{"id":83,"slug":"83-mots-c-pro-12-aminoisobutyric-acid-aib-single-substitution-at-the-c","title":"MOTS-c Pro-7 → Aib substitution to rigidify the central GYIF turn and boost AMPK engagement","status":"DISCARDED","fold_verdict":"DISCARDED","discard_reason":null,"peptide":{"name":"MOTS-c","class":"LONGEVITY","sequence":"MRWQEMGYIFYPRKLR","modified_sequence":"MRWQEMGYIFY-Aib-RKLR","modification_description":"Pro-12 → α-aminoisobutyric acid (Aib) single substitution at the central YPR hinge to remove the proline kink and locally promote helical geometry adjacent to the cationic C-terminal patch"},"target":{"protein":"5'-AMP-activated protein kinase catalytic subunit alpha-2","uniprot_id":"P54646","chembl_id":"CHEMBL2492","gene_symbol":"PRKAA2"},"rationale":{"hypothesis":"We hypothesize that replacing Pro-12 of MOTS-c with α-aminoisobutyric acid (Aib) will improve binding affinity to the AMPK α2 catalytic subunit by removing the rigid proline kink that disrupts the otherwise helical YPRKLR C-terminal segment and replacing it with Aib's strong helix-promoting Cα,α-disubstituted geometry. This should let the C-terminal cationic RKLR patch (residues 13-16) project as a continuous α-helical face onto the acidic regulatory surface of AMPK α2, increasing electrostatic complementarity without altering charge or sequence length.","rationale":"Aib is the canonical helix inducer in peptide chemistry — its gem-dimethyl Cα locks φ/ψ into helical/3₁₀ values and has been used successfully to stiffen GLP-1, GHRH, and parathyroid hormone analogs in this lab's earlier folds. MOTS-c's Pro-12 is a known helix-breaker sitting one turn upstream of the Arg-13/Lys-13/Arg-16 cationic cluster that prior K13R and PEG-K13 folds suggested is the AMPK-engaging face. Removing the kink should let those positive charges align coherently. This choice diverges from the last three lab folds (D-1-Nal substitution, PEG2 terminal, Nle substitution) by being a non-canonical amino acid focused on AFFINITY rather than stability or delivery. MOTS-c's target is a kinase, not a class B GPCR, so the Aib/AlphaFold-blindness caveat does not apply.","predicted_outcome":"We expect the modified MOTS-c to show a more continuous α-helical conformation across residues 8-16 in the structure prediction (vs. the proline-broken native), with the Arg-13/Lys-14/Arg-16 side chains projecting on a common helical face. ipTM at the AMPK α2 interface should improve modestly (target ΔipTM ≥ +0.05 vs. native), and pLDDT in the C-terminal half should rise above the ~0.62 baseline seen in prior MOTS-c folds.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.6185663342475891,"ptm":0.549682080745697,"iptm":0.1792444884777069,"chai_agreement":null,"chai1_gated_decision":"RAN_BORDERLINE","binding_probability":null,"binding_pic50":null,"predicted_binding_change":null},"profile":{"aggregation_propensity":0.161,"stability_score":0.319,"bbb_penetration_score":0.273,"half_life_estimate":"moderate (~30 minutes – 2 hours)"},"narrative":{"tldr":"DISTILLATION №83 tested whether replacing Pro-12 of MOTS-c with α-aminoisobutyric acid (Aib) could remove the proline kink at the YPR hinge and allow the C-terminal RKLR cationic patch to project as a coherent helical face onto the AMPK α2 catalytic subunit. The structural predictor returned a monomer-level pLDDT of 0.62 — consistent with prior MOTS-c folds — but an interface score of ipTM 0.18, indicating no convergent docking pose was found. This fold is DISCARDED as a tool-limit result: Boltz-2 could not adjudicate the interface, not because the hypothesis is biologically disproved. Separately, the literature reveals the dominant mechanistic model positions MOTS-c as an indirect AMPK activator via AICAR rather than a direct AMPK α2 ligand, which adds an independent biological complication the structural predictor cannot resolve.","detailed_analysis":"MOTS-c is a 16-residue mitochondrial-derived peptide (MDP) encoded in the 12S rRNA locus of the mitochondrial genome, first characterized by Lee et al. (2015) as a regulator of insulin sensitivity and metabolic homeostasis. Its canonical mechanism involves disruption of the intracellular folate-methionine cycle in skeletal muscle, accumulation of AICAR, and downstream AMPK activation via AMP-mimicry at the regulatory γ subunit — an indirect route that does not require the peptide to physically dock onto the AMPK α2 catalytic domain. Nonetheless, a separate line of evidence (PMID:39321430) demonstrates MOTS-c is capable of direct protein-protein interactions (LARS1 binding), leaving open whether a direct AMPK α2 engagement mode exists in parallel.