The event in question is N-terminal methionine cleavage, one of the most common co-translational modifications in the cell. When a protein is being built by the ribosome, translation almost always starts with the amino acid methionine. In many proteins, an enzyme called methionine aminopeptidase snips that first residue off almost as soon as it emerges, a process governed by the identity of the second amino acid in the chain. For proteins destined for mitochondria, the situation is more complicated, because the N-terminus often carries the targeting information that directs the protein to the organelle and then must be processed again inside. What the new study shows is that this single-residue trimming, far from being an incidental byproduct of maturation, is globally consequential: proteins that undergo this cleavage are collectively stabilized as components of mitochondrial complexes, and blocking the pathway destabilizes assemblies across the organelle.

To reach that conclusion, the researchers combined quantitative proteomics, which measures the abundance of thousands of proteins at once, with methods for assessing how those proteins behave in their native complexes. The central observation is a correlation with mechanistic weight: mitochondrial proteins whose N-termini are processed by removal of the initiating methionine show a distinctive stabilization signature once they are incorporated into their resident complexes, whereas proteins that retain their full N-terminus do not benefit in the same way. In other words, the cleavage event is not merely decorative. It appears to mark, and in some sense license, the transition of a newly imported polypeptide from a vulnerable, unincorporated state into the stable architecture of the respiratory chain, the contact sites and the metabolic assemblies that give mitochondria their structure and function.

The significance of this becomes clearer when one considers the scale of the protein traffic involved. Mitochondrial biogenesis requires the coordinated synthesis of proteins encoded by two genomes: the nuclear genome produces the overwhelming majority of mitochondrial proteins in the cytosol, while the small mitochondrial genome contributes a handful of essential components of the oxidative phosphorylation system. These two streams must converge with remarkable precision. Subunits of complex I, complex III, complex IV and the ATP synthase are assembled in a defined order, with assembly factors ushering each new piece into place and quality-control proteases disposing of surplus or damaged components. Any imbalance in this choreography — too much of one subunit, too little of another, or a subunit that fails to mature properly — can clog assembly lines and generate reactive oxygen species. A maturation step that applies broadly to imported proteins, and that measurably affects their stability, therefore touches nearly every major assembly pathway in the organelle.

Technically, the study’s strength lies in its systems-level view. Rather than isolating one complex and asking how its subunits mature, the authors surveyed the entire imported proteome and asked which proteins are subject to N-terminal processing and how processing correlates with protein stability and complex incorporation. This global approach revealed that the effect is not confined to a single pathway or a single respiratory complex. Instead, matured N-termini are a shared feature across many mitochondrial protein families, and the stabilizing consequence of cleavage emerges as a general principle of mitochondrial proteostasis — the collective term for the networks that keep the organelle’s protein complement correctly folded, correctly assembled and correctly turned over.

Why would removing one amino acid matter so much? Protein stability at the molecular level is governed by how well a polypeptide’s residues pack against one another and against binding partners. The N-terminus occupies a special position in this calculus: it is the beginning of the chain, it often carries a charged or bulky methionine, and in complexes its position can sit at a subunit interface or near a cofactor-binding site. An untrimmed methionine can create steric clash, alter local charge or interfere with the assembly contacts that hold multisubunit machines together. Conversely, once the residue is removed, the new N-terminal residue can engage in stabilizing interactions or even undergo further modifications, such as N-acetylation, that lock the protein into its mature conformation. The new work suggests that for many mitochondrial proteins, the cleaved state is the state that fits the assembled complex — the final piece of a molecular jigsaw that only clicks into place once the edge has been trimmed.

The findings also connect to a broader theme in mitochondrial biology: the organelle’s extreme sensitivity to defects in protein maturation. Mutations in mitochondrial processing peptidases and related maturation factors have been linked to cardiomyopathy, encephalopathy and other severe human diseases, and defects in N-terminal processing are known to impair the respiratory chain in model organisms. By showing that a single-residue cleavage has global consequences for complex integrity, the study offers a mechanistic framework for understanding why the maturation machinery is so essential. It is not simply that each protein needs its N-terminus trimmed to work; it is that the entire population of imported proteins depends on the process to reach the stable, assembled state that keeps the respiratory chain and other complexes running.

