https://orcid.org/0000-0001-7782-054X
ABSTRACT
If cellular reactive oxygen species (ROS) production surpasses the intracellular antioxidant capacity, thus altering the ROS homeostasis, the cell needs to eradicate faulty mitochondria responsible for these excessive ROS. We have shown that even moderate ROS production breaks the KEAP1-PGAM5 complex, inhibiting the proteasomal removal of PGAM5. This leads to an accumulation of PGAM5 interfering with PINK1 processing that sensitizes mitochondria to autophagic removal. We propose that such a negative feedback system maintains cell ROS homeostasis.
Mitophagy is essential to remove “worn-out” mitochondria and to improve the mitochondrial population quality within the cell. It is well known how depolarized mitochondria that have reached the end of their life cycle are removed by PRKN-dependent mitophagy. However, inner membrane depolarization is not the only prerequisite for mitophagy. Dysfunctional mitochondria could keep their membrane potential even at the expense of cytosolic ATP, but this potential favors excessive ROS production damaging the host cell. Thus, to survive, the cell should have a mechanism(s) to remove these internal killers, which are not yet depolarized, although they have (partially) lost their function. There were many speculations on how excessive ROS activate mitochondrial removal by autophagy, but the real mechanism has not been shown yet. The present study unravels a mechanism of mitophagy regulation by cell ROS level.
In a recent work [Citation1], we demonstrated that a moderate mitochondrial superoxide-hydrogen peroxide overproduction (below levels inducing mitochondrial membrane depolarization) can induce KEAP1-dependent mitophagy.
KEAP1 is one of the primary cellular ROS sensors; when it becomes oxidized, it loses its interaction with NFE2L2/NRF2. The latter then migrates to the nucleus and activates several antioxidant enzyme genes as well as some pro-mitophagy genes. We, therefore, expected that knocking out (or activating) NFE2L2 should also modulate the KEAP1-dependent mitophagy induced by moderate ROS production. Surprisingly, this is not the case. Then, we tested various proteins that possess the KEAP1 binding site and, at the same time, were known to interact with or stabilize PINK1. Among them, only PGAM5 (PGAM family member 5, mitochondrial serine/threonine protein phosphatase) accumulates in response to moderate mitochondrial ROS production and, at the same time, induces PINK1-PRKN-dependent mitophagy.
PGAM5 is found in cells in two forms: a full-length and a short form. After being imported into mitochondria, PGAM5 is either inserted into the mitochondrial outer membrane (full-length form) or cleaved by the inner mitochondrial membrane (IMM)-resident proteases and then released back to the cytosol (short form). In our experiments, only the full-length PGAM5 is co-immunoprecipitated with KEAP1, suggesting that KEAP1, being localized only in the cytosol, should interact with PGAM5 before it is imported to the mitochondria or/and when it is already inserted into the outer mitochondrial membrane. To dissect these two possibilities, we overexpressed fluorescently tagged PGAM5 and followed its fate in cells. Interestingly, although the fluorescent PGAM5 is normally localized in the mitochondria, it starts to accumulate in the cytosol when its proteasomal degradation is blocked. Furthermore, Western blot analysis demonstrated that it is specifically the full-length form of PGAM5 accumulating in the cytosol in response to proteasomal inhibition. Taken together, this suggests that KEAP1 controls the degradation of cytosolic full-length PGAM5 before it is inserted into the outer mitochondrial membrane.
We also tried to understand how PGAM5 could lead to mitophagy. Initially, we expected that PGAM5 would dephosphorylate and activate the mitophagy receptor FUNDC1 or DNM1L, promoting mitochondrial fragmentation. However, to our surprise, neither knocking down FUNDC1 or DNM1L nor suppressing its phosphatase activity abolishes PGAM5-induced mitophagy. We then speculated that PGAM5 might directly affect PINK1 processing. PINK1 and PGAM5 are both cleaved by the same IMM-resident proteases and competition between the substrates would favor PINK1 stabilization (followed by PRKN recruitment) if PGAM5 accumulates.
