细胞NAD+在静止条件下连续降解和合成。在哺乳动物中,NAD+的合成主要是由烟酰胺(Nam)启动的Nam磷酸核糖基转移酶,而聚(ADP-核糖)聚合酶1(PARP1)和2(PARP2),sirtuin1(SIRT1),CD38和含有1(SARM1)的无菌α和TIR基序参与NAD+分解。使用2H标记的Nam的通量分析,我们发现,当哺乳动物细胞在没有Nam的情况下培养时,细胞NAD+水平得以维持,NAD+分解被完全抑制。在Nam面前,NAD+分解率(RB)在PARP1、PARP2、SIRT1或SARM1缺失时没有显著变化,而CD38的稳定表达并没有增加RB。然而,与野生型细胞相比,PARP1缺失细胞中的RB高得多,其中PARP1活性被选择性抑制剂阻断。相比之下,与对照细胞相比,在特异性CD38抑制剂存在下,CD38过表达细胞中的RB要低得多。结果表明,PARP1缺失上调了其他NAD酶的活性,而CD38表达下调内源性NAD酶的活性,包括PARP1和PARP2。细胞NAD+分解的速率和产生的NAD+浓度可以保持在恒定水平。尽管NAD+降解酶表达发生变化,通过对NAD酶活性的补偿调节。
Cellular NAD+ is continuously degraded and synthesized under resting conditions. In mammals, NAD+ synthesis is primarily initiated from nicotinamide (Nam) by Nam phosphoribosyltransferase, whereas poly(ADP-ribose) polymerase 1 (PARP1) and 2 (PARP2), sirtuin1 (SIRT1),
CD38, and sterile alpha and TIR motif containing 1 (SARM1) are involved in NAD+ breakdown. Using flux analysis with 2H-labeled Nam, we found that when mammalian cells were cultured in the absence of Nam, cellular NAD+ levels were maintained and NAD+ breakdown was completely suppressed. In the presence of Nam, the rate of NAD+ breakdown (RB) did not significantly change upon PARP1, PARP2, SIRT1, or SARM1 deletion, whereas stable expression of
CD38 did not increase RB. However, RB in PARP1-deleted cells was much higher compared with that in wild-type cells, in which PARP1 activity was blocked with a selective inhibitor. In contrast, RB in
CD38-overexpressing cells in the presence of a specific
CD38 inhibitor was much lower compared with that in control cells. The results indicate that PARP1 deletion upregulates the activity of other NADases, whereas
CD38 expression downregulates the activity of endogenous NADases, including PARP1 and PARP2. The rate of cellular NAD+ breakdown and the resulting NAD+ concentration may be maintained at a constant level, despite changes in the NAD+-degrading enzyme expression, through the compensatory regulation of NADase activity.