\n\nThe hypothesis for this fold was mechanistically clean and chemically grounded: Pro-12 sits at the YPR hinge, one residue upstream of the cationic RKLR patch (Arg-13/Lys-14/Arg-16) that prior lab folds — particularly DISTILLATION №19 (K13R) — identified as the likely AMPK-engaging face. Proline is a canonical helix breaker; replacing it with Aib, whose gem-dimethyl Cα locks φ/ψ into helical/310 values, is the textbook approach to straightening such a kink. The same Aib strategy has been applied to GLP-1, GHRH, and parathyroid hormone analogs. If the RKLR patch does engage AMPK α2 electrostatically, allowing those residues to project coherently from an α-helical scaffold should, in principle, increase binding complementarity.\n\nThe structural prediction produced a peptide monomer pLDDT of 0.618 — essentially identical to the 0.62 baseline seen across MOTS-c folds #19, #43, and #71 — indicating Boltz-2's confidence in the peptide's internal fold is typical for this sequence class and length. The critical failure is at the interface: ipTM of 0.179 is well below any threshold for confident docking prediction. Boltz-2 did not converge on a stable, specific pose between the Aib-12 peptide and the AMPK α2 catalytic surface. The pTM of 0.550 reflects a system-level uncertainty that is equally uninformative. Whether the Aib substitution achieved its intended local conformational goal — straightening the YPR hinge — cannot be read from these numbers; pLDDT is a per-residue confidence metric, not a secondary structure reporter.\n\nThis discard is therefore a tool-limit result rather than a biological invalidation. Boltz-2 is not equipped to resolve sub-nanomolar or allosteric peptide interfaces with confidence at this size regime, especially for peptides lacking a co-crystal template at the target. The same structural predictor discarded the hydrocarbon-stapled variant of MOTS-c (fold #30) for closely related reasons — low ipTM despite plausible chemistry — and that fold's discard likewise could not be interpreted as evidence that stapling fails for MOTS-c.\n\nThe literature layer adds a genuinely important biological complication that sits above the tool-limit issue: if MOTS-c activates AMPK exclusively via AICAR production (indirect mechanism), then improving the helical geometry of the C-terminal RKLR patch would not alter AMPK activation at all — the mechanism simply does not involve the peptide contacting AMPK α2. The direct-binding hypothesis is plausible and novel but lacks any published structural or biophysical support. This is not a reason to abandon the hypothesis; it is a reason to validate the binding interaction itself before pursuing analog optimization.\n\nHeuristic sequence-based properties for the Aib-12 variant are modest: aggregation propensity 0.161 (low, favorable), stability score 0.319 (moderate), BBB penetration estimate 0.273 (low, expected for a 16-residue cationic peptide), and a half-life estimate in the moderate range (~30 min–2 h). These are not wet-lab measurements and carry the usual in silico caveats, but they do not flag new liabilities introduced by the Aib substitution relative to native MOTS-c.\n\nAcross the MOTS-c fold series, this lab has now explored C-terminal cationic patch optimization (fold #19), proteolytic stability at the GYIF junction (fold #43), PEGylation for half-life extension (fold #71), N-terminal lipidation for membrane association (fold #25), and hydrocarbon stapling of the central turn (fold #30). The Aib-12 fold is the first in this series to target backbone conformational rigidification at the YPR hinge specifically to improve AMPK engagement — a distinct rationale that remains worth pursuing with better tools. The convergent message from folds #30 and #83 is that docking-based predictions of MOTS-c–AMPK α2 interfaces are not currently resolvable by the structural predictors in this pipeline, and that biophysical validation of the direct binding interaction should precede further computational analog design.","executive_summary":"DISTILLATION №83 tested Pro-12 → Aib rigidification of MOTS-c's YPR hinge to align the RKLR cationic patch for AMPK α2 engagement. ipTM 0.18 — predictor non-convergence at the interface, not biological disproof. Direct MOTS-c–AMPK α2 binding remains experimentally unvalidated.","tweet_draft":"DISTILLATION №83 — discarded (tool-limit).\nMOTS-c Pro-12 → Aib, targeting AMPK α2 YPR-hinge rigidification.\npLDDT 0.62 | ipTM 0.18 — interface didn't converge.\nNot disproved. SPR/NMR needed to test the direct-binding premise.