There is also an evolutionary dimension worth savoring. The endosymbiotic origin of mitochondria means the organelle inherited its proteome largely from a bacterial ancestor, yet the bacterial proteins did not need targeting sequences to reach their destination — their modern descendants do. The evolution of the mitochondrial import system, with its cleavable presequences and its two-step processing, created new opportunities for regulation at the N-terminus. The new results imply that evolution exploited this: what began as a targeting requirement became a quality-control and stability checkpoint, in which successful maturation signals that a protein is ready to be committed to a complex. Proteostasis networks inside the organelle can then discriminate between fully matured, import-competent proteins and stalled or incomplete intermediates, directing the latter toward degradation before they can interfere with assembly.

For researchers working on mitochondrial disease, aging and cancer — fields in which mitochondrial dysfunction is central — the study opens concrete lines of inquiry. If N-terminal cleavage stabilizes complexes globally, then defects in the cleavage machinery should be detectable as characteristic destabilization patterns in patient cells or disease models, potentially providing biomarkers. Conversely, understanding the structural rules that make a cleaved N-terminus stabilizing could eventually inform strategies to shore up fragile protein complexes in degenerative conditions. And because the respiratory chain is a major source of cellular energy and a major site of drug targeting in oncology, the observation that its integrity is tuned by a maturation step adds a new variable to any account of how cells regulate their energy supply.

What makes the finding so striking is its economy. Biology is full of elaborate regulatory cascades, yet here a single cut — one residue removed from the tip of a growing protein — turns out to underpin the structural coherence of an entire organelle. Kücükköse, Luzarowski and colleagues have transformed what looked like routine housekeeping into a central principle of mitochondrial proteostasis, reminding us that in the crowded interior of a mitochondrion, even the smallest molecular edits can have consequences measured across the whole complex machinery of life.

Subject of Research: N-terminal amino acid cleavage of imported mitochondrial proteins and its global role in stabilizing mitochondrial protein complexes

Article Title: A single-amino-acid cleavage controls global mitochondrial complex integrity

Article References: Kücükköse, C., Luzarowski, M., Stockert, F., Flotho, A., Cosenza-Contreras, M., Demir, F., Gilbert, M., Dengjel, J., Drepper, F., Jeske, M., Koch, H.-G., Huesgen, P. F., & Vögtle, F.-N. (2026). A single-amino-acid cleavage controls global mitochondrial complex integrity. Nature Structural & Molecular Biology. https://doi.org/10.1038/s41594-026-01876-7

Image Credits: AI Generated

DOI: 10.1038/s41594-026-01876-7

Keywords: mitochondria, N-terminal methionine cleavage, protein import, proteostasis, protein complex stability, oxidative phosphorylation, protein maturation, proteomics, respiratory chain, mitochondrial diseases, single-amino-acid, cleavage

In recent years, a range of mitochondrial base editors (BEs) emerged, including DddA-derived cytosine base editors (DdCBEs) and TALE-linked deaminases (TALEDs). Our own laboratory introduced a new type of mitochondrial base editor, abbreviated mitoBE, capable of cytosine-to-thymine (C-to-T) and adenine-to-guanine (A-to-G) conversions in the mitochondrial genome. These constructs combine a double-stranded or single-stranded DNA deaminase with a DNA nickase, harnessing the potential of transcription activator-like effectors (TALEs) that are customized to bind specific sequences in mtDNA. The original version of mitoBEs, described previously, showed minimal off-target editing, primarily because the TALE design confers strong strand specificity. Nonetheless, the fervent push for generating mouse models to replicate the precise mutations implicated in human mitochondrial disorders called for an even higher level of accuracy and efficiency, particularly when microinjecting these tools as RNA into zygotes.

