Overzet K, Gensler T J, Kim S J, Geiger M E, van Venrooij W J, Pollard K M, Anderson P, Utz P J
Denver Arthritis Clinic, Colorado, USA.
Arthritis Rheum. 2000 Jun;43(6):1327-36. doi: 10.1002/1529-0131(200006)43:6<1327::AID-ANR15>3.0.CO;2-S.
Proteins that are phosphorylated during apoptosis are commonly precipitated by autoantibodies found in the sera of patients with systemic lupus erythematosus. We sought to determine whether scleroderma autoantigens such as small nucleolar RNPs (snoRNP) also associate with phosphoproteins in response to various cellular stressors.
We screened a panel of monoclonal antibodies derived from mice exposed to mercury, a well-characterized murine model of the anti-snoRNP autoimmune response, for the ability to selectively precipitate phosphoproteins from radiolabeled lysates prepared from Jurkat T cells subjected to stressful stimuli.
Monoclonal antibodies reactive with snoRNPs precipitated a phosphoprotein complex (pp42, pp34, and pp23) from lysates prepared from apoptotic cells. Several novel phosphoproteins (pp62 and pp18) were also observed. The phosphorylation and/or recruitment of these proteins to the snoRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, anisomycin, or ultraviolet irradiation), an effect that is blocked by overexpression of Bcl-2. We were unable to demonstrate an association of the phosphoprotein complex with snoRNPs in cells treated with the xenobiotic agent mercury. The snoRNP-associated phosphoprotein complex is composed of serine/arginine (SR) splicing factors, including SRp40.
The association of phosphorylated SR proteins with snoRNPs in cells undergoing apoptosis suggests that the immune response to fibrillarin that characterizes a subset of patients with scleroderma may be related to cell death induced by apoptotic stimuli (e.g., Fas ligation, irradiation, or chemical toxins), or by exposure to mercury.
在细胞凋亡过程中发生磷酸化的蛋白质通常会被系统性红斑狼疮患者血清中的自身抗体沉淀。我们试图确定硬皮病自身抗原，如小核仁核糖核蛋白（snoRNP），是否也会在各种细胞应激源作用下与磷酸化蛋白发生关联。
我们筛选了一组源自暴露于汞的小鼠的单克隆抗体，汞是一种已充分表征的抗snoRNP自身免疫反应的小鼠模型，以检测其从经应激刺激的Jurkat T细胞制备的放射性标记裂解物中选择性沉淀磷酸化蛋白的能力。
与snoRNP反应的单克隆抗体从凋亡细胞制备的裂解物中沉淀出一种磷酸化蛋白复合物（pp42、pp34和pp23）。还观察到了几种新的磷酸化蛋白（pp62和pp18）。这些蛋白向snoRNP复合物的磷酸化和/或募集是由多种凋亡刺激（如Fas连接、茴香霉素或紫外线照射）诱导的，这种效应可被Bcl-2的过表达所阻断。在用外源性物质汞处理的细胞中，我们未能证明磷酸化蛋白复合物与snoRNP有关联。与snoRNP相关的磷酸化蛋白复合物由丝氨酸/精氨酸（SR）剪接因子组成，包括SRp40。
在经历凋亡的细胞中，磷酸化的SR蛋白与snoRNP的关联表明，硬皮病患者亚群所特有的针对纤维蛋白原的免疫反应可能与凋亡刺激（如Fas连接、照射或化学毒素）或汞暴露诱导的细胞死亡有关。