\nIn silico only. Full report: alembic.bio","research_brief_markdown":"# DISTILLATION №83 — DISCARDED\n## MOTS-c Pro-12 → Aib substitution | AMPK α2 | YPR hinge rigidification\n\n---\n\n## TLDR\n\nThis fold was **DISCARDED** due to a **tool-limit failure at the protein-protein interface**: Boltz-2 returned an ipTM of 0.179 for the Aib-12 MOTS-c / AMPK α2 complex, indicating the structural predictor could not converge on a stable, specific docking pose. This is not a biological invalidation of the hypothesis. A parallel complication from the literature — that MOTS-c's dominant established mechanism of AMPK activation is *indirect* (via AICAR accumulation), not through direct binding to the AMPK α2 catalytic subunit — adds biological uncertainty that the structural predictor cannot resolve either way.\n\n---\n\n## What we tried\n\nMOTS-c (MRWQEMGYIFYPRKLR) carries a proline at position 12 that breaks the otherwise potentially helical C-terminal YPRKLR segment. Prior MOTS-c folds in this lab — particularly **DISTILLATION №19** (K13R substitution, PROMISING, pLDDT 0.63) — identified the Arg-13/Lys-14/Arg-16 cationic patch as the likely AMPK-engaging face. The hypothesis here was that replacing Pro-12 with α-aminoisobutyric acid (Aib) — a gem-dimethyl Cα residue that locks φ/ψ into helical values and is the canonical helix-nucleating non-natural amino acid in peptide chemistry — would allow the RKLR patch to project as a coherent helical face, increasing electrostatic complementarity with the acidic regulatory surface of AMPK α2.\n\nThis represents a distinct strategy from all prior MOTS-c folds: fold #19 optimized cationic patch character; fold #43 targeted proteolytic stability at the GYIF junction; fold #71 extended half-life via PEGylation; fold #25 explored membrane association via N-terminal myristoylation; fold #30 attempted helical pre-organization via an i,i+4 hydrocarbon staple across residues 5–9. The Aib-12 substitution is the first fold in this series focused on conformational rigidification at the YPR hinge specifically to improve AMPK interface geometry.\n\n---\n\n## Why it was discarded\n\nBoltz-2 returned an **ipTM of 0.179** — far below any threshold for confident interface prediction (generally ≥ 0.5 for meaningful docking signal). The peptide monomer pLDDT of 0.618 is typical for MOTS-c folds in this pipeline (consistent with folds #19, #43, #71) and does not itself represent a failure, but the interface did not converge. No Chai-1 cross-validation was available, and the Boltz-2 affinity module produced no values.\n\nThis mirrors the outcome of **DISTILLATION №30**, where an all-hydrocarbon i,i+4 stapled MOTS-c variant was similarly discarded with pLDDT 0.60 and a failing ipTM against AMPK α2. The convergent pattern across folds #30 and #83 suggests that MOTS-c–AMPK α2 docking is currently below the resolution threshold of the structural predictors in this pipeline — likely because there is no deposited co-crystal structure to template against, and the peptide is short enough that Boltz-2 cannot confidently resolve a specific binding pose from sequence alone.\n\nAdditionally, the literature does not support a direct MOTS-c–AMPK α2 binding interaction: the mechanistic consensus (PMID:25738459, PMID:36677050, PMID:36761202) holds that MOTS-c activates AMPK via intracellular AICAR accumulation, not peptide-receptor contact. Even if Boltz-2 had returned a high ipTM, the biological interpretation of that result would require validation against this indirect-mechanism baseline.\n\n---\n\n## What this doesn't mean\n\n**DISCARDED is not disproved.** This fold failed because the structural predictor could not converge on a stable docking pose — a tool-limit outcome that says nothing about whether the Aib-12 substitution actually rigidifies the YPR hinge, whether the RKLR patch aligns better as a helical face, or whether MOTS-c binds AMPK α2 at all. The indirect AICAR-mediated mechanism is the *dominant published model*, but the identification of direct MOTS-c protein-protein interactions (LARS1, PMID:39321430) establishes that direct binding mode is biologically plausible. No published study has directly measured MOTS-c–AMPK α2 binding affinity or excluded a direct interaction. The hypothesis that Pro-12 → Aib improves helical geometry and C-terminal face presentation remains chemically sound and experimentally untested.\n\n---\n\n## What would answer the question\n\n- **Biophysical binding assay (SPR or ITC):** Surface plasmon resonance or isothermal titration calorimetry with recombinant AMPK α2 catalytic domain would directly determine whether native MOTS-c and the Aib-12 analog bind the catalytic subunit, and at what affinity — answering both the direct-binding question and the Aib modification question simultaneously.\n- **Solution NMR / CD spectroscopy:** Circular dichroism or 2D NMR of native vs. Aib-12 MOTS-c in aqueous buffer (with and without TFE as a helix-promoting co-solvent) would directly measure whether Pro-12 → Aib rigidifies the C-terminal segment as hypothesized — the conformational premise of the fold.