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: 10413213
• 负责人: CHARLES E. CHALFANT
• 金额: $ 36.14万
• 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/10413213
• 关键词: Ablation; Apoptosis; Autophagocytosis; BAX gene; Binding; Binding Sites; Bioinformatics; Biological; Biosensor; CRISPR/Cas technology; Cardiac; Cardiolipins; Cell Death; Cell Survival; Cell physiology; Cells; Cellular Membrane; Cellular Stress; Ceramides; Consensus Sequence; Coupled; Data; Enzymes; Event; Excision; Functional disorder; Generations; Genetic; Inflammatory Response; Inner mitochondrial membrane; Location; Malignant Neoplasms; Mediating; Membrane; Mitochondria; Molecular; Neurodegenerative Disorders; Outer Mitochondrial Membrane; Peptide Initiation Factors; Phosphorylation; Play; Process; Production; Proteins; Proteomics; Regulation; Resistance; Role; Signal Induction; Signal Transduction; Site; Site-Directed Mutagenesis; Small Interfering RNA; Sodium Selenite; Sphingolipids; Stimulus; Stress; Therapeutic; biophysical techniques; cardiolipin synthase; ceramide 1-phosphate; ceramide kinase; dihydroceramide desaturase; genetic manipulation; inducible gene expression; mitochondrial membrane; mutant; recruit; response; sensor; sphingomyelin phosphodiesterase D; tool; wound; wound healing

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.

神经酰胺1-磷酸盐（C1P）是通过神经酰胺激酶（CERK）磷酸化来产生的。 尽管在炎症反应和伤口愈合的关键方面已隐含C1P，但这种生物活性 鞘脂仍然保持不明，对Cerk的调节知之甚少。确实，激活 Cerk（CL）的Cerk（50倍）被忽略了 线粒体膜（OMM）。随着破坏事件从内部线粒体膜（IMM）移动CL 到OMM，我们检查了Cerk是否充当Cl依赖性线粒体损伤的生物传感器 产生C1P。在初步研究中，我们发现线粒体损伤诱导线粒体C1P水平。 灭活CERK表达的遗传操作阻碍了这种作用以及增强的细胞死亡。我们也是 确定了CERK中可能的CL结合位点，以及一个结构完整的CL结合缺陷的CERK突变体 证明CL关联/激活缺乏。为了响应线粒体应力剂，重新表达 这个Cerk突变体未能“营救”线粒体C1P的产生和对Cerk细胞中细胞死亡的抗性 - / - 与野生型（WT）Cerk相反。那我们假设Cerk充当线粒体的传感器 通过其CL结合域的损坏诱导C1P，从而降低神经酰胺水平以减轻固有 凋亡/细胞死亡可以由OMM中的神经酰胺水平升高引起。 我们进一步提出，Cerk通过信号发出线粒体的诱导来充当细胞存活的变阻器 特定的自噬降解过程，用于清除损坏的线粒体。我们的初步数据表明 CERK的遗传去除阻滞线粒体。因此，我们假设Cerk在刺激中起着重要作用 线粒体神经酰胺产生C1P，线粒体和减轻细胞死亡。 UVRAG是自噬诱导的关键起始因子，据报道通过调节Bax来抑制凋亡 与线粒体的关联。我们的初步研究表明，紫外线与C1P专门相互作用。 此外，在CERK - / - 细胞中，UVRAG与细胞膜的相互作用显着改善 对线粒体刺激的反应。这是我们进一步假设CERK调节线粒体和细胞存活 通过C1P的产生以及随后将UVRAG招募到OMM。为了验证我们的假设，我们 提案三个特定的目的是确定塞克作为细胞“变阻器”和线粒体驱动力的作用。我们 还建议在介导这些生物学机制的细胞中鉴定关键的C1P传感器（例如UVRAG）。 意义：这些研究有可能描述线粒体和细胞中的新信号范式 在癌症治疗，心脏病生理学和神经退行性疾病中的生存。