Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.

富含亮氨酸的 alpha-2-glycoprotein-1 (LRG1) 已被证明与细胞凋亡激活因子-1 (Apaf-1) 竞争结合细胞色素c (Cyt c ) 并可能在抑制细胞凋亡中发挥作用。使用 MCF-7 乳腺癌细胞，我们报告细胞内 LRG1 确实可以防止细胞凋亡。因此，转染lrg1基因并表达更高水平LRG1的细胞比亲代细胞更能抵抗过氧化氢诱导的细胞凋亡，而LRG mRNA被短发夹（sh）RNA诱导的降解敲低的细胞更敏感。凋亡细胞胞质溶胶中与 Apaf-1 共免疫沉淀的 Cyt c量与 LRG1 表达水平呈负相关。在lrg1转染的细胞在胞质溶胶中发现了部分糖基化的 LRG1，并且相对于亲本细胞，在转染 lrg1的活细胞中胞质 Cyt c增加。然而，细胞凋亡不是自发诱导的，因为 Cyt c与 LRG1 而不是 Apaf-1 结合。Cyt c是唯一可检测到的与 LRG1 共免疫沉淀的蛋白质。在过氧化氢处理后，LRG1 的降解允许诱导细胞凋亡。我们提出细胞内 LRG1 提高了诱导细胞凋亡所需的细胞质 Cyt c的阈值，因此，阻止了细胞内 Cyt c的内在途径的发生。线粒体的释放不是由承诺的凋亡信号引起的。LRG1 提供的这种生存机制可能不同于几个研究小组报告的其细胞外生存功能。