EVIDENCE FOR INVOLVEMENT OF PROTEASOME COMPLEX

白细胞介素-1（IL-1）被认为是一种效应分子， 自身免疫性糖尿病中β-细胞破坏。 IL-1抑制胰岛素 通过刺激胰腺β细胞的表达， 产生一氧化氮的诱导型一氧化氮合酶（iNOS）。 IL-1还诱导IL-1的诱导型同种型的共表达。 环氧合酶（考克斯-2）与促炎性因子过度产生 拟南芥素和它们的前体花生四烯酸， 同位素稀释质谱法 目前的研究 表征蛋白酶参与信号通路 IL-1诱导的iNOS和考克斯-2表达。 生化和分子 使用大鼠胰岛素瘤β细胞系RINm 5 F进行研究。 一种丝氨酸蛋白酶抑制剂，N（-对甲苯磺酰基-L-赖氨酸氯甲基酮 （TLCK）和蛋白酶体复合物（26 S）抑制剂MG 132可抑制 IL-1诱导的一氧化氮氧化产物亚硝酸盐的形成 由iNOS以浓度依赖性方式产生。 TLCK和MG 132也抑制了iNOS基因表达。 mRNA和蛋白质水平。 TLCK和MG 132对IL-1也有抑制作用 诱导的考克斯-2酶活性（PGE 2形成）和考克斯-2基因 在mRNA和蛋白质水平表达。 在人类胰岛中， 蛋白酶体抑制剂MG 132也抑制了 iNOS和考克斯-2酶活性产物、亚硝酸盐和PGE 2 分别 这些发现表明， TLCK和MG 132对iNOS和考克斯-2表达的影响先于转录。 转录因子NF（B）是激活转录因子A的必需因子。 的数量，并被评估为可能的 IL-1诱导共表达所必需的蛋白酶作用位点 iNOS和考克斯-2。 TLCK和MG 132均抑制IL-1诱导的IL-12表达。 通过胰岛和RINm 5 F细胞活化NF β B和降解1 β B。 这些结果暗示蛋白酶激活作为早期信号 IL-1诱导的β-细胞功能抑制事件。

Interleukin-1( (IL-1) has been implicated as an effector molecule of (-cell destruction in autoimmune diabetes. IL-1 inhibits insulin secretion from pancreatic (-cells by stimulating the expression of inducible nitic oxice synthase (iNOS) that generates nitric oxide. IL-1 also induces co-expression of the inducible isoform of cyclooxygenase (COX-2) and overproduction of proinflammatory prostaglandins and their precursor arachidonic acid, as demonstrated by isotope dilution mass spectrometry. The current studies characterize the involvement of protease(s) in the signaling pathway of IL-1-induced iNOS and COX-2 expression. Biochemical and molecular studies were performed using the rat insulinoma (-cell line, RINm5F. A serine protease inhibitor, N(-p-tosyl-L-lysine chloromethyl ketone (TLCK) and a proteasome complex (26S) inhibitor, MG 132, inhibited Il-1-induced nitrite formation, an oxidation product of nitric oxide produced by iNOS, in a concentration-dependen t manner. Both TLCK and MG 132 also inhibited iNOS gene expression at the level of mRNA and protein. TLCK and MG 132 also inhibited IL-1 induced COX-2 enzyme activity (PGE2 formation) and COX-2 gene expression at the level of mRNA and protein. In human islets, the proteasome inhibitor, MG 132, also inhibited the formation of the products of iNOS and COX-2 enzyme activity, nitrite and PGE2 respectively. These findings suggested that the inhibitory action of TLCK and MG 132 on iNOS and COX-2 expression precedes transcription. The transcription factor, NF(B, is essential for activation of a number of cytokine-inducible enzymes and was evaluated as a possible site of protease action necessary for IL-1 induced co-expression of iNOS and COX-2. TLCK and MG 132 inhibited both IL-1 induced activation of NF(B and degradatin of 1(B( by islets and RINm5F cells. These results implicate protease activation as an early signaling event in IL-1-induced inhibition of (-cell function.