Pathological mutations promote proteolysis of mitochondrial tRNA-specific 2-thiouridylase 1 (MTU1) via mitochondrial caseinolytic peptidase (CLPP).

MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.

Regulation of mitochondrial iron homeostasis by sideroflexin 2.

Mitochondrial iron is indispensable for heme biosynthesis and iron-sulfur cluster assembly. Several mitochondrial transmembrane proteins have been implicated to function in the biosynthesis of heme and iron-sulfur clusters by transporting reaction intermediates. However, several mitochondrial proteins related to iron metabolism remain uncharacterized. Here, we show that human sideroflexin 2 (SFXN2), a member of the SFXN protein family, is involved in mitochondrial iron metabolism. SFXN2 is an evolutionarily conserved protein that localized to mitochondria via its transmembrane domain. SFXN2-knockout (KO) cells had an increased mitochondrial iron content, which was associated with decreases in the heme content and heme-dependent enzyme activities. By contrast, the activities of iron-sulfur cluster-dependent enzymes were unchanged in SFXN2-KO cells. Moreover, abnormal iron metabolism impaired mitochondrial respiration in SFXN2-KO cells and accelerated iron-mediated death of these cells. Our findings demonstrate that SFXN2 functions in mitochondrial iron metabolism by regulating heme biosynthesis.

Transactivator protein: An alternative for delivery of recombinant proteins for safer reprogramming of induced Pluripotent Stem Cell.

Induced pluripotent stem cells (iPSC) are somatic cells reprogrammed to pluripotency by forced expression of pluripotency factors. These cells are shown to have the same pluripotent potential as embryonic stem cells (ESC) and considered as an alternative to the much controversial usage of ESC which involved human embryos. However, the traditional method in reprogramming cells into iPSC using genome-integrating retro- or lenti- viruses remains an obstacle for its application in clinical setting. Although numerous studies have been conducted for a safer DNA-based reprogramming, reprogramming of iPSC by genetic modifications may raise the possibility of malignant transformation and has been a major limitation for its usage in clinical applications. Therefore, there is a need for an alternative method to reprogram the cells without the use of gene editing and a much safer way to deliver transcription factors to induce pluripotency on target cells. Using protein transduction approach, a number of studies have demonstrated the generation of human iPSCs from human fibroblasts and mouse embryonic fibroblasts by direct delivery of reprogramming proteins. In this review, the definition and mechanism of HIV-TAT protein (a type of protein transduction domain) in delivering recombinant proteins, including the potential of protein-based delivery to induce iPSC were further discussed.