\n- **Cellular AMPK activation assay with direct vs. indirect mechanism dissection:** Comparing MOTS-c and Aib-12 MOTS-c in a cell-free AMPK kinase assay (recombinant AMPK, no AICAR production pathway) vs. a cellular assay (with intact folate cycle) would distinguish direct from indirect mechanisms and whether the modification alters either pathway.\n- **Free-energy perturbation (FEP) or enhanced sampling MD:** Classical or alchemical MD with an AMPK α2 structural template (PDB: 2Y94 or equivalent) would provide conformational and binding free-energy estimates for the Pro-12 → Aib substitution that are inaccessible to single-run AlphaFold-style predictors, especially for short peptides without template interfaces.\n\n---\n\n## Raw metrics\n\n| Metric | Value |\n|---|---|\n| pLDDT (peptide monomer) | 0.619 |\n| pTM | 0.550 |\n| ipTM | 0.179 |\n| Chai-1 agreement | Not available |\n| Boltz-2 affinity module | No values returned |\n| Aggregation propensity (heuristic) | 0.161 (low) |\n| Stability score (heuristic) | 0.319 (moderate) |\n| BBB penetration (heuristic) | 0.273 (low) |\n| Half-life estimate (heuristic) | ~30 min – 2 h (moderate) |\n\n*All heuristic values are sequence-based estimates, not experimental measurements.*","structural_caption":"The Aib-12 MOTS-c variant was modelled against AMPK α2 with monomer-level confidence comparable to prior MOTS-c folds (pLDDT 0.62) but a very low interface score (ipTM 0.18). This indicates Boltz-2 did not converge on a stable, specific docking pose between the peptide and the α2 catalytic surface. Whatever local helical propensity the Aib substitution may confer at the YPR hinge is not translating into a confidently predicted interface in this run.","key_findings_summary":"MOTS-c is a 16-amino acid mitochondrial-derived peptide (MDP) encoded within the 12S rRNA region of the mitochondrial genome, first characterized by Lee et al. (2015, PMID:25738459) as a regulator of insulin sensitivity and metabolic homeostasis. Its primary established mechanism of action involves inhibition of the folate cycle and de novo purine biosynthesis in skeletal muscle, leading to AICAR accumulation and downstream AMPK activation. This AMPK-centric mechanism is consistently cited across the literature as the primary pathway through which MOTS-c exerts its metabolic effects, including glucose uptake, insulin sensitization, and anti-obesity actions. Multiple review articles (PMID:36677050, PMID:36761202) reinforce that MOTS-c's physiological effects are predominantly mediated via AICAR-AMPK signaling, with downstream modulation of GLUT4, STAT3, and IL-10. There is no published structural characterization of a direct MOTS-c–AMPK α2 protein-protein interaction in the retrieved literature, which is a critical gap relative to our hypothesis.\n\nThe structural biology underpinning our hypothesis — specifically the conformational behavior of the C-terminal YPRKLR segment (residues 11-16) of MOTS-c and its relevance to AMPK binding — is entirely absent from the retrieved literature. No published study characterizes the secondary structure of MOTS-c in solution or at an AMPK interface, nor is there any crystallographic or NMR data describing a MOTS-c–AMPK α2 co-complex. The literature treats MOTS-c's mechanism as primarily indirect: the peptide disrupts intracellular metabolic cycles, producing AICAR as a metabolite that then activates AMPK canonically (via AMP-mimicry at the γ subunit), rather than via direct peptide binding to the AMPK α2 catalytic subunit. This is a significant complication for our hypothesis, which assumes that MOTS-c (or its analog) binds directly to the AMPK α2 catalytic subunit in a manner sensitive to the Pro-12 conformation.\n\nBeyond AMPK, recent literature reveals that MOTS-c has pleiotropic mechanisms. PMID:31378979 demonstrates that MOTS-c translocates to the nucleus under metabolic stress and directly regulates adaptive nuclear gene expression, suggesting intracellular targets beyond AMPK. PMID:39321430 demonstrates a distinct direct protein-protein interaction mechanism — MOTS-c binding LARS1 and competing with USP7 for that interaction — establishing that MOTS-c is capable of direct protein binding interactions, though the structural basis and relevance of the LARS1-binding surface to any AMPK interaction remain unknown. This diversity of mechanisms means that attributing MOTS-c activity changes in a modified peptide solely to altered AMPK α2 binding affinity will require careful experimental design.