Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells.
Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells.
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在培养细胞中的合成和拓扑的动力学方面，crap和细胞prion蛋白不同。
DOI：
10.1083/jcb.110.3.743
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发表时间：
1990-03
期刊：
影响因子：
--
通讯作者：
Prusiner SB
中科院分区：
文献类型：
--
作者：
Borchelt DR;Scott M;Taraboulos A;Stahl N;Prusiner SB
PrP的细胞和痒病亚型PrPc和PrPSc都由一个单拷贝染色体基因编码，似乎是从相同的2.1 kb mRNA翻译而来。在PrPC被水解和PrPSc具有抗性的条件下，PrPC可以通过有限的蛋白水解来与PrPSc区分。我们在这里报道，PrPC可以通过磷脂酰肌醇特异性磷脂酶C （PIPLC）消化从正常对照和瘙痒病感染的小鼠神经母细胞瘤（N2a）细胞表面释放出来，并且可以选择性地用磺胺生物素（一种膜非必需试剂）标记。相比之下，PrPSc既没有被PIPLC释放，也没有被标记为磺胺生物素。脉冲追逐实验表明，[35S]蛋氨酸几乎立即与PrPC结合，而仅在追逐期间才与PrPSc分子结合。PrPC的合成和降解相对较快（t1/2约为5 h），而PrPSc的合成较慢（t1/2约为15 h），且呈积累状。这些结果与之前在啮齿动物大脑中观察到的PrP mRNA和PrPC水平在整个痒病感染过程中没有变化，但PrPSc积累的水平超过了PrPC。我们的动力学研究表明，PrPSc来源于蛋白酶敏感前体，蛋白酶K抗性的获得是翻译后事件的结果。作为朊病毒疾病的主要特征，潜伏期延长是否反映了PrPSc合成缓慢仍有待确定。
Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie- infected murine neuroblastoma (N2a) cells by phosphatidylinositol- specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo- NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.