在PEX1缺乏的HeLa细胞中，一种新的TBK1-MARCHF7-PXMP4-NBR1轴调控pexophagy（自噬性吞噬）的过程

《Autophagy》：Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 Axis in PEX1-depleted HeLa cells

通过功能筛选发现E3泛素连接酶MARCHF7调控PEX1缺失诱导的过氧化物酶体自噬，其机制涉及TBK1磷酸化激活MARCHF7，进而泛素化PXMP4蛋白，招募自噬受体NBR1介导过氧化物酶体降解。研究揭示了PEX1缺失通过ROS积累激活TBK1-MARCHF7-PXMP4-NBR1通路调控自噬的分子机制。

　　
Peroxisomes are critical organelles involved in lipid metabolism, redox balance, and cellular defense mechanisms. Their dysfunction is linked to peroxisomal biogenesis disorders (PBDs) and neurodegenerative diseases, where impaired pexophagy—selective autophagy for peroxisome degradation—exacerbates cellular damage. Recent studies have highlighted the role of E3 ubiquitin ligases in organelle-selective autophagy, such as MARCHF5 mediating mitophagy and MARCHF6 regulating ER homeostasis. However, the molecular mechanisms underlying pexophagy remain poorly understood, particularly the link between peroxisomal biogenesis factors and autophagy signaling.

A key discovery in this study is the identification of MARCHF7 as a novel E3 ligase orchestrating pexophagy under conditions of PEX1 depletion. PEX1, a core component of peroxisomal assembly machinery, is frequently mutated in PBDs like Zellweger syndrome. When PEX1 is knocked down, peroxisomes accumulate ROS due to defective β-oxidation of very-long-chain fatty acids (VLCFAs), which triggers pexophagy to eliminate malfunctioning organelles. Notably, bafilomycin A1—a blocker of autophagosome-lysosome fusion—inhibits PEX1 depletion-induced peroxisome loss, confirming the involvement of macroautophagy. This observation aligns with prior reports showing that peroxisomal ROS accumulation activates stress responses, including mitophagy and ER phagy.

The study reveals a hierarchical signaling cascade where PEX1 depletion activates TBK1 kinase through ROS-mediated phosphorylation at serine 172. Activated TBK1 subsequently phosphorylates MARCHF7, enhancing its ability to ubiquitize PXMP4—a peroxisomal membrane protein at lysine 20. This ubiquitination tags PXMP4 for recognition by NBR1, an autophagy receptor that mediates peroxisome degradation. Key experimental findings include:
1. **MARCHF7 dependency**: Depletion of MARCHF7 completely blocks PEX1-induced peroxisome loss, while reconstitution with wild-type PXMP4 restores pexophagy. Mutations at PXMP4's lysine 20 (K20R) abolish ubiquitination and prevent peroxisome degradation, demonstrating K20 as the critical ubiquitination site.
2. **TBK1 activation role**: Chemical inhibitors of TBK1 (GSK8612, MRT67307) and genetic knockout of TBK1 suppress peroxisome loss, while TBK1 overexpression exacerbates it. This establishes TBK1 as a central regulator linking peroxisomal stress to pexophagy.
3. **PXMP4 as a signaling hub**: PXMP4's K20 residue serves as a molecular tag for MARCHF7-mediated ubiquitination. Disrupting this interaction (via PXMP4 depletion or K20R mutation) or inhibiting MARCHF7 (siRNA) prevents NBR1 recruitment to peroxisomes, visualized through reduced colocalization of NBR1 with ABCD3—a peroxisomal marker.
4. **ROS as upstream signal**: N-acetyl-L-cysteine (NAC), a ROS scavenger, abrogates PEX1 depletion-induced TBK1 phosphorylation, PXMP4 ubiquitination, and peroxisome loss. This confirms ROS as the primary driver of the TBK1-MARCHF7-PXMP4 axis.

Mechanistically, the TBK1-MARCHF7-PXMP4-NBR1 axis operates through three coordinated steps: (1) PEX1 deficiency disrupts peroxisomal biogenesis, causing ROS accumulation and peroxisomal dysfunction; (2) ROS activate TBK1 through unknown downstream mediators, leading to its phosphorylation at Ser172; (3) Phosphorylated TBK1 enhances MARCHF7's ability to ubiquitize PXMP4 at K20, creating a peroxisomal "eat-me" signal that guides NBR1 recruitment. This process is dynamically regulated, as NAC treatment not only neutralizes ROS but also prevents TBK1 activation and subsequent signaling downstream.

The study advances understanding of pexophagy by identifying a previously uncharacterized E3 ligase-substrate relationship. MARCHF7 directly interacts with PXMP4, with computational predictions and site-directed mutagenesis confirming K20 as the primary ubiquitination site. This contrasts with prior reports where MARCHF5 and MARCHF6 target different organelles, highlighting functional diversity within the MARCH family. Notably, the TBK1-MARCHF7 axis parallels mechanisms in mitophagy (PINK1/PRKN) and ER phagy (KEAP1), suggesting conserved strategies for organelle turnover across eukaryotic cells.

Therapeutic implications are significant. Since 65% of Zellweger spectrum disorders involve PEX1 mutations, targeting this pathway could mitigate peroxisomal loss. For example, TBK1 inhibitors could theoretically reduce pexophagy in PEX1-deficient cells, preserving peroxisomes. However, global autophagy suppression is risky due to its roles in stress responses and organelle biogenesis. Therefore, specific inhibitors targeting the TBK1-MARCHF7-PXMP4 axis or stabilizing non-ubiquitinated PXMP4 variants might offer safer therapeutic options. The study also raises questions about crosstalk with other peroxisomal stress sensors, such as ATM kinase, which was shown to phosphorylate PEX5 under similar conditions [30]. Future research should explore whether TBK1 interacts with ATM or other kinases to coordinate pexophagy.

Methodologically, the study combines genetic screening (siRNA library), biochemical assays (immunoprecipitation, ubiquitination assays), and imaging (confocal microscopy with mCherry-SEpHluorin-SKL constructs). The double-fluorescent system elegantly distinguishes peroxisomes (mCherry+) from pexolysosomes (SEpHluorin-), allowing real-time visualization of pexophagy flux. Colocalization studies with NBR1 further confirm the role of ubiquitinated PXMP4 in autophagosome recruitment.

Clinical relevance is underscored by the study's findings. PEX1 mutations are prevalent in PBDs, and excessive pexophagy contributes to metabolic deficits. For instance, chloroquine, an autophagy enhancer, was previously reported to rescue peroxisomes in PEX1-mutant cells [34]. However, this study identifies a specific target (PXMP4 K20) for modulating pexophagy. Future trials could combine TBK1 inhibitors with PXMP4 stabilizers to balance peroxisomal turnover and function.

Limitations include the reliance on cell culture models and the need for in vivo validation. Additionally, the role of MARCHF7 in other organelle-selective autophagies remains unclear. Future work should investigate whether this ligase family成员 have specialized roles in different organelle degradation pathways.

In summary, this study uncovers a precise molecular circuit for pexophagy regulation. By linking PEX1 deficiency to ROS-driven TBK1 activation, MARCHF7 phosphorylation, and PXMP4 ubiquitination, it provides a framework for understanding peroxisomal homeostasis. The discovery of K20 as a critical ubiquitination site not only clarifies MARCHF7's substrate specificity but also opens avenues for targeted therapies in peroxisomal disorders and neurodegenerative diseases where pexophagy is dysregulated. The integration of kinase activity, E3 ligase substrate targeting, and receptor recruitment offers a blueprint for future studies on organelle-selective autophagy mechanisms.