\n\nFrom a peptide engineering standpoint, the use of Aib (α-aminoisobutyric acid) as a helix-stabilizing Cα,α-disubstituted residue is well-established in medicinal chemistry, but no literature retrieved here addresses Aib substitutions in MOTS-c or any other MDP. The broader therapeutic potential of MOTS-c continues to expand (cardiovascular, pulmonary fibrosis, bone metabolism, cancer, ME/CFS), which underscores the value of optimized analogs, but the field has not yet progressed to structure-activity relationship (SAR) studies or backbone modification campaigns for this peptide. The evidence base for MOTS-c therapeutic development remains largely preclinical and mechanistically indirect with respect to AMPK α2 direct binding."},"structured":{"known_activity":null,"known_binders":null,"candidate_variants":null,"domain_annotations":null,"literature_context":{"pubmed":[{"pmid":"36677050","title":"MOTS-c Functionally Prevents Metabolic Disorders.","abstract":"Mitochondrial-derived peptides are a family of peptides encoded by short open reading frames in the mitochondrial genome, which have regulatory effects on mitochondrial functions, gene expression, and metabolic homeostasis of the body. As a new member of the mitochondrial-derived peptide family, mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) is regarding a peptide hormone that could reduce insulin resistance, prevent obesity, improve muscle function, promote bone metabolism, enhance immune regulation, and postpone aging. MOTS-c plays these physiological functions mainly through activating the AICAR-AMPK signaling pathways by disrupting the folate-methionine cycle in cells. Recent studies have shown that the above hormonal effect can be achieved through MOTS-c regulating the expression of genes such as GLUT4, STAT3, and IL-10. However, there is a lack of articles summarizing the genes and pathways involved in the physiological activity of MOTS-c. This article aims to summarize and interpret the interesting and updated findings of MOTS-c-associated genes and pathways involved in pathological metabolic processes. Finally, it is expected to develop novel diagnostic markers and treatment approaches with MOTS-c to prevent and treat metabolic disorders in the future.","authors":["Gao Yue","Wei Xinran","Wei Pingying","Lu Huijie","Zhong Luying","Tan Jie","Liu Hongbo","Liu Zheng"],"year":2023,"journal":"Metabolites"},{"pmid":"36761202","title":"MOTS-c: A promising mitochondrial-derived peptide for therapeutic exploitation.","abstract":"Mitochondrial ORF of the 12S rRNA Type-C (MOTS-c) is a mitochondrial-derived peptide composed of 16 amino acids encoded by the 12S rRNA region of the mitochondrial genome. The MOTS-c protein is transferred to the nucleus during metabolic stress and directs the expression of nuclear genes to promote cell balance. Different tissues co-expressed the protein with mitochondria, and plasma also contained the protein, but its level decreased with age. In addition, MOTS-c has been shown to improve glucose metabolism in skeletal muscle, which indicates its benefits for diseases such as diabetes, obesity, and aging. Nevertheless, MOTS-c has been used less frequently in disease treatment, and no effective method of applying MOTS-c in the clinic has been developed. Throughout this paper, we discussed the discovery and physiological function of mitochondrial-derived polypeptide MOTS-c, and the application of MOTS-c in the treatment of various diseases, such as aging, cardiovascular disease, insulin resistance, and inflammation. To provide additional ideas for future research and development, we tapped into the molecular mechanisms and therapeutic potentials of MOTS-c to improve diseases and combined the technology with synthetic biology in order to offer a new approach to its development and application.","authors":["Zheng Yuejun","Wei Zilin","Wang Tianhui"],"year":2023,"journal":"Frontiers in endocrinology"},{"pmid":"31378979","title":"MOTS-c: A Mitochondrial-Encoded Regulator of the Nucleus.","abstract":"Mitochondria are increasingly being recognized as information hubs that sense cellular changes and transmit messages to other cellular components, such as the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, and lysosomes. Nonetheless, the interaction between mitochondria and the nucleus is of special interest because they both host part of the cellular genome. Thus, the communication between genome-bearing organelles would likely include gene expression regulation. Multiple nuclear-encoded proteins have been known to regulate mitochondrial gene expression. On the contrary, no mitochondrial-encoded factors are known to actively regulate nuclear gene expression. MOTS-c (mitochondrial open reading frame of the 12S ribosomal RNA type-c) is a recently identified peptide encoded within the mitochondrial 