Ubiquitinated aldolase B accumulates during starvation-induced lysosomal proteolysis.
Ubiquitinated aldolase B accumulates during starvation-induced lysosomal proteolysis.
复制标题
泛素化醛缩酶 B 在饥饿诱导的溶酶体蛋白水解过程中积累。
DOI：
10.1002/(sici)1097-4652(199901)178:1
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发表时间：
1999
影响因子：
5.6
通讯作者：
DunnJr,WA
中科院分区：
文献类型：
--
作者：
Lenk,SE;Susan,PP;Hickson,I;Jasionowski,T;DunnJr,WA
我们之前已经表明，应激诱导的蛋白质降解需要功能性泛素活化酶和自噬-溶酶体途径。在这项研究中，我们研究了在营养饥饿期间形成的泛素-蛋白质缀合物的发生。肾脏和肝脏上皮细胞通过增强自噬和蛋白质降解来响应营养应激。我们已经证明，这种降解反应在非分裂培养物中更为显著。此外，自噬的发生被抑制由pactamycin，放线菌酮，嘌呤霉素。我们观察到一个积累的泛素化蛋白质一致的降解反应氨基酸饥饿。应激诱导的蛋白质泛素化不受放线菌酮的影响，表明蛋白质合成是不需要的。泛素化的蛋白质定位于细胞质和亚细胞组分丰富的自噬体和溶酶体。自噬抑制剂3-甲基腺嘌呤显著减少了泛素化蛋白质掺入自溶酶体中。有证据表明，泛素化的蛋白质被自噬隔离降解。接下来，我们着手鉴定60 kDa和68 kDa的主要泛素化蛋白。制备了针对这些蛋白质的多克隆抗体，这些蛋白质已从大鼠肝溶酶体中免疫纯化。针对那些68 kDa蛋白质制备的抗体也识别胞质组分中的40 kDa蛋白质。从该40 kDa蛋白质的两个溴化氰片段获得的内部氨基酸序列与肝脏果糖-1，6-二磷酸醛缩酶B中的序列相同。抗Ub 68抗体识别纯化的醛缩酶A和醛缩酶B。相反，针对醛缩酶B制备的抗体识别40 kDa的醛缩酶以及4至5种高分子量形式，包括68 kDa的蛋白质。最后，我们已经表明，在氨基酸和血清饥饿过程中，醛缩酶B的降解增强。这种降解被氯喹和3-甲基腺嘌呤抑制，表明醛缩酶B在自溶酶体内降解。我们认为，醛缩酶B是泛素化的细胞质内，然后运输到自噬体和自溶酶体降解营养胁迫。J Cell Physiol 178：17-27，1999.© 1999 Wiley利斯公司
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 fructose‐1,6‐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. J Cell Physiol 178:17–27, 1999. © 1999 Wiley‐Liss, Inc.