Mitochondrial autophagy (mitophagy) is an important mechanism for regulating mitochondrial quality and quantity, and is highly relevant to physiological and pathological conditions such as development, cancer, aging and neurodegenerative diseases. Two types of mammalian mitophagy pathways are known: the ubiquitin-dependent pathway mediated by PINK1-Parkin and the receptor-dependent pathway mediated by BNIP3, NIX and FUNDC1. However, the mechanisms of mitophagy regulation and their physiopathologic significance remain to be explored. Prominently, mice lacking known mitophagy pathways (e.g., Pink1-/-, Parkin-/-, Bnip3-/-, etc.) are phenotypically weak, with a clear gap from the commonly recognized importance of mitophagy.

On December 26, 2023, the laboratory of Hui Jiang at the Institute for Biomedical Interdisciplinary Research/Beijing Institute of Life Sciences, Tsinghua University, published online in Molecular Cell a paper entitled A mitophagy sensor PPTC7 controls BNIP3 and NIX degradation to regulate mitochondrial mass. The paper identifies a mitophagy-sensing and inhibiting factor PPTC7, elucidates the molecular mechanism by which PPTC7 regulates mitophagy, and reveals its role in the regulation of mitochondrial mass and metabolic homeostasis. As a key factor monitoring mitophagy levels, PPTC7 senses the protein levels of mitophagy receptors BNIP3 and NIX, which in turn initiates the assembly of the BNIP3/NIX-PPTC7-SCFFBXL4 intact complex, promotes ubiquitination and degradation of BNIP3 and NIX in SCFFBXL4, and thus inhibits mitophagy. . This pathway is essential for the maintenance of cellular mitochondrial number, metabolic homeostasis, and individual survival.

FBXL4-mediated mitophagy regulation and degenerative diseases MTDPS13

The laboratory of Hui Jiang has long been engaged in the study of mitochondrial protein homeostatic regulatory mechanisms, and previously reported that a mitochondrial outer membrane complex, UBXD8-VCP, mediates the degradation of BNIP3, which inhibits mitophagy (cited in NIBS 2022-08-20). To further explore the mechanisms of mitophagy regulation, graduate student Yu Cao identified multiple mitophagy repressors, including FBXL4 and PPTC7, through a genetic screen targeting mitochondria (citing NIBS 2023-3-13 report). Mutations in FBXL4 are known to cause the lethal degenerative disease mitochondrial DNA depletion syndrome 13 (MTDPS13)^{1, 2}， but the mechanism is unknown. The authors found that FBXL4 localizes to the mitochondrial outer membrane and, together with Skp1 and Cullin1, forms the SCFFBXL4 E3 ubiquitin ligase. SCFFBXL4 inhibits mitophagy by ubiquitinating degradation of BNIP3 and NIX, whereas patient-derived FBXL4 mutations inhibit SCFFBXL4 assembly, leading to BNIP3 and NIX protein accumulation and mitophagy hyperactivation. patients with FBXL4 mutations exhibit severe symptoms such as mtDNA depletion, reduced mitochondrial numbers, acidosis, and degenerative neurologic and muscular lesions. Similar to FBXL4 mutant patients, Fbxl4-/- mice exhibit severe phenotypes of BNIP3 and NIX protein accumulation, uncontrolled mitophagy, reduced mitochondrial number, metabolic disorders, and perinatal death. More importantly, knockdown of Bnip3 or Nix in Fbxl4-/- mice rescued mitochondrial number, restored metabolic homeostasis, and saved mouse lifespan. These results suggest that reduced mitochondrial number due to excessive mitophagy is the causative agent of MTDPS13 and provide ideas for treatment.