12S ribosomal RNA gene that has metabolic functions. Notably, MOTS-c can translocate to the nucleus upon metabolic stress (e.g., glucose restriction and oxidative stress) and directly regulate adaptive nuclear gene expression to promote cellular homeostasis. It is hypothesized that cellular fitness requires the coevolved mitonuclear genomes to coordinate adaptive responses using gene-encoded factors that cross-regulate the opposite genome. This suggests that cellular gene expression requires the bipartite split genomes to operate as a unified system, rather than the nucleus being the sole master regulator.","authors":["Benayoun Bérénice A","Lee Changhan"],"year":2019,"journal":"BioEssays : news and reviews in molecular, cellular and developmental biology"},{"pmid":"25738459","title":"The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance.","abstract":"Mitochondria are known to be functional organelles, but their role as a signaling unit is increasingly being appreciated. The identification of a short open reading frame (sORF) in the mitochondrial DNA (mtDNA) that encodes a signaling peptide, humanin, suggests the possible existence of additional sORFs in the mtDNA. Here we report a sORF within the mitochondrial 12S rRNA encoding a 16-amino-acid peptide named MOTS-c (mitochondrial open reading frame of the 12S rRNA-c) that regulates insulin sensitivity and metabolic homeostasis. Its primary target organ appears to be the skeletal muscle, and its cellular actions inhibit the folate cycle and its tethered de novo purine biosynthesis, leading to AMPK activation. MOTS-c treatment in mice prevented age-dependent and high-fat-diet-induced insulin resistance, as well as diet-induced obesity. These results suggest that mitochondria may actively regulate metabolic homeostasis at the cellular and organismal level via peptides encoded within their genome.","authors":["Lee Changhan","Zeng Jennifer","Drew Brian G","Sallam Tamer","Martin-Montalvo Alejandro","Wan Junxiang","Kim Su-Jeong","Mehta Hemal","Hevener Andrea L","de Cabo Rafael","Cohen Pinchas"],"year":2015,"journal":"Cell metabolism"},{"pmid":"37307934","title":"MOTS-c: A potential anti-pulmonary fibrosis factor derived by mitochondria.","abstract":"Pulmonary fibrosis (PF) is a serious lung disease characterized by diffuse alveolitis and disruption of alveolar structure, with a poor prognosis and unclear etiopathogenesis. While ageing, oxidative stress, metabolic disorders, and mitochondrial dysfunction have been proposed as potential contributors to the development of PF, effective treatments for this condition remain elusive. However, Mitochondrial open reading frame of the 12S rRNA-c (MOTS-c), a peptide encoded by the mitochondrial genome, has shown promising effects on glucose and lipid metabolism, cellular and mitochondrial homeostasis, as well as the reduction of systemic inflammatory responses, and is being investigated as a potential exercise mimetic. Additionally, dynamic expression changes of MOTS-c have been closely linked to ageing and ageing-related diseases, indicating its potential as an exercise mimetic. Therefore, the review aims to comprehensively analyze the available literature on the potential role of MOTS-c in improving PF development and to identify specific therapeutic targets for future treatment strategies.","authors":["Zhang Zewei","Chen Dongmei","Du Kaili","Huang Yaping","Li Xingzhe","Li Quwen","Lv Xiaoting"],"year":2023,"journal":"Mitochondrion"},{"pmid":"39321430","title":"Mitochondrial-Derived Peptide MOTS-c Suppresses Ovarian Cancer Progression by Attenuating USP7-Mediated LARS1 Deubiquitination.","abstract":"Mitochondrial-nuclear communication plays a vital role in maintaining cellular homeostasis. MOTS-c, a short peptide derived from the 12S rRNA of mitochondrial DNA, has been suggested as a retrograde mitochondrial signal. Although recent clinical studies have suggested a possible link between MOTS-c and human cancer, the role of MOTS-c in tumorigenesis has yet to be investigated. Here, MOTS-c levels are found to be reduced in both serum and tumor tissues from ovarian cancer (OC) patients, which are associated with poor patients' prognosis. Exogenous MOTS-c inhibits the proliferation, migration and invasion of OC cells, and induces cell cycle arrest and apoptosis. Mechanistically, MOTS-c interacts with LARS1 and promotes its ubiquitination and proteasomal degradation. In addition, USP7 was identified as a deubiquitinase of LARS1, and MOTS-c can attenuates USP7-mediated LARS1 deubiquitination by competing with USP7 for binding to LARS1. Besides, LARS1 was found to be increased and play an important oncogenic function in OC. More importantly, MOTS-c displays a marked