ的磷酸化，这表明ANXA7间接调节HP1γ磷酸化。然后，基于我们先前发现的ANXA7与AMP激活的蛋白激酶（AMPK）相互作用，我们研究了AMPK/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路对ABO增加的HP1γ磷酸化的影响。我们发现ABO降低了AMPK的磷酸化，增加了mTOR的磷酸化水平和活性。在存在AMPK激活剂或mTOR抑制剂的情况下，ABO不能增加HP1γ磷酸化。因此,ABO抑制人真皮成纤维细胞(HDFs)的衰老。在这项研究中,我们发现ANXA7是SAHF的新调节剂，它可以通过AMPK/mTOR通路调节SAHF的形成。数据表明，ABO可以用作抑制HDF复制衰老的有力工具。
