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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
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NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia

Chih-Wei Chen1, Chi Su1, Chang-Yu Huang1

  • 1Institute of Molecular Medicine, College of Medicine, National Taiwan University, 10002, Taipei, Taiwan.

Nature Communications
|March 14, 2024
PubMed

Insights

Nucleoside diphosphate kinase 3 (NME3) regulates mitophagy via histidine phosphorylation, not its kinase activity. This process is crucial for protecting against ischemia/reperfusion injury and maintaining cerebellar function.

Area of Science:

  • Mitochondrial Biology
  • Cellular Stress Response
  • Neuroscience

Background:

  • Nucleoside diphosphate kinase 3 (NME3) is a mitochondrial outer membrane protein.
  • Mitophagy, the selective degradation of mitochondria, plays a role in cellular homeostasis and stress response.
  • Hypoxia and ischemia/reperfusion injury are significant physiological stressors impacting cellular function and survival.

Purpose of the Study:

  • To investigate the role of NME3 in hypoxia-induced mitophagy.
  • To elucidate the mechanism by which NME3 regulates mitophagy, focusing on its active site histidine phosphorylation and phosphatidic acid binding.
  • To determine the impact of NME3 dysfunction on physiological outcomes, such as ischemia/reperfusion injury and cerebellar function.

Main Methods:

  • Utilized knock-in mice with a mutated NME3 gene affecting histidine phosphorylation.
  • Performed mechanistic analysis involving hypoxia, phosphatidic acid (PA) localization, and interactions between NME3, DRP1, and MUL1.
  • Assessed mitophagy levels and DRP1 ubiquitination status under various experimental conditions, including MUL1 overexpression and DRP1 mutant expression.

Main Results:

  • NME3's role in hypoxia-induced mitophagy depends on its active site phosphohistidine, not its nucleoside diphosphate kinase (NDPK) activity.
  • Mice with impaired NME3 histidine phosphorylation exhibited vulnerability to ischemia/reperfusion-induced infarction and cerebellar abnormalities.
  • Hypoxia-induced mitochondrial PA is essential for mitophagy and NME3-DRP1 interaction; NME3 binding stabilizes DRP1 against MUL1-mediated ubiquitination, promoting mitophagy.

Conclusions:

  • NME3, through its phosphohistidine-dependent PA binding, acts as a critical regulator of hypoxia-induced mitophagy.
  • This NME3 function is vital for cellular protection against ischemia/reperfusion injury and maintaining normal cerebellar function.
  • The interaction between active NME3 and DRP1 provides a protective microenvironment for DRP1, facilitating mitophagy.

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