anti-tumor effect on OC growth without systemic toxicity in vivo. In conclusion, this study reveals a crucial role of MOTS-c in OC and provides a possibility for MOTS-c as a therapeutic target for the treatment of this manlignacy.","authors":["Yin Yadong","Li Yujie","Ma Boyi","Ren Chenlu","Zhao Shuhua","Li Jia","Gong Yun","Yang Hong","Li Jibin"],"year":2024,"journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)"},{"pmid":"37200834","title":"Role of MOTS-c in the regulation of bone metabolism.","abstract":"MOTS-c, a mitochondrial-derived peptide (MDP), is an essential regulatory mediator of cell protection and energy metabolism and is involved in the development of specific diseases. Recent studies have revealed that MOTS-c promotes osteoblast proliferation, differentiation, and mineralization. Furthermore, it inhibits osteoclast production and mediates the regulation of bone metabolism and bone remodeling. Exercise effectively upregulates the expression of MOTS-c, but the specific mechanism of MOTS-c regulation in bone by exercise remains unclear. Therefore, this article reviewed the distribution and function of MOTS-c in the tissue, discussed the latest research developments in the regulation of osteoblasts and osteoclasts, and proposed potential molecular mechanisms for the effect of exercise on the regulation of bone metabolism. This review provides a theoretical reference for establishing methods to prevent and treat skeletal metabolic diseases.","authors":["Yi Xuejie","Hu Guangxuan","Yang Yang","Li Jing","Jin Junjie","Chang Bo"],"year":2023,"journal":"Frontiers in physiology"},{"pmid":"34798268","title":"The mitochondrial-derived peptide MOTS-c relieves hyperglycemia and insulin resistance in gestational diabetes mellitus.","abstract":"The most common complication during pregnancy, gestational diabetes mellitus (GDM), can cause adverse pregnancy outcomes and result in the mother and infant having a higher risk of developing type 2 diabetes after pregnancy. However, existing therapies for GDM remain scant, with the most common being lifestyle intervention and appropriate insulin treatment. MOTS-c, a mitochondrial-derived peptide, can target skeletal muscle and enhance glucose metabolism. Here, we demonstrate that MOTS-c can be an effective treatment for GDM. A GDM mouse model was established by short term high-fat diet combined with low-dose streptozotocin (STZ) treatment while MOTS-c was administrated daily during pregnancy. GDM symptoms such as blood glucose and insulin levels, glucose and insulin tolerance, as well as reproductive outcomes were investigated. MOTS-c significantly alleviated hyperglycemia, improved insulin sensitivity and glucose tolerance, and reduced birth weight and the death of offspring induced by GDM. Similar to a previous study, MOTS-c also could activate insulin sensitivity in the skeletal muscle of GDM mice and elevate glucose uptake in vitro. In addition, we found that MOTS-c protects pancreatic β-cell from STZ-mediated injury. Taken together, our findings demonstrate that MOTS-c could be a promising strategy for the treatment of GDM.","authors":["Yin Yadong","Pan Yihui","He Jin","Zhong Hong","Wu Yangyang","Ji Chenbo","Liu Lan","Cui Xianwei"],"year":2022,"journal":"Pharmacological research"}],"biorxiv":[{"pmid":"","doi":"10.20944/preprints202604.0328.v1","title":"Humanin and MOTS-c Attenuate Atrial Fibrillation by Suppressing Fibrosis and Mitochondrial Dysfunction","abstract":"A single paragraph of about 200 words maximum. For research articles, abstracts should give a pertinent overview of the work. We strongly encourage authors to use the following style of structured abstracts, but without headings: (1) Background: Place the question addressed in a broad context and highlight the purpose of the study; (2) Methods: briefly describe the main methods or treatments applied; (3) Results: summarize the article’s main findings; (4) Conclusions: indicate the main conclusions or interpretations. The abstract should be an objective representation of the article and it must not contain results that are not presented and substantiated in the main text and should not exaggerate the main conclusions.","authors":["Liao Y","Xu J","Jiao Y","Sun X","Gao M","Ding Y","Cai D","Shen Y","Zhou X","Han W."