Sundararaj Kamala P, Wood Rachel E, Ponnusamy Suriyan, Salas Arelis M, Szulc Zdzislaw, Bielawska Alicia, Obeid Lina M, Hannun Yusuf A, Ogretmen Besim
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2004 Feb 13;279(7):6152-62. doi: 10.1074/jbc.M310549200. Epub 2003 Nov 20.
Ceramide has been demonstrated as one of the upstream regulators of telomerase activity. However, the role for ceramide in the control of telomere length remains unknown. It is shown here that treatment of the A549 human lung adenocarcinoma cells with C(6)-ceramide results in rapid shortening of telomere length. During the examination of ceramide-regulated telomere-binding proteins, nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified to associate with both single- and double-stranded telomeric DNA with high specificity in vitro. The association of nuclear GAPDH with telomeres in interphase nuclei was also demonstrated by co-fluorescence in situ hybridization and chromatin immunoprecipitation analysis. Further data demonstrated that the nuclear localization of GAPDH is regulated by ceramide in a cell cycle-dependent manner parallel with the inhibition of its telomere binding activity in response to ceramide. In addition, the results revealed that nuclear GAPDH is distinct from its cytoplasmic isoform and that telomere binding function of nuclear GAPDH is strikingly higher than the cytoplasmic isoform. More importantly, the functional role for nuclear GAPDH in the maintenance and/or protection of telomeric DNA was identified by partial inhibition of the expression of GAPDH using small interfering RNA, which resulted in rapid shortening of telomeres. In contrast, overexpression of nuclear GAPDH resulted in the protection of telomeric DNA in response to exogenous ceramide as well as in response to anticancer drugs, which have been shown to induce endogenous ceramide levels. Therefore, these results demonstrate a novel function for nuclear GAPDH in the maintenance and/or protection of telomeres and also show that mechanisms of the rapid degradation of telomeres in response to ceramide involve the inhibition of the telomere binding activity of nuclear GAPDH.
神经酰胺已被证明是端粒酶活性的上游调节因子之一。然而，神经酰胺在控制端粒长度方面的作用仍不清楚。本文表明，用C(6)-神经酰胺处理A549人肺腺癌细胞会导致端粒长度迅速缩短。在检测神经酰胺调节的端粒结合蛋白时，发现核甘油醛-3-磷酸脱氢酶（GAPDH）在体外与单链和双链端粒DNA具有高度特异性结合。共荧光原位杂交和染色质免疫沉淀分析也证实了间期细胞核中核GAPDH与端粒的结合。进一步的数据表明，GAPDH的核定位受神经酰胺以细胞周期依赖性方式调节，这与它响应神经酰胺时端粒结合活性的抑制是平行的。此外，结果显示核GAPDH与其细胞质异构体不同，并且核GAPDH的端粒结合功能明显高于细胞质异构体。更重要的是，通过使用小干扰RNA部分抑制GAPDH的表达，确定了核GAPDH在维持和/或保护端粒DNA中的功能作用，这导致了端粒的迅速缩短。相反，核GAPDH的过表达导致在响应外源性神经酰胺以及响应已被证明可诱导内源性神经酰胺水平的抗癌药物时对端粒DNA的保护。因此，这些结果证明了核GAPDH在维持和/或保护端粒方面的新功能，并且还表明响应神经酰胺时端粒快速降解的机制涉及对核GAPDH端粒结合活性的抑制。
