Pathogenic roles of the IMNEPD-associated PTRH2 mutants in aggravating mitochondrial dynamics and its rescue.

backgroundMitochondrial dynamics, orchestrated by a finely tuned balance between fusion and fission, are critical for cellular homeostasis and development. Dysregulation of these processes has been increasingly implicated in various diseases, including developmental disorders. Although core components, such as mitofusin 1 and 2 (MFN1/2), have been characterized, the broader regulatory network remains incompletely defined.

methodsGain-of-function screening of mitochondrial proteins was performed in human cell lines to identify regulators of mitochondrial morphology. Deletion mutants of PTRH2, MFN1/2, and FKBP8, as well as disease-related PTRH2 truncation mutants (A90fs, W108*), were generated by site-directed mutagenesis. Protein–protein interactions were examined by co-immunoprecipitation and TurboID-based proximity labeling, while mitochondrial morphology was assessed by confocal microscopy. Functional consequences of PTRH2 modulation were evaluated using CRISPR/Cas9 knockout and overexpression models. Mitochondrial function was analyzed by assays of ATP production, membrane potential, and reactive oxygen species, and mitophagy activity was monitored using mito-Keima–based fluorescence imaging.

resultsHere, we identify peptidyl-tRNA hydrolase 2 (PTRH2) as a novel modulator of mitochondrial dynamics through a gain-of-function screening approach. PTRH2 interacts with MFN1/2, interfering with MFN dimerization and thereby suppressing mitochondrial fusion. Notably, the PTRH2 truncation mutants A90fs and W108*, implicated in infantile multisystem neurologic, endocrine, and pancreatic disease (IMNEPD), exhibit an enhanced binding to MFN1/2, leading to mitochondrial fragmentation. This inhibitory interaction is alleviated under amino acid deprivation, a condition known to promote mitochondrial fusion. Furthermore, sustained PTRH2-induced mitochondrial fragmentation results in perinuclear aggregation of the mitochondria via recruiting FK506-binding protein 8 (FKBP8) and impairing mitophagy (Fig. 1).

conclusionsThese findings establish PTRH2 as a crucial negative regulator of mitochondrial fusion, providing new insights into mitochondrial dynamics and the molecular pathology of IMNEPD.