PPTC7 regulates FBXL4-mediated degradation of BNIP3 and NIX

Given the powerful and dangerous mitochondrial clearance of BNIP3 and NIX, cells must keep their levels firmly in check, but the molecular mechanisms by which cells monitor and regulate the degradation of BNIP3 and NIX remain unclear. Graduate student Yuqiu Sun studies another screened discovery of the mitophagy inhibitor PPTC7. Previous studies have shown that PPTC7 is a phosphatase localized in the mitochondrial matrix. However, surprisingly, similar to knockdown of FBXL4, knockdown of PPTC7 also resulted in the accumulation of BNIP3 and NIX and excessive mitophagy; Pptc7-/- mice exhibited a phenotype of uncontrolled mitophagy, reduced mitochondrial number, and perinatal death, and the phenotype could be rescued by Nix knockdown (Figure 1). Furthermore, PPTC7 overexpression promoted the degradation of endogenous BNIP3 and NIX by FBXL4, whereas FBXL4 overexpression had no effect, suggesting that PPTC7 acts as an upstream rate-limiting factor to regulate FBXL4-mediated degradation of BNIP3 and NIX. But how does the mitochondrial matrix protein PPTC7 regulate protein degradation on the mitochondrial outer membrane?

Figure 1. PPTC7 deletion overactivates BNIP3- and NIX-dependent mitophagy, resulting in reduced mitochondrial numbers and mouse death. A, Pptc7 -KO elevates protein levels of the mitophagy receptors. B, NIP3 and NIX in mouse livers (red boxes) and leads to a decrease in other mitochondrial proteins (blue boxes).B, Mouse survival curves.

PPTC7 precursors linger at the mitochondrial outer membrane through direct interaction with BNIP3/NIX

Normally, the precursor form of mitochondrial matrix proteins (precursor) enters the mitochondria guided by the N-terminal targeting sequence and excises the targeting sequence at the mitochondrial matrix to complete the maturation process. The authors found that in wild-type cells, PPTC7 exists in the mitochondrial matrix in a mature form with the targeting sequence excised, whereas in FBXL4-KO cells, PPTC7 has both a mature form localized in the mitochondrial matrix and a precursor form localized in the mitochondrial outer membrane. Further analysis showed that accumulation of BNIP3 and NIX in FBXL4-KO cells resulted in the appearance of PPTC7 precursors at the outer mitochondrial membrane; direct overexpression of BNIP3 or NIX in wild-type cells also induced the appearance of PPTC7 precursors localized at the outer mitochondrial membrane.

Through in vitro recombinant protein experiments, the authors found that BNIP3 and NIX directly interacted with PPTC7 and analyzed the NIX-PPTC7 interaction region using crosslinking mass spectrometry. After truncating the direct interaction region between NIX and PPTC7, NIX was unable to induce the outer membrane localization of PPTC7 precursors, suggesting that BNIP3 and NIX localize PPTC7 precursors to the mitochondrial outer membrane through direct interaction with PPTC7.

Weakened PPTC7 Localization Sequences Help PPTC7 Retain at the Mitochondrial Outer Membrane

Interestingly, the authors found that the PPTC7 localization sequence was short in length and poorly guided compared to the classical mitochondrial matrix localization sequence, a feature that presumably weakened the ability of the PPTC7 precursor to enter the mitochondrial matrix and favored the retention of the PPTC7 precursor by the outer membrane BNIP3/NIX. Therefore, the investigators replaced the PPTC7 localization sequence with a classical sequence and found that it allowed all of PPTC7 to enter the mitochondrial matrix, where it could not be retained at the outer membrane and lost its ability to regulate BNIP3 and NIX degradation.

Outer membrane PPTC7 initiates assembly of the BNIP3/NIX-PPTC7-SCFFBXL4 complex

So how does outer membrane PPTC7 promote SCFFBXL4-mediated degradation of BNIP3 and NIX? The authors found that in addition to direct interaction with BNIP3/NIX, PPTC7 also interacts directly with Cullin1 in the SCFFBXL4 complex. The authors expressed and purified recombinant proteins of NIX, PPTC7, FBXL4,Cullin1-Rbx1, and Skp1, and found that these proteins could only assemble into the complete NIX-PPTC7-SCFFBXL4 complex if all of the proteins were present, and that the absence of any of the components of NIX, PPTC7, and FBXL4 resulted in the fragmentation of the complex.

The above results provide a model for negative feedback regulation of mitophagy: mitophagy receptors BNIP3 and NIX, which accumulate in the mitochondrial outer membrane, interact directly with the PPTC7 precursor, retaining it in the outer membrane and initiating the assembly of the BNIP3/NIX-PPTC7-SCFFBXL4 complex, thereby ubiquitinating and degrading BNIP3 and NIX and preventing mitophagy overactivation (Figure 2).

