Mitochondria, as crucial semi-autonomous organelles within cells, are primarily responsible for energy production. They are central to aerobic respiration and oxidative phosphorylation and play a core role in metabolic and homeostatic regulation. Mitochondrial functional states are influenced by various factors, including mitochondrial membrane potential, membrane channels, intra-mitochondrial calcium ion concentration, the activity of respiratory complexes, reactive oxygen species (ROS) production, and mutations in mitochondrial DNA. To maintain mitochondria's functional and structural integrity, cells employ several mechanisms to control mitochondrial quality, among which mitophagy is a key regulatory means. Mitophagy is a selective autophagy process aimed at clearing damaged or dysfunctional mitochondria to maintain cellular health and homeostasis. The mitophagy pathway includes the following major steps:

1. PINK1 Stabilization: Under mitochondrial stress/depolarization, the TOM and TIM complexes prevent PINK1 from entering the mitochondria, resulting in the stabilization of full-length PINK1 on the mitochondrial outer membrane and subsequent cleavage by the inner membrane protease PARL.

2. PINK1 Activation: When stabilized on the mitochondrial surface, PINK1 is activated through autophosphorylation at the Ser228 site.

3. Ubiquitin Phosphorylation: Activated PINK1 phosphorylates ubiquitin on substrates at the Ser65 site on the mitochondrial outer membrane.

4. Parkin Recruitment: Phosphorylated ubiquitin at the Ser65 site recruits cytosolic Parkin (a cytosolic E3 ubiquitin ligase) to the mitochondria.

5. Parkin Phosphorylation: Once at the mitochondria, PINK1 phosphorylates Parkin at the Ser65 site, thereby activating Parkin, which also binds to phosphorylated ubiquitin.

6. Parkin-Dependent Ubiquitination of Substrates: Activated Parkin ubiquitinates numerous substrates on the mitochondrial outer membrane. Increased ubiquitin chains result in higher levels of phosphorylated ubiquitin, thus amplifying the recruitment of Parkin in a feed-forward loop.

7. Recruitment of Autophagy Receptors: Ubiquitin molecules on the mitochondrial surface act as "eat-me signals," promoting the recruitment of the ubiquitin-proteasome system (UPS) mechanism and the autophagy mechanism. Autophagy receptors then bridge ubiquitinated mitochondria to LC3-positive autophagosomes. TBK1 phosphorylation of autophagy receptors enhances the interaction between LC3 and ubiquitin.

8. Autophagosome Formation: Ubiquitinated mitochondria are engulfed by autophagosomes.

9. Fusion with Lysosomes: Autophagosomes fuse with lysosomes, where lysosomal hydrolases degrade the autophagosomal contents.

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Molecular Mechanisms of Mitophagy

Mitophagy mechanisms can be classified into ubiquitin (Ub)-dependent and Ub-independent pathways.

• Ubiquitin-Dependent Pathway

In the Ub-dependent mitophagy pathway, PINK1 (PTEN-induced kinase 1) and Parkin are key proteins. This pathway is also known as the PINK1–Parkin-mediated mitophagy pathway. When the mitochondrial membrane potential is impaired, PINK1 accumulates on the outer mitochondrial membrane and activates Parkin. Parkin initiates the autophagy process by ubiquitinating proteins in the mitochondria. Furthermore, PINK1 recruits autophagy receptor proteins (e.g., NIX, BNIP3, and FUNDC1) through direct ubiquitin phosphorylation, facilitating the autophagic sequestration and clearance of mitochondria.

Figure 1. Mechanisms of mitophagy regulation: pink1-parkin-independent pathways and key proteins involved. (Wang S, et al., 2023)

• Ubiquitin-Independent Pathway

Mitophagy that does not depend on ubiquitin is primarily driven by mitochondrial autophagy receptors such as NIX, BNIP3, and FUNDC1. These receptors contain a conserved LC3-interacting region (LIR) that can directly bind to autophagy-related proteins, initiating the autophagy process.

Role of Mitophagy in Diseases

• Neurodegenerative Diseases

Mitophagy plays a crucial role in neurons, particularly in managing damaged mitochondria. If these damaged mitochondria are not timely removed, they can produce harmful substances such as ROS, which may further trigger or exacerbate neurodegenerative diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic Lateral Sclerosis (ALS). For instance, studies have found that ALS-related genes (e.g., OPTN) and the TBK1 kinase are pivotal in regulating mitophagy. Modulating these genes and proteins might potentially lead to new therapeutic strategies for these neurodegenerative conditions.

• Cardiovascular Diseases

Enhanced mitophagy contributes to improved cardiovascular health by lowering blood pressure and protecting the cardiovascular system. Research indicates that mutations in the PARK2 gene (which encodes Parkin) are associated with hypertension. Astaxanthin, a natural antioxidant, promotes mitophagy by increasing PINK1 and Parkin expression, consequently reducing hypertension-induced vascular remodeling. This discovery provides new avenues for hypertension prevention and treatment.

• Cancers

Mitophagy suppresses tumor growth by removing dysfunctional mitochondria. In gastric cancer, the loss of PINK1 leads to the Warburg effect and impaired mitophagy, while Parkin affects metabolic remodeling and glycolysis in cancer cells. Research suggests that by modulating these key factors, tumor occurrence and progression can be effectively inhibited, offering a novel anti-cancer strategy.

• Metabolic Diseases

Mitophagy is closely linked to diabetes. Parkin-mediated mitophagy can maintain pancreatic β-cell function, thereby preventing Type 1 diabetes (T1D). High glucose levels promote ROS production, which can damage tissues. Thus, the expression levels of mitophagy-associated proteins significantly change in diabetic patients. By regulating these proteins, diabetic symptoms and prognosis can be improved, providing new directions for diabetes treatment.

Detection Methods for Mitophagy-Related Proteins

Mitophagy levels are relatively low under normal homeostatic conditions, making protein detection challenging. Chemical stimulation of samples can enhance mitophagy, facilitating protein detection. For instance, with PINK1 and BNIP3 as examples:

PINK1: Under homeostatic conditions, PINK1 exists in a cleaved form, with the full-length form being scarce under normal physiological conditions. Under autophagy-inducing conditions, PINK1 accumulates on the mitochondrial outer membrane, observable as enhanced bands in Western Blot (WB) experiments.

BNIP3L/NIX and BNIP3: Under hypoxic conditions, BNIP3L and BNIP3 are transcriptionally activated, anchoring as homodimers on the mitochondrial outer membrane to induce mitophagy. Similar effects can be achieved using cobalt chloride to mimic hypoxia.

Mitophagy plays a crucial role in maintaining cellular homeostasis and preventing diseases. In-depth research into the mechanisms of mitophagy and detection methods for related proteins is significant for understanding its role in diseases. These studies will not only help reveal how cells maintain health through autophagy mechanisms but also provide potential disease prevention and treatment strategies. By studying mitophagy, we can better understand the dynamic balance of the intracellular environment and its impact on disease development, laying the foundation for future medical research and clinical therapies.

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