The next-generation variant, termed mitoBEs v2, arose from the thorough redesign of adenine and cytosine deaminases to mitigate unwanted off-target edits. When expressed in cells, the original versions of these mitochondrial base editors were shown to cause occasional undesired edits in the mitochondrial genome and, more significantly, in transcripts, because certain cytosine and adenine deaminases can inadvertently bind and deaminate RNA. This risk becomes more pronounced when the editors are delivered in the form of mRNA, which yields higher expression levels and correspondingly higher on-target editing but also riskier interactions with off-target substrates. By strategically mutating and screening key residues within TadA, a bacterial adenine deaminase commonly used for adenine base editing, the new version of mitoABE included the substitution V28F in TadA8e-V106W, leading to substantially improved specificity and reduced RNA off-target edits. In parallel, our team explored a library of cytosine deaminases for an alternative to APOBEC1—the widely used but often promiscuous deaminase—ultimately selecting CBE6d, a TadA-derived cytosine deaminase that delivered a higher degree of efficiency and a narrower editing window.

Having validated mitoBEs v2 in cultured cells, we systematically screened 70 pathogenic point mutations in the mouse mitochondrial genome that mirror known human mutations. Among these, we found 68 to be editable by mitoBEs v2, indicating that the modified editors maintain broad applicability for disease modeling. The editing efficiency often exceeded 10%, and in some loci, including mt-Rnr1 A978G, mt-TrnV G1029A, mt-Atp6 T8576C, mt-Atp6 T8591C, mt-Nd5 T12499C, and mt-Nd5 A12784G, we observed rates of up to 20–25% in cultured neuroblastoma cells. While such cell-line testing provides only an initial screening for the feasibility of each target site, it also reveals the interplay between TALE binding, deaminase activity, and the architecture of the target sequence in shaping how effectively any given site can be mutated.

The next challenge was to translate this high efficiency to live animals, where editing has to happen at the zygote stage so that every cell in the adult mouse carries the mtDNA change, making it possible to recapitulate human disease phenotypes. We chose two specific positions for in vivo proof-of-concept studies: mt-Atp6 T8591C, corresponding to the human m.T9191C mutation implicated in Leigh syndrome, and mt-Nd5 A12784G, mirroring the human m.A13379G mutation implicated in LHON. When we delivered mRNA or circRNA encoding the new mitoBEs v2 into one-cell mouse embryos, the editing efficiencies reached unprecedented levels. Notably, circRNA-encoded mitoBEs v2 proved more effective than mRNA-encoded versions, often doubling the mutation rates. In some embryos, we attained editing efficiency upward of 60% at T8591C or 62% at A12784G when analyzed at the blastocyst stage. Extending this success to live offspring, many F0 mice carried mutation loads of 40–50% or even up to 82% at their respective loci, underscoring the ability of mitoBEs v2 to create highly heteroplasmic or near-homoplasmic conditions.

This level of editing in F0 mice is particularly important for studying mitochondrial disorders because disease phenotypes often manifest only when the proportion of mutated mtDNA surpasses a threshold. In LHON, for instance, the typical threshold is around 60% for the mutant mtDNA to induce the visual impairments commonly observed in patients, though lower loads can sometimes be sufficient. By generating mice in which the majority of their mtDNA is mutated, we produce more faithful analogs of human disease states, facilitating a clearer understanding of pathological mechanisms and the development of treatments. Importantly, we verified the specificity of these edits. Whole-genome sequencing revealed no significant off-target editing within the nuclear genome at relevant sequencing depths, and a thorough survey of potential off-target sites, including computational predictions with TALENoffer, showed the background to be effectively clean. Even in the mitochondrial genome, where the original version of mitoCBEs occasionally introduced off-target conversions, the new cytosine deaminase variant CBE6d displayed minimal detectable bystander or off-target edits.