Hepatic induction of PPTC7 under fasting to inhibit mitophagy and maintain metabolic homeostasis

An important question is whether PPTC7 responds to physiological signals in addition to negative feedback regulation of mitophagy? Given that PPTC7 overexpression promotes BNIP3 and NIX degradation, the authors focused on the regulation of PPTC7 expression by physiological signals and found that fasting did not alter skeletal muscle PPTC7 expression but strongly induced PPTC7 in the liver. in contrast, fasting strongly induced skeletal muscle mitophagy but did not alter hepatic mitophagy levels, and the authors hypothesized that hepatic mitophagy was suppressed by the upregulation of PPTC7.

Therefore, the authors knocked out PPTC7 in mouse liver and found that fasting resulted in the loss of large numbers of mitochondria in PPTC7-deficient livers. Under fasting, the liver has an important mission - to maintain blood glucose levels through gluconeogenesis. Gluconeogenesis requires two important supports from mitochondria: ATP and pyruvate carboxylase. The authors found that knockdown of hepatic PPTC7 resulted in a lack of hepatic ATP and impaired gluconeogenesis under fasting, causing severe hypoglycemia. Thus, under fasting, liver-specific upregulation of PPTC7 to repress mitophagy maintains mitochondrial number and metabolic homeostasis (Figure 2)

Figure 2. working model of PPTC7 regulation of mitophagy.

1. negative feedback homeostatic regulation of mitophagy. Under normal conditions, PPTC7 is expressed and enters the mitochondrial matrix to exercise normal functions. When mitophagy receptors BNIP3 and NIX accumulate, BNIP3/NIX retains PPTC7 precursors in the outer membrane, initiating the assembly of the BNIP3/NIX-PPTC7-SCFFBXL4 complex, which degrades BNIP3 and NIX and prevents overactivation of mitophagy and loss of mitochondria.2. Under fasting, the liver upregulates PPTC7 to suppress mitophagy, thereby maintaining hepatic energy supply and gluconeogenic pathway viability and maintaining blood glucose levels.

Taken together, this work reports a PPTC7-SCFFBXL4 pathway that senses and suppresses mitophagy. PPTC7 acts as a key regulator that integrates homeostatic maintenance and physiological signals to modulate mitophagy levels to maintain mitochondrial number and cellular metabolic homeostasis. The discovery of this pathway has significantly improved our understanding of mitophagy and mitochondrial mass control. Differential regulation of mitophagy and its function in liver and skeletal muscle under fasting also enriched our knowledge of tissue-specific mitophagy regulation under physiological conditions.

Yuqiu Sun, a PhD student at the Institute of Biomedical Interdisciplinary Research, Tsinghua University, is the first author of the paper, and Dr. Hui Jiang is the corresponding author. Other authors of the paper include Yu Cao and Huayun Wan from Hui Jiang's group, Drs. Adalaiti Ÿbuytiming and Mengqiu Dong from Mengqiu Dong's lab, Drs. Yan Ma and Yang Cao from the Metabolomics Center, Drs. Shibu Chen and Lin Li from the Proteomics Center, Drs. Qi Li and Chongyang Wu from the Genetic Screening Center, and Meng Wang from Qinghua Liu's lab. The study was funded by the Ministry of Science and Technology, Beijing Municipal Government and Tsinghua University, and was done at the Institute of Biomedical Crossroads/Beijing Institute of Life Sciences, Tsinghua University.

1 Bonnen, Penelope E. et al. Mutations in FBXL4 Cause Mitochondrial Encephalopathy and a Disorder of Mitochondrial DNA Maintenance. The American Journal of Human Genetics 93, 471-481, doi:https://doi.org/10.1016/j.ajhg.2013.07.017 (2013).

2 Gai, X. et al. Mutations in FBXL4, Encoding a Mitochondrial Protein, Cause Early-Onset Mitochondrial Encephalomyopathy. Human Genetics 93, 482-495, doi:https://doi.org/10.1016/j.ajhg.2013.07.016 (2013).