Knockdown of mu-calpain in Fanconi anemia, FA-A, cells by siRNA restores alphaII spectrin levels and corrects chromosomal instability and defective DNA interstrand cross-link repair.
Knockdown of mu-calpain in Fanconi anemia, FA-A, cells by siRNA restores alphaII spectrin levels and corrects chromosomal instability and defective DNA interstrand cross-link repair.
复制标题
通过 siRNA 敲低范可尼贫血 (FA-A) 细胞中的 mu-钙蛋白酶可恢复 αII 血影蛋白水平，并纠正染色体不稳定性和有缺陷的 DNA 链间交联修复。
DOI：
10.1021/bi100656j
复制
发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Lambert,MurielW
中科院分区：
文献类型：
--
作者：
Zhang,Pan;Sridharan,Deepa;Lambert,MurielW
我们先前已经证明，在范可尼贫血（FA）患者的细胞中，存在结构蛋白非红细胞α血影蛋白（αIISp）的缺陷。这些研究表明，这种缺陷是由于αIISp稳定性降低所致，并与FA细胞中DNA链间交联修复水平降低和染色体不稳定性相关。αIISp稳定性的一个重要因素是其对蛋白酶μ-钙蛋白酶切割的敏感性。我们假设FA细胞中αIISp的μ-钙蛋白酶切割水平增加导致αIISp分解水平增加，并且FA细胞中μ-钙蛋白酶表达敲低应恢复αIISp水平并纠正观察到的许多表型缺陷。结果表明，FA-A、FA-C、FA-D2、FA-F和FA-G细胞中μ-calpain活性增高，可能是这些FA细胞中αIISp缺乏的原因。蛋白质相互作用研究表明FANCA和FANCG直接与μ-钙蛋白酶结合。我们推测这种结合可能导致正常细胞中μ-钙蛋白酶活性的抑制。在FA-A细胞中通过siRNA敲低μ-calpain使αIISp水平恢复到正常水平，并逆转了这些细胞中的许多细胞缺陷。它纠正了DNA修复缺陷和暴露于DNA链间交联剂后观察到的染色体不稳定性。这些研究表明，FA蛋白可能通过调节μ-calpain对αIISp的切割，在维持αIISp在细胞内的稳定性方面发挥重要作用。因此，通过降低FA细胞中αIISp的分解水平，我们可能能够逆转在这种疾病中观察到的许多细胞缺陷。
We have previously shown that there is a deficiency in the structural protein, nonerythroid α spectrin (αIISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of αIISp and correlates with a decreased level of repair of DNA interstrand cross-links and chromosomal instability in FA cells. An important factor in the stability of αIISp is its susceptibility to cleavage by the protease, μ-calpain. We hypothesized that an increased level of μ-calpain cleavage of αIISp in FA cells leads to an increased level of breakdown of αIISp and that knocking down expression of μ-calpain in FA cells should restore levels of αIISp and correct a number of the phenotypic defects observed. The results showed that there is increased μ-calpain activity in FA-A, FA-C, FA-D2, FA-F, and FA-G cells that could account for the deficiency in αIISp in these FA cells. Protein interaction studies indicated that FANCA and FANCG bind directly to μ-calpain. We hypothesize that this binding may lead to inhibition of μ-calpain activity in normal cells. Knocking down μ-calpain by siRNA in FA-A cells restored levels of αIISp to normal and reversed a number of the cellular deficiencies in these cells. It corrected the DNA repair defect and the chromosomal instability observed after exposure to a DNA interstrand cross-linking agent. These studies indicate that FA proteins may play an important role in maintaining the stability of αIISp in the cell by regulating its cleavage by μ-calpain. Thus, by reducing the level of breakdown of αIISp in FA cells, we may be able to reverse a number of the cellular deficiencies observed in this disorder.