This mechanism may serve as fine-tuned feedback allowing cells to sense when the quality of their mitochondrial population is declining even before the mitochondrial membrane potential loss, and it is time to step in and increase the sensitivity of its mitophagy machinery. Moreover, such mitophagy can eliminate dysfunctional mitochondria having elevated potential that would favor excessive ROS production. Globally, this feedback could be an effective mechanism to keep cellular ROS homeostasis that is necessary for many cell functions ().
Figure 1. The KEAP1-PGAM5 complex serves as a sensor for mitochondria-produced ROS to induce mitophagy. Under low, physiological ROS production, PGAM5 binds to KEAP1, a substrate adaptor for the CUL3-based E3 ubiquitin ligase, leading to its permanent ubiquitination. As a result, most of the PGAM5 is targeted to proteasomal degradation, and only a small fraction goes to mitochondria. However, elevated ROS (mitochondrially produced superoxide converted to hydrogen peroxide that diffuses across the mitochondrial membranes) oxidize the cysteine residues in KEAP1. As a result, PGAM5 dissociates from KEAP1 and avoids proteasomal degradation, thus leading to its accumulation in the cytosol and in mitochondria. In the mitochondria, PGAM5 interferes with PINK1 processing, presumably by affecting IMM proteases, PARL and OMA1, leading to the stabilization of PINK1 at the mitochondrial outer membrane. In turn, stabilized PINK1 sensitizes the PINK1-PRKN mitophagy machinery, thus improving the quality of the mitochondrial population and decreasing ROS production.
Our findings also have an important technical aspect. The usual media used for neuronal cultures (RPMI or B27 supplemented Neurobasal) contain different antioxidants. In our experiments, moderate ROS production fails to induce any mitophagy until all antioxidants are removed from the media and supplements used by us. This stresses the importance of antioxidant activity control in experiments studying mitophagy.
In addition, exogenous KEAP1 inhibitors of various chemical classes (some of which are currently at different stages of clinical trial) could activate the KEAP1-PGAM5 pathway and induce mitophagy. These substances include electrophilic KEAP1 inhibitors that covalently modify cysteine residues in KEAP1 and non-electrophilic inhibitors that disrupt the protein-protein interaction between KEAP1 and NFE2L2.
We first noticed that electrophilic inhibitors such as dimethyl fumarate/DMF, omaveloxone/RTA408, and RA839 lead to abrupt dissipation of the mitochondrial membrane potential. This suggests that their effect on mitophagy is mixed: although they might also induce PGAM5 accumulation, their main mechanism, by which they induce mitophagy, is likely through the mitochondrial membrane depolarization inducing PINK1 accumulation.
In contrast, non-electrophilic inhibitors that disrupt the protein-protein interaction between KEAP1 and NFE2L2 similarly break down the interaction between KEAP1 and PGAM5. This blocks the proteasomal degradation of PGAM5, leading to its accumulation, and sensitizes the mitophagy machinery of the cell. Our study demonstrated that the amplitude of these effects is relatively moderate under control conditions, and such limited effect could be an advantage for avoiding a massive mitochondrial loss following therapeutic interventions. Such pharmacological control of the KEAP1-PGAM5 system as a tool for mitophagy modulation could open exciting therapeutic perspectives because many disorders (including neurological disorders) are linked to weakened or, conversely, excessive mitophagy.
Acknowledgments
We are grateful for funding from the Estonian Research Council (PRG400) and the European Regional Development Fund (Project No. 2014-2020.4.01.15-0012). A.K. was supported by Chan Zuckerberg Initiative and A.K. and V.V. by Estonian - French Research Program PARROT.
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No potential conflict of interest was reported by the author(s).
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Reference
- Zeb A, Choubey V, Gupta R, et al. A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy. Redox Biol. 2021;48:102186.