Paillard S, Strauss F
Institut Jacques Monod, Paris, France.
Proteins. 1993 Mar;15(3):330-7. doi: 10.1002/prot.340150310.
Ku protein, a relatively abundant nuclear protein associated with DNA of mammalian cells, is known to be a heterodimer with subunits of 85 and 72 kDa which binds in vitro to DNA ends and subsequently translocates along the molecule. The functional role played by this protein in the cell, however, remains to be elucidated. We have observed here that Ku protein, purified from cultured monkey cells, is the target of specific endoproteolysis in vitro, by which the 85 kDa subunit is cleaved at a precise site while the 72 kDa subunit remains intact. This cleavage releases an 18 kDa polypeptide and converts Ku protein into a heterodimer composed of the 72 kDa subunit associated with a 69 kDa fragment from the 85 kDa subunit. The proteolyzed form of Ku protein, denoted Ku', has DNA binding properties similar to those of Ku protein. The proteolytic mechanism, which is inhibited by leupeptin and chymostatin, is extremely sensitive to ionic conditions, in particular to pH, being very active at pH 7.0 and completely inhibited at pH 8.0. In addition, cleavage occurs only when Ku protein is bound to DNA, not free in solution. We suggest that in vivo, such proteolysis might be necessary for Ku protein function at some stage of the cell cycle.
Ku蛋白是一种与哺乳动物细胞DNA相关的相对丰富的核蛋白，已知它是一种由85 kDa和72 kDa亚基组成的异二聚体，在体外可与DNA末端结合并随后沿分子移位。然而，这种蛋白在细胞中所起的功能作用仍有待阐明。我们在此观察到，从培养的猴细胞中纯化的Ku蛋白是体外特异性内切蛋白酶解的靶标，通过这种酶解，85 kDa亚基在一个精确位点被切割，而72 kDa亚基保持完整。这种切割释放出一个18 kDa的多肽，并将Ku蛋白转化为由72 kDa亚基与来自85 kDa亚基的一个69 kDa片段组成的异二聚体。Ku蛋白的蛋白水解形式（称为Ku'）具有与Ku蛋白相似的DNA结合特性。这种蛋白水解机制受到亮抑蛋白酶肽和抑糜蛋白酶肽的抑制，对离子条件，特别是pH极为敏感，在pH 7.0时非常活跃，在pH 8.0时完全被抑制。此外，切割仅在Ku蛋白与DNA结合时发生，而不是在溶液中游离时发生。我们认为，在体内，这种蛋白水解在细胞周期的某些阶段可能是Ku蛋白发挥功能所必需的。
