The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study selective macroautophagy in mammalian cells.

通过监测溶酶体中谷胱甘肽S-转移酶-甜菜碱-同型半胱氨酸S-甲基转移酶（GST-BHMT）报告蛋白的片段化过程，既往研究已建立GST-BHMT检测体系，作为一种基于底物的终点检测方法，用于分析饥饿诱导的基本非选择性自噬。本研究发现，在营养充足条件下，通过药物或基因操作实现的蛋白酶体抑制，可诱导类似的依赖自噬的GST-BHMT切割加工过程。但从机制上看，这种蛋白酶体抑制诱导的自噬与饥饿诱导的自噬存在显著差异：它不依赖于细胞营养与能量状态的上游核心感受器——雷帕霉素机制性靶点（MTOR，mechanistic target of rapamycin [serine/threonine kinase]）与AMP活化蛋白激酶α催化亚基（PRKAA/AMPK，protein kinase, AMP-activated, α catalytic subunit）的调控，而是需要通常参与选择性自噬通路的底物受体SQSTM1/p62（Sequestosome 1）与BRCA1基因1邻蛋白（NBR1，neighbor of BRCA1 gene 1）的存在。此外，该过程依赖于内质网（ER，endoplasmic reticulum）应激信号通路，尤其是内质网到细胞核信号1（ERN1/IRE1，endoplasmic reticulum to nucleus signaling 1）及其主要下游效应分子丝裂原活化蛋白激酶8（MAPK8/JNK1，mitogen-activated protein kinase 8），而非X盒结合蛋白1（XBP1，X-box binding protein 1），其具体调控方式为通过磷酸化依赖的机制，解离B细胞CLL/淋巴瘤2（BCL2，B-cell CLL/lymphoma 2）与自噬相关蛋白Beclin 1（BECN1，Beclin 1, autophagy related）之间的结合。进一步研究显示，GST-BHMT的多聚化结构域对其响应蛋白酶体抑制的切割加工过程是必需的，这与其在饥饿诱导的切割加工中发挥的非必需作用形成鲜明对比。综上，本研究结果支持如下模型：在营养充足条件下，蛋白酶体失活通过一种独特的机制诱导GST-BHMT报告蛋白的自噬依赖型切割加工，该机制与酵母细胞质到液泡靶向（Cvt，cytoplasm-to-vacuole targeting）通路具有显著相似性；同时本研究提示，GST-BHMT报告基因可作为一种便捷的检测手段，用于研究哺乳动物细胞中的选择性巨自噬。