Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
Biochem J. 2011 Jul 15;437(2):313-22. doi: 10.1042/BJ20110226.
Granzyme-mediated cell death is the main pathway for cytotoxic lymphocytes to kill virus-infected and tumour cells. A major player in this process is GrB (granzyme B), which triggers apoptosis in both caspase-dependent and caspase-independent pathways. A caspase-independent substrate of GrB is the highly conserved transmembrane receptor Notch1. The GrB cleavage sites in Notch1 and functional consequences of Notch1 cleavage by GrB were unknown. In the present study, we confirmed that Notch1 is a direct and caspase-independent substrate of GrB. We demonstrate that GrB cleaved the intracellular Notch1 domain at least twice at two distinct aspartic acids, Asp1860 and Asp1961. GrB cleavage of Notch1 can occur in all subcellular compartments, during maturation of the receptor, at the membrane, and in the nucleus. GrB also displayed perforin-independent functions by cleaving the extracellular domain of Notch1. Overall, cleavage of Notch1 by GrB resulted in a loss of transcriptional activity, independent of Notch1 activation. We conclude that GrB disables Notch1 function, probably resulting in anti-cellular proliferation and cell death signals.
颗粒酶介导的细胞死亡是细胞毒性淋巴细胞杀伤病毒感染和肿瘤细胞的主要途径。在这个过程中，GrB（颗粒酶 B）是一个主要的参与者，它在依赖半胱天冬酶和不依赖半胱天冬酶的途径中触发细胞凋亡。GrB 的一个不依赖半胱天冬酶的底物是高度保守的跨膜受体 Notch1。Notch1 的 GrB 切割位点和 GrB 切割 Notch1 的功能后果尚不清楚。在本研究中，我们证实 Notch1 是 GrB 的直接和不依赖半胱天冬酶的底物。我们证明 GrB 在两个不同的天冬氨酸残基，Asp1860 和 Asp1961 处至少两次切割了细胞内的 Notch1 结构域。GrB 对 Notch1 的切割可以发生在所有亚细胞区室中，包括受体成熟过程中、在膜上以及在核内。GrB 还通过切割 Notch1 的细胞外结构域表现出穿孔素非依赖性功能。总的来说，GrB 对 Notch1 的切割导致转录活性丧失，而不依赖于 Notch1 的激活。我们得出结论，GrB 使 Notch1 失活，可能导致抗细胞增殖和细胞死亡信号。