One of the most intriguing discoveries about these newly engineered mice was the extent to which the mutations were stably propagated across various somatic tissues and over time. By sampling 26 tissues at 2 months of age in two different F0 mice, we saw that editing levels remained relatively stable in many tissues. Some variations were detected, which could be due to the interplay between environmental factors, tissue-specific energetic demands, and potential selective pressures on certain mtDNA variants. Indeed, such tissue-specific segregation is part of the broader story of the mitochondrial genetic bottleneck, wherein different subpopulations of mtDNA can get amplified or suppressed depending on energetic or developmental constraints.

The question of heritability was addressed by mating female mice carrying the edited mtDNA with wild-type males. mtDNA is well known to be maternally inherited in mammals. We observed that mutation loads fluctuated in F1 and F2 generations, sometimes increasing, sometimes decreasing, a classic manifestation of the mitochondrial bottleneck effect. Remarkably, some F1 offspring attained 100% mutation load at the A12784G site, showcasing the potential to generate fully homoplasmic lines within just one generation. In contrast, the T8591C variants seemed detrimental to embryonic development or gamete maturation, as illustrated by lower birth rates in those lines and a gradual diminution of the T8591C mutation over subsequent generations. By the F2 or F3 generation, T8591C either was substantially reduced or disappeared in most offspring, pointing to a powerful selective force that eliminates highly deleterious mtDNA variants.

These lineage-tracking observations in mice are emblematic of the broader phenomenon experienced by human carriers of pathogenic mtDNA. Some families are known to spontaneously lose or reduce detrimental variants over generations, presumably because these variants compromise oocyte or embryo viability. Conversely, other variants, even if pathogenic, can persist or expand under certain circumstances. The ability to control and quantify these effects in a laboratory mouse population is an invaluable asset for dissecting exactly how mitochondrial heteroplasmy shifts occur, what molecular signals lead to selection for or against certain mtDNA haplotypes, and how to potentially intervene in disease scenarios.

Phenotypic characterization of the F0 mice revealed disease-relevant symptoms that mirror clinical data in humans. Mice with high editing levels at T8591C in mt-Atp6 had significantly reduced heart rates and a notably diminished left ventricular ejection fraction as assessed by echocardiography. Leigh syndrome is often associated with severe cardiovascular and neurological abnormalities. The phenotypic data in these mice strengthen the link between that particular point mutation and the observed phenotype, consistent with human clinical observations that T9191C can lead to Leigh syndrome with pronounced cardiac manifestations. Similarly, the mice carrying the A12784G mutation in mt-Nd5 displayed impaired visual function evidenced by electroretinography. Under dark-adapted conditions, both a-wave and b-wave responses were suppressed in these animals, and under light-adaptation conditions, the b-wave responses were notably depressed. LHON in humans is characterized by central vision loss, optic atrophy, and deficits in the photoreceptor signals, which aligns with these findings. By recapitulating such phenotypes, these new mouse lines represent crucial platforms for future interventions, drug testing, and mechanistic analyses of mitochondrial diseases.

Despite these accomplishments, a challenge remains in achieving truly single-base alterations without introducing secondary edits in the adjacent window. Because TALE-based editors typically incorporate a small window in which deamination can occur, multiple bases within that window can be converted if the sequence context permits. While the presence of bystander mutations is tolerable in certain therapeutic contexts, disease modeling demands the highest precision to unambiguously link genotype to phenotype. In the present study, we addressed this by shifting the TALE-binding sites in the mt-Nd5 A12784G system, effectively narrowing the potential editing window so that only the base at position 12784 is selectively edited. Indeed, some screening in cell lines identified pairs of TALE monomers that yield cleaner edits, and embryo injections of these improved pairs gave rise to F0 mice whose only edit was at the target site. Phenotypic tests using electroretinography on these single-mutation mice confirmed that even in the absence of neighboring bystander mutations, the A12784G change was sufficient to cause LHON-like visual defects. Therefore, rational design of TALE binding, coupled with the improved catalytic specificity of deaminases, can yield nearly perfect single-base mitochondrial edits.

