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AMBRA1 通过控制其泛素化负调控癌症干细胞标志物 ALDH1B1 的功能
发表日期:2021-11-11


AMBRA1 Negatively Regulates the Function of ALDH1B1, a Cancer Stem Cell Marker, by Controlling Its Ubiquitination
AMBRA1 通过控制其泛素化负调控癌症干细胞标志物 ALDH1B1 的功能

 

Activating molecule in Beclin-1-regulated autophagy (AMBRA1), a negative regulator of tumorigenesis, is a substrate receptor of the ubiquitin conjugation system. ALDH1B1, an aldehyde dehydrogenase, is a cancer stem cell (CSC) marker that is required for carcinogenesis via upregulation of the β-catenin pathway. Although accumulating evidence suggests a role for ubiquitination in the regulation of CSC markers, the ubiquitination-mediated regulation of ALDH1B1 has not been unraveled. While proteome analysis has suggested that AMBRA1 and ALDH1B1 can interact, their interaction has not been validated. Here, we show that AMBRA1 is a negative regulator of ALDH1B1. The expression of ALDH1B1-regulated genes, including PTEN, CTNNB1 (β-catenin), and CSC-related β-catenin target genes, is inversely regulated by AMBRA1, suggesting a negative regulatory role of AMBRA1 in the expression of ALDH1B1-regulated genes. We found that the K27- and K33-linked ubiquitination of ALDH1B1 is mediated via the cooperation of AMBRA1 with other E3 ligases, such as TRAF6. Importantly, ubiquitination site mapping revealed that K506, K511, and K515 are important for the K27-linked ubiquitination of ALDH1B1, while K33-linked ubiquitination occurs at K506. A ubiquitination-defective mutant of ALDH1B1 increased the self-association ability of ALDH1B1, suggesting a negative correlation between the ubiquitination and self-association of ALDH1B1. Together, our findings indicate that ALDH1B1 is negatively regulated by AMBRA1-mediated noncanonical ubiquitination.

Beclin-1 调节的自噬 (AMBRA1) 中的激活分子是肿瘤发生的负调节因子,是泛素结合系统的底物受体。ALDH1B1 是一种醛脱氢酶,是一种癌症干细胞 (CSC) 标志物,通过上调 β-catenin 途径,是致癌作用所必需的。尽管越来越多的证据表明泛素化在 CSC 标志物调控中的作用,但泛素化介导的 ALDH1B1 调控尚未解开。虽然蛋白质组分析表明 AMBRA1 ALDH1B1 可以相互作用,但它们的相互作用尚未得到验证。在这里,我们表明 AMBRA1 ALDH1B1 的负调节因子。ALDH1B1 调控基因的表达,包括PTENCTNNB1(β-catenin) CSC 相关的 β-catenin 靶基因受 AMBRA1 的反向调控,表明 AMBRA1 ALDH1B1 调控基因的表达中具有负调控作用。我们发现 ALDH1B1 K27 K33 连接的泛素化是通过 AMBRA1 与其他 E3 连接酶(如 TRAF6)的合作介导的。重要的是,泛素化位点映射显示 K506K511 K515 K27 连接的 ALDH1B1 泛素化很重要,而 K33 连接的泛素化发生在 K506ALDH1B1 泛素化缺陷突变体增加了 ALDH1B1 的自缔合能力,表明 ALDH1B1 的泛素化和自缔合之间呈负相关。总之,我们的研究结果表明 ALDH1B1 受到 AMBRA1 介导的非经典泛素化的负调控。

 10层细胞工厂
10层细胞工厂

Activating molecule in Beclin-1-regulated autophagy (AMBRA1) is a highly intrinsically disordered protein. As with other intrinsically disordered proteins [1], AMBRA1 is involved in various biological processes and regulates proteinprotein interactions [2]. AMBRA1 is involved in the induction of autophagy via interactions with ULK1 [3] and elongin B [4] and in the induction of mitophagy via interactions with PARKIN [5] and LC3 [6]. Furthermore, AMBRA1 is crucial for nervous system development in mouse embryos [7] and skeletal muscle development in zebrafish [8].

Recently, AMBRA1 has emerged as a tumor suppressor that mediates the degradation of proto-oncogene c-Myc [9] and D-type cyclins [10]. AMBRA1-deficient tumor cells were more likely to grow when injected into nude mice than wild-type tumor cells, implying a negative relationship between AMBRA1 and tumorigenesis [11]. In addition, AMBRA1 expression inversely correlates with the stemness signature in lung cancer [12]. Despite the increasing evidence of the functional role of AMBRA1 in cancer stem cell (CSC) properties, the underlying mechanisms remain unknown.

Aldehyde dehydrogenases (ALDHs) are members of a superfamily of NAD(P)+-dependent enzymes and catalyze the oxidation of aldehydes to their respective acids. ALDH1B1, which is one of the ALDH isozymes, is reported to be strictly expressed in the SC compartments of normal human colon and is highly expressed in human colonic adenocarcinoma [13]. In a clinical study [14], the upregulation of ALDH1B1 was correlated with high-grade colorectal carcinoma and the presence of lymph-node metastases, implying a potential role of ALDH1B1 in carcinogenesis. The depletion of ALDH1B1 in colon cancer cells resulted in the downregulation of Wnt/β-catenin, Notch, and PI3K/Akt pathway-related genes such as CTNNB1, Akt, and Notch1 [15]. Negative regulation of ALDH1B1 by microRNA-761 in osteosarcoma cells suppresses tumor cell proliferation by regulating TGF-β signaling and cell adhesion [16]. Although several lines of evidence suggest the importance of the modification and regulation of CSC markers by ubiquitination [17], few studies have investigated the regulators modifying ALDH1B1.

