Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats
Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats
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DOI：
10.1172/jci7300
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发表时间：
1999-11-01
影响因子：
15.9
通讯作者：
Goldberg, AL
中科院分区：
文献类型：
--
作者：
Lecker, SH;Solomon, V;Goldberg, AL
胰岛素缺乏（如急性糖尿病或空腹）与骨骼肌中蛋白质分解增强导致肌肉萎缩有关。由于最近的研究表明，这种增加的蛋白质水解是由于泛素-蛋白酶体（Ub-蛋白酶体）途径的激活，我们研究了糖尿病是否与Ub结合到肌肉蛋白的速度增加有关。链脲佐菌素诱导的胰岛素缺乏大鼠的肌肉提取物比对照动物的提取物含有更多的Ub结合蛋白，并且I-125- Ub与内源性肌肉蛋白的结合率高出40-50%。这种增强的ub偶联主要通过涉及E2（14k）和E3 α的n端规则通路发生。该途径的特定底物α -乳清蛋白在糖尿病提取物中被更快地泛素化，而E214k的显性阴性形式抑制了泛素化率的增加。E2（14k）和E3 α都被证明对Ub偶联具有限速作用，因为在提取物中加入少量的任何一种都能刺激Ub偶联。此外，E2（14k）和E3 α （E1除外）mRNA在糖尿病大鼠肌肉中升高了2倍，尽管免疫印迹或活性测定未检测到E2（14k）和E3 α含量的显著增加。对这些结果最简单的解释是，胰岛素缺乏动物肌肉中E2（14k）和E3 α的少量增加共同加速了n端规则途径的Ub结合和蛋白质降解，该途径在癌症恶病质、败血症和甲状腺功能亢进中被激活。
Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of I-125- Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3 alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E214k inhibited this increase in ubiquitination rates. Both E2(14k) and E3 alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3 alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3 alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3 alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.