The success of mitoBEs v2 in generating robust mouse models of disease points the way to further possibilities in therapeutic development. Gene therapy approaches using adeno-associated virus or lipid-based nanoparticle systems could, in principle, deliver these editors to adult tissues. However, the inefficiency of delivering proteins or RNAs specifically to mitochondria in vivo remains a formidable obstacle. The impetus to solve such challenges is growing, given that more than 90 disease-related point mutations in human mtDNA have been identified. Of these, around 85 are theoretically addressable by some form of base editor that converts A-to-G or C-to-T. Although the present study focuses on using mitoBEs v2 to produce heritable changes in mouse zygotes, one can envision analogous methods, refined delivery vehicles, or direct in vivo injections that eventually correct pathogenic variants in patients.

Moreover, the principle of generating clean backgrounds for disease modeling fosters confidence that the observed phenotypes reflect the intended single mutation or cluster of mutations, rather than confounding off-target effects in the nuclear genome. As we scaled from single-blastocyst analyses to whole-litter screens and then entire F0 and F1 cohorts, we saw no evidence of spurious large-scale nuclear edits or integration events, which historically have bedeviled certain gene-editing tools. This is crucial not only for basic science but also for any translational endeavor where specificity is key to meeting regulatory standards for safety.

Another intriguing aspect of the new mitochondrially targeted editors is their compatibility with circular RNA (circRNA). CircRNA vectors are more stable than linear mRNAs, conferring prolonged expression. This was reflected in the heightened editing efficiencies in mouse embryos injected with circRNA constructs, where sustained editor expression presumably facilitated more comprehensive editing of mtDNA. The successful translation of circRNA in mitochondria-targeted editing underscores the broader potential of circular RNA technologies across various domains of gene therapy, from disease modeling in preclinical species to future therapeutic interventions in humans.

In sum, the research on mitoBEs v2 ushers in a new era of mitochondrial disease modeling, bridging the gap between theoretical constructs of disease-causing mutations and fully realized mouse lines that recapitulate human syndromes. The capacity to achieve editing efficiencies of up to 82% in F0 mice, combined with the successful demonstration of maternal inheritance and phenotypic manifestations closely matching known human conditions, is a definitive testament to the power of these refined genome-editing tools. These mouse models have already yielded insights into how pathogenic mtDNA influences embryonic and tissue-specific viability, how the mutation load can shift dramatically from mother to offspring, and how single-point changes in critical mitochondrial proteins directly provoke the cardinal features of Leigh syndrome or LHON. For the research community, the newly minted mouse lines that mimic other mitochondrial disorders—be it through tRNA mutations or protein-coding gene alterations—offer an expansive toolkit to elucidate unknown mechanisms and test novel therapeutic strategies.

The quest for single-base editing in mitochondrial DNA, once limited by a dearth of tools and hampered by the intricacy of delivering effectors to mitochondria, is now coming to fruition. The lessons learned from the iterative optimization of mitoBEs v2—especially the efforts to minimize RNA off-target edits and reduce promiscuous deaminase activity—will undoubtedly inform future developments, perhaps enabling the next generation of mitochondrially targeted editors to be even safer and more precise. Ultimately, the synergy between robust mitochondrial base editing and advanced in vivo delivery systems could herald genuine clinical interventions for a wide spectrum of mitochondrial disorders, enabling the possibility that familial burdens associated with debilitating mutations can be alleviated by precise reprogramming of the mitochondrial genome. Far from being a mere technical feat, these breakthroughs embody a promising leap toward conquering inherited metabolic conditions that have long stood as insurmountable clinical challenges.

Subject of Research: Mitochondrial genome base editing to create mouse models of human mitochondrial diseases

Article Title : Precise modelling of mitochondrial diseases using optimized mitoBEs

News Publication Date : 22 January 2025

Keywords : Mitochondrial diseases, base editing, mitoBEs v2, genome editing, animal models, TALE-fused deaminases, off-target effects, maternal inheritance, disease phenotypes, Leigh syndrome, LHON, circRNA technology