According to the Biological General Repository for Interaction Datasets (thebiogrid.org, Accessed 15 December 2020), ALDH1B1 has been identified as one of the AMBRA1-interacting proteins via affinity purification-mass spectrometry analysis of Flag-AMBRA1-transfected HEK293T human embryonic kidney cells [18]. However, the biological significance of the interaction between AMBRA1 and ALDH1B1 is not yet understood. Here, for the first time, we show that AMBRA1 downregulates the self-association and enzyme activity of ALDH1B1, and subsequently regulates CSC-associated Wnt/β-catenin signaling in an ALDH1B1-dependent manner. Mechanistically, AMBRA1 controls ALDH1B1 function via the ubiquitination of ALDH1B1 with K27- and K33-linked ubiquitin chains. Further, we found that TRAF6 and DDB1, an adaptor protein of the CRL4 complex, are able to ubiquitinate ALDH1B1 in an AMBRA1-independent fashion.

 AMBRA1 通过控制其泛素化负调控癌症干细胞标志物 ALDH1B1 的功能
血清培养基瓶500ml

Beclin-1 调节的自噬 (AMBRA1) 中的激活分子是一种高度内在无序的蛋白质。与其他本质上无序的蛋白质 [ 1 ] 一样,AMBRA1 参与各种生物过程并调节蛋白质-蛋白质相互作用 [ 2 ]AMBRA1 通过与 ULK1 [ 3 ] elongin B [ 4 ] 的相互作用参与诱导自噬,并通过与 PARKIN [ 5 ] LC3 [ 6 ] 的相互作用参与诱导线粒体自噬。此外,AMBRA1 对小鼠胚胎的神经系统发育 [ 7 ] 和斑马鱼的骨骼肌发育 [ 8 ]至关重要。

最近,AMBRA1 已成为介导原癌基因 c-Myc [ 9 ] D 型细胞周期蛋白 [ 10 ]降解的肿瘤抑制因子。与野生型肿瘤细胞相比,AMBRA1 缺陷型肿瘤细胞在注射到裸鼠体内时更容易生长,这意味着 AMBRA1 与肿瘤发生之间存在负相关关系 [ 11 ]。此外,AMBRA1 表达与肺癌中的干细胞特征呈负相关 [ 12 ]。尽管越来越多的证据表明 AMBRA1 在癌症干细胞 (CSC) 特性中的功能作用,但其潜在机制仍然未知。

醛脱氢酶 (ALDH) NAD(P) +依赖性酶超家族的成员,可催化醛氧化为其各自的酸。据报道,ALDH1B1 ALDH 同工酶之一,在正常人结肠的 SC 区室中严格表达,在人结肠腺癌中高度表达 [ 13 ]。在一项临床研究 [ 14 ] 中,ALDH1B1 的上调与高级别结直肠癌和淋巴结转移的存在相关,这意味着 ALDH1B1 在致癌作用中的潜在作用。结肠癌细胞中 ALDH1B1 的缺失导致 Wnt/β-cateninNotch PI3K/Akt 通路相关基因(如CTNNB1Akt和缺口 1 [ 15 ]。骨肉瘤细胞中微小RNA-761ALDH1B1的负调节通过调节TGF-β信号传导和细胞粘附来抑制肿瘤细胞增殖[ 16 ]。尽管有几条证据表明通过泛素化修饰和调节 CSC 标志物的重要性 [ 17 ],但很少有研究调查调节 ALDH1B1 的调节剂。


三角细胞培养摇瓶250ml

根据相互作用数据集的生物通用知识库(thebiogrid.org2020 12 15 日访问),通过对 Flag-AMBRA1 转染的 HEK293T 人胚胎肾细胞的亲和纯化质谱分析,ALDH1B1 已被鉴定为 AMBRA1 相互作用蛋白之一[ 18]。然而,AMBRA1 ALDH1B1 之间相互作用的生物学意义尚不清楚。在这里,我们首次表明 AMBRA1 下调 ALDH1B1 的自缔合和酶活性,随后以 ALDH1B1 依赖性方式调节 CSC 相关的 Wnt/β-连环蛋白信号传导。从机制上讲,AMBRA1 通过 K27 K33 连接的泛素链泛素化 ALDH1B1 来控制 ALDH1B1 的功能。此外,我们发现 TRAF6 DDB1CRL4 复合物的衔接蛋白)能够以不依赖 AMBRA1 的方式泛素化 ALDH1B1


来源:MDPI  https://www.mdpi.com/1422-0067/22/21/12079/htm

关键词:colorectal cancer stem cell; E3 ubiquitin ligase; substrate receptor; aldehyde dehydrogenase 1B1; noncanonical ubiquitination

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