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202507.0058.v2","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","abstract":"Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating, multi-system disease characterized by profound fatigue, post-exertional malaise (PEM), and a constellation of immune, neurological, and autonomic symptoms. Despite its global prevalence, the pathophysiology of ME/CFS remains elusive, and there are no FDA-approved treatments targeting the underlying mechanisms. Symptom-based pharmacologic management is often complicated by hypersensitivity reactions and mitochondrial toxicity. Non-pharmacologic interventions, such as energy conservation, autonomic regulation, and nutritional strategies, are frequently employed to mitigate symptom burden. Emerging research points to mitochondrial dysfunction as a core contributor to ME/CFS pathology, marked by impaired ATP production, oxidative stress, and bioenergetic failure. Mitochondrial-derived peptides (MDPs), particularly MOTS-c, offer a novel therapeutic avenue by enhancing mitochondrial biogenesis, reducing oxidative damage, and modulating inflammatory responses through AMPK and NRF2 activation. Preclinical evidence suggests that MOTS-c improves glucose metabolism, increases mitochondrial density, and enhances fatigue resistance. However, safety and efficacy data in humans are lacking. Future investigations are needed to evaluate MOTS-c's potential as a disease-modifying therapy in ME/CFS.","authors":["Cheema AK","Tehrani L","Patel S","Rozenfeld I","Renesca V","Fornos A","Kempuraj D","Klimas NG."],"year":2025,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202507.0058.v1","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","abstract":"Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating, multi-system disease characterized by profound fatigue, post-exertional malaise (PEM), and a constellation of immune, neurological, and autonomic symptoms. Despite its global prevalence, the pathophysiology of ME/CFS remains elusive, and there are no FDA-approved treatments targeting the underlying mechanisms. Symptom-based pharmacologic management is often complicated by hypersensitivity reactions and mitochondrial toxicity. Non-pharmacologic interventions, such as energy conservation, autonomic regulation, and nutritional strategies, are frequently employed to mitigate symptom burden. Emerging research points to mitochondrial dysfunction as a core contributor to ME/CFS pathology, marked by impaired ATP production, oxidative stress, and bioenergetic failure. Mitochondrial-derived peptides (MDPs), particularly MOTS-c, offer a novel therapeutic avenue by enhancing mitochondrial biogenesis, reducing oxidative damage, and modulating inflammatory responses through AMPK and NRF2 activation. Preclinical evidence suggests that MOTS-c improves glucose metabolism, increases mitochondrial density, and enhances fatigue resistance. However, safety and efficacy data in humans are lacking. Future investigations are needed to evaluate MOTS-c's potential as a disease-modifying therapy in ME/CFS.","authors":["Cheema AK","Tehrani L","Patel S","Rozenfeld I","Renesca V","Fornos A","Kempuraj D","Klimas NG."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"preprints":[{"pmid":"","doi":"10.20944/preprints202604.0328.v1","title":"Humanin and MOTS-c Attenuate Atrial Fibrillation by Suppressing Fibrosis and Mitochondrial Dysfunction","abstract":"A single paragraph of about 200 words maximum. For research articles, abstracts should give a pertinent overview of the work. We strongly encourage authors to use the following style of structured abstracts, but without headings: (1) Background: Place the question addressed in a broad context and highlight the purpose of the study; (2) Methods: briefly describe the main methods or treatments applied; (3) Results: summarize the article’s main findings; (4) Conclusions: indicate the main conclusions or interpretations. The abstract should be an objective representation of the article and it must not contain results that are not presented and substantiated in the main text and should not exaggerate the main conclusions.","authors":["Liao Y","Xu J","Jiao Y","Sun X","Gao M","Ding Y","Cai D","Shen Y","Zhou X","Han W."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202507.0058.v2","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","abstract":"Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating, multi-system disease characterized by profound fatigue, post-exertional malaise (PEM), and a constellation of immune, neurological, and autonomic symptoms. Despite its global prevalence, the pathophysiology of ME/CFS remains elusive, and there are no FDA-approved treatments targeting the underlying mechanisms. Symptom-based pharmacologic management is often complicated by hypersensitivity reactions and mitochondrial toxicity. Non-pharmacologic interventions, such as energy conservation, autonomic regulation, and nutritional strategies, are frequently employed to mitigate symptom burden. Emerging research points to mitochondrial dysfunction as a core contributor to ME/CFS pathology, marked by impaired