背景:真核细胞膜囊泡含有特定的蛋白质组,这些蛋白质决定囊泡的功能并以特定的目的地穿梭。蓝氏贾第鞭毛虫含有未知的胞质囊泡,这些囊泡与鉴定人类骨髓性白血病因子(MLF)的同源物(MLFV)有关。以前的研究表明,MLF也与两种自噬机制共定位,FYVE和ATG8样蛋白,MLFV是响应雷帕霉素的蛋白酶体或自噬底物的应激诱导区室,MG132和氯喹处理。细胞周期蛋白依赖性激酶2的突变蛋白CDK2m3用于了解异常蛋白是否靶向降解成分。有趣的是,MLF被CDK2m3上调,并且它们都共定位在相同的囊泡内。自噬是一种自我消化过程,被激活以去除受损的蛋白质,以防止细胞响应各种压力而死亡。因为缺乏一些自噬机制,自噬的机制尚不清楚。
结果:在这项研究中,我们测试了哺乳动物细胞中的六种自噬体和应激诱导剂,包括MG132,雷帕霉素,氯喹,诺考达唑,DTT,和G418,并发现它们的处理增加了活性氧的产生和囊泡的数量和MLF的水平,FYVE,还有G.Lamblia中的ATG8样蛋白.五种应激诱导剂也增加了CDK2m3蛋白水平和囊泡。使用压力诱导剂和敲低系统进行MLF,我们发现,CDK2m3的应激诱导受MLF的正调控。一种自噬体还原剂,3-甲基腺嘌呤,可以减少MLF和CDK2m3囊泡和蛋白质。此外,用CRISPR/Cas9系统敲低MLF降低了用应激诱导剂处理后的细胞存活率。我们新开发的CRISPR/Cas9互补系统表明,MLF的互补可以恢复细胞对应激诱导物的存活。此外,人类MLF2,像贾第虫MLF,可以增加蓝氏酵母囊壁蛋白的表达和囊肿的形成,它可以与MLFV共定位并与MLF相互作用。
结论:我们的结果表明,MLF家族蛋白在进化中是功能保守的。我们的结果还表明,MLF在应激条件下的存活中起着重要作用,并且MLFV与自噬区室具有相似的应激诱导特征。
The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia.
In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and
G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF.
Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments.