Cross-species evaluation of TANGO2 homologs, including HRG-9 and HRG-10 in Caenorhabditis elegans, challenges a proposed role in heme trafficking

Our data, coupled with clinical observations in TDD, do not support a primary role for TANGO2 as a heme chaperone. In C. elegans lacking TANGO2 homologs, we were unable to fully replicate prior findings of defective heme transport using toxic and fluorescent heme analogs. Parallel studies in yeast and zebrafish failed to reproduce previously reported phenotypes in growth and muscle fiber integrity, respectively.

Exposing worms to GaPP, a toxic heme analog, we observed that nematodes deficient in HRG-9 and HRG-10 displayed increased survival compared to WT worms, consistent with prior work (Sun et al., 2022), though the between-group difference was markedly smaller in our study. We required higher GaPP concentrations to induce lethality, potentially due to product vendor differences, but did observe a clear dose-dependent effect across strains. Although it was previously proposed that the survival benefit seen in worms lacking HRG-9 and HRG-10 resulted from reduced transfer from intestinal cells after GaPP ingestion, our data suggest the reduced lethality is more likely due to decreased environmental GaPP uptake. Supporting this notion, DKO worms exhibited lawn avoidance, reduced pharyngeal pumping, and modestly lower intestinal ZnMP accumulation when exposed to this fluorescent heme analog on agar plates. In liquid media, DKO worms demonstrated higher fluorescence, but only in ZnMP-free conditions, suggesting the presence of gut granule autofluorescence. Furthermore, survival following exposure to GaPP was highest in eat-2 mutants, despite heme trafficking being unaffected in this strain.

In addition to altered pharyngeal pumping, DKO worms displayed multiple previously unreported phenotypic features, suggesting a broader metabolic impairment reminiscent of some clinical manifestations observed in patients with TDD. Elucidating the mechanisms underlying this phenotype, and whether they reflect a core bioenergetic defect, is an active area of investigation in our lab. Several C. elegans heme-responsive genes have been characterized, revealing relatively specific defects in heme uptake or utilization rather than broad organismal dysfunction. For example, hrg-1 and hrg-4 mutants exhibit impaired growth only under heme-limited conditions (Yuan et al., 2012), and hrg-3 loss affects brood size and embryonic viability specifically when maternal heme is scarce (Chen et al., 2011). By contrast, hrg-9 and hrg-10 mutants exhibit the most severe phenotypes of the hrg family to date, including reduced pharyngeal pumping, decreased motility, shortened lifespan, and smaller broods, even when fed a heme-replete diet.

Laboratory abnormalities in human patients TDD include abnormal acylcarnitine profiles, hyperammonemia, and elevated creatinine kinase levels during metabolic crises (Powell et al., 2021; Miyake et al., 2023), while abnormalities associated with defective heme transport (e.g., erythrocyte membrane defects, low hemoglobin levels) have not been observed in patients with this condition. Strikingly, retrospective data suggest that patients with TDD receiving B-vitamin supplementation inclusive of pantothenic acid, a precursor of CoA, do not experience metabolic crises (Miyake et al., 2023) and show substantial improvement in other domains as well. Pantothenic acid supplementation also yielded full phenotypic rescue in a Drosophila model of TDD (Asadi et al., 2023). It is difficult to reconcile how this treatment would be beneficial in a condition characterized by dysregulated heme trafficking.

Heme is a hydrophobic molecule; thus, it is plausible that if TANGO2 and its homologs are involved in lipid binding, as was recently demonstrated in Hep2G cells (Lujan et al., 2025), these proteins may also weakly bind heme. Han et al., 2023 demonstrated that a bacterial heme homolog, HtpA, directly binds heme and is necessary for cytochrome c function. We would note, however, that TANGO2 was not among the 378 heme-binding proteins identified on a recent proteomic screen of three separate cell lines (Homan et al., 2022). Jayaram et al., 2025 recently proposed that TANGO2 may instead interact with the mitochondrial heme exporter FLVCR1b to release mitochondrial heme without directly binding to heme itself, though this interaction was observed only after heme synthesis was potentiated via d-ALA and iron supplementation, raising questions about the role of TANGO2 under basal heme conditions. Furthermore, the investigators identified GAPDH as the exclusive binding partner of heme upon export through FLVCR1b. How GAPDH, a protein important for multiple cellular functions, including glycolysis, is affected in TDD remains unknown.

In this study, we demonstrated that hrg-9 expression is strongly induced by paraquat, a generator of superoxide free radical. Prior work has also identified hrg-9 as a major transcriptional target of the mitochondrial unfolded protein response (mtUPR) through ATFS-1 transcription factor binding (Soo and Van Raamsdonk, 2021; Di Pede et al., 2025). Given that heme is an essential component of cytochromes in the electron transport chain, heme deficiency could plausibly activate hrg-9 through the induction of mitochondrial stress. Conversely, artificially stimulating heme synthesis may exert a similar transcriptional effect, as excess heme is mitotoxic and can itself trigger a stress response. Indeed, hrg-9 was also shown to be modestly upregulated in the high-heme state, unlike other genes in the hrg family (Figure 3A), and our reanalysis of an RNA-seq dataset examining transcription under low-heme conditions revealed broad induction of stress-responsive genes with no established role in heme trafficking (Figure 3B).

In summary, our findings challenge the notion that TANGO2 functions as a heme chaperone. Instead, and consistent with growing clinical evidence, they support a model in which TANGO2 may help mitigate cellular stress and maintain mitochondrial function under conditions of redox imbalance (Figure 5). Clearly, further work is needed to define the precise role of this highly conserved protein as we work to develop effective treatments for patients with TDD.

Proposed model of TANGO2 and its homologs acting as stress-responsive mediators of mitochondrial dysfunction.

(A) Paraquat exposure and heme deficiency each induce reactive oxygen species (ROS) formation, mitochondrial stress, and enrichment of hrg-9 in C. elegans. TANGO2 and its homologs may help mitigate mitochondrial stress under these conditions, though the exact function of these proteins remains unknown. (B) Physiological triggers such as fasting or illness also precipitate oxidative stress in the absence of TANGO2. Abnormal lipid profiles have been observed in multiple models of TANGO2 deficiency in the setting of impaired lipid mobilization and reduced fatty acid oxidation. In humans, loss of TANGO2 results in a complex clinical syndrome reminiscent of multiple secondary mitochondrial disorders. In C. elegans, loss of hrg-9 and hrg-10 induces a phenotype previously observed in nematode strains exhibiting mitochondrial dysfunction. Created with BioRender.com.