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首页> 外文期刊>Journal of Cellular Physiology >Ubiquitinated aldolase B accumulates during starvation-induced lysosomal proteolysis.
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Ubiquitinated aldolase B accumulates during starvation-induced lysosomal proteolysis.

机译：Ubiquitinated醛缩酶B中积累starvation-induced溶酶体蛋白水解作用。

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We have previously shown that stress-induced protein degradation requires a functional ubiquitin-activating enzyme and the autophagic-lysosomal pathway. In this study, we examined the occurrence of ubiquitin-protein conjugates that form during nutrient starvation. Kidney and liver epithelial cells respond to nutrient stress by enhancing autophagy and protein degradation. We have shown that this degradative response was more dramatic in nondividing cultures. In addition, the onset of autophagy was suppressed by pactamycin, cycloheximide, and puromycin. We observed an accumulation of ubiquitinated proteins coincident with the degradative response to amino acid starvation. The stress-induced protein ubiquitination was not affected by cycloheximide, indicating that protein synthesis was not required. The ubiquitinated proteins were localized to the cytosol and subcellular fractions enriched with autophagosomes and lysosomes. The incorporation of the ubiquitinated proteins into autolysosomes was dramatically reduced by 3-methyladenine, an inhibitor of autophagy. The evidence suggests that ubiquitinated proteins are sequestered by autophagy for degradation. We next set out to identify those primary ubiquitinated proteins at 60 kDa and 68 kDa. Polyclonal antibodies were prepared against these proteins that had been immunopurified from rat liver lysosomes. The antibodies prepared against those 68 kDa proteins also recognized a 40 kDa protein in cytosolic fractions. Internal amino acid sequences obtained from two cyanogen bromide fragments of this 40 kDa protein were shown to be identical to sequences in liver fructose1,6-bisphosphate aldolase B. Anti-Ub68 antibodies recognized purified aldolase A and aldolase B. Conversely, antibodies prepared against aldolase B recognized the 40 kDa aldolase as well as four to five high molecular weight forms, including a 68 kDa protein. Finally, we have shown that the degradation of aldolase B was enhanced during amino acid and serum starvation. This degradation was suppressed by chloroquine and 3-methyladenine, suggesting that aldolase B was being degraded within autolysosomes. We propose that aldolase B is ubiquitinated within the cytosol and then transported into autophagosomes and autolysosomes for degradation during nutrient stress.
机译：我们先前已经表明,应激蛋白质降解需要功能ubiquitin-activating酶和autophagic-lysosomal途径。检查ubiquitin-protein的发生轭合物形成期间营养饥饿。肾和肝上皮细胞响应营养加强自噬和压力蛋白质降解。使下降的反应是更戏剧性的:文化。自噬被pactamycin压制,环己酰亚胺和嘌呤霉素。积累ubiquitinated蛋白质重合与氨基酸的降解反应饥饿。泛素化不受环己酰亚胺,表明蛋白质合成并不是必需的。本地化细胞溶质和亚细胞分数丰富与自噬小体溶酶体。蛋白自溶酶体是戏剧性的减少3-methyladenine的抑制剂自噬。ubiquitinated蛋白质是隔离的自噬降解。识别这些主要ubiquitinated蛋白质60 kDa, 68 kDa。准备对这些蛋白质immunopurified从鼠肝脏溶酶体。抗体准备对这68 kDa蛋白质也认识到一个40 kDa蛋白在胞质分数。从两个溴化氰40的碎片kDa蛋白质被证明是相同的序列在肝脏fructose1 6-bisphosphate醛缩酶b Anti-Ub68抗体识别纯化醛缩酶A和醛缩酶b。相反,抗体制备与醛缩酶B认可40 kDa醛缩酶以及四到五高分子量的形式,包括一个68 kDa蛋白质。醛缩酶B的降解中增强氨基酸和血清饥饿。抑制了氯喹和3-methyladenine,表明醛缩酶B自吞噬泡内被降解。醛缩酶B是ubiquitinated内胞质,然后运送到自噬小体在营养和自溶酶体降解压力。

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