The role of ceramide kinase in mitophagy

神经酰胺激酶在线粒体自噬中的作用

• 批准号: 10593576
• 负责人: CHARLES E. CHALFANT
• 金额: $ 21.58万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-20 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10593576
• 关键词: role; ceramide; kinase; mitophagy

Ceramide-1-phosphate (C1P) is produced via the phosphorylation of ceramide by ceramide kinase (CERK). Although C1P has been implicated in key aspects of inflammatory responses and wound healing, this bioactive sphingolipid still remains understudied and little is known about the regulation of CERK. Indeed, the activation of CERK (50-fold) by cardiolipin (CL) has been overlooked as well as the enzyme’s location at the outer mitochondrial membrane (OMM). As damaging events move CL from the inner mitochondrial membrane (IMM) to the OMM, we examined whether CERK acts as a biosensor of CL-dependent mitochondrial damage via producing C1P. In preliminary studies, we found that mitochondrial damage induces mitochondrial C1P levels. Genetic manipulations that inactivate CERK expression block this effect as well as enhanced cell death. We also identified a possible CL-binding site in CERK, and a structurally intact, CL-binding deficient mutant of CERK demonstrated deficiency in CL association/activation. In response to mitochondrial stress agents, re-expression of this CERK mutant failed to “rescue” mitochondrial C1P generation and resistance to cell death in CERK-/- cells in contrast to wild-type (WT) CERK. Thus, we hypothesize that CERK acts as a sensor for mitochondrial damage via its CL-binding domain to induce C1P, thereby, reducing ceramide levels to mitigate intrinsic apoptosis/cell death that can be triggered by elevated ceramide levels in the OMM. We further propose that CERK acts as a rheostat for cell survival by signaling the induction of mitophagy, a specific autophagic degradation process for clearing damaged mitochondria. Our preliminary data show that the genetic removal of CERK blocks mitophagy. We, thus, hypothesize that CERK plays a major role in stimulating mitophagy and mitigating cell death by utilizing mitochondrial ceramide to generate C1P. UVRAG, a key initiation factor for autophagy induction, reportedly suppresses apoptosis by regulating BAX association with the mitochondria. Our preliminary studies show that UVRAG specifically interacts with C1P. Moreover, the interaction of UVRAG with cellular membranes was dramatically decreased in CERK-/- cells in response to mitophagic stimuli. Thus, we further hypothesize that CERK regulates mitophagy and cell survival via the generation of C1P and subsequent recruitment of UVRAG to the OMM. To validate our hypotheses, we propose three specific aims to determine the role of CERK as a cellular “rheostat” and driver of mitophagy. We also propose to identify key C1P-sensors (e.g., UVRAG) in cells that mediate these biological mechanisms. Significance: These studies have the potential to delineate a new signaling paradigm in mitophagy and cell survival with implications in cancer therapeutics, cardiac pathophysiologies, and neurodegenerative diseases.

1-磷酸神经酰胺 (C1P) 是通过神经酰胺激酶 (CERK) 磷酸化神经酰胺而产生的。 尽管 C1P 与炎症反应和伤口愈合的关键方面有关，但这种生物活性 鞘脂仍处于研究之中，并且对 CERK 的激活的调节知之甚少。 心磷脂 (CL) 对 CERK 的影响（50 倍）以及酶在外层的位置都被忽略了。 当损伤事件将 CL 从线粒体内膜 (IMM) 移出时。 对于 OMM，我们通过以下方式检查了 CERK 是否充当 CL 依赖性线粒体损伤的生物传感器： 在初步研究中，我们发现线粒体损伤会诱导线粒体 C1P 水平。 我们还通过基因操作使 CERK 表达失活来阻止这种效应并增强细胞死亡。 鉴定出 CERK 中可能的 CL 结合位点，以及结构完整、CL 结合缺陷的 CERK 突变体 CL 关联/激活中表现出缺陷，以响应线粒体应激剂，重新表达。 这种CERK突变体未能“拯救”CERK-/-细胞中线粒体C1P的生成和对细胞死亡的抵抗力 与野生型 (WT) CERK 相比，我们发现 CERK 充当线粒体传感器。 通过其 CL 结合域诱导 C1P，从而减少损伤，降低神经酰胺水平以减轻内在的 OMM 中神经酰胺水平升高可引发细胞凋亡/细胞死亡。 我们进一步提出，CERK 通过诱导线粒体自噬（一种线粒体自噬）信号，充当细胞存活的变阻器。 我们的初步数据表明，清除受损线粒体的特定自噬降解过程。 CERK 的基因去除会阻止线粒体自噬。因此，我们认为 CERK 在刺激中发挥着重要作用。 利用线粒体神经酰胺生成 C1P 进行线粒体自噬并减轻细胞死亡。 UVRAG 是自噬诱导的关键启动因子，据报道可通过调节 BAX 抑制细胞凋亡 我们的初步研究表明 UVRAG 与 C1P 发生特异性相互作用。 此外，在 CERK-/- 细胞中，UVRAG 与细胞膜的相互作用显着降低。 因此，我们进一步证实 CERK 调节线粒体自噬和细胞存活。 通过生成 C1P 和随后将 UVRAG 招募到 OMM 中，我们验证了我们的假设。 我们提出了三个具体目标来确定 CERK 作为细胞“变阻器”和线粒体自噬驱动程序的作用。 还建议鉴定细胞中介导这些生物机制的关键 C1P 传感器（例如 UVRAG）。 意义：这些研究有可能描绘线粒体自噬和细胞的新信号传导范式 生存对癌症治疗、心脏病理生理学和神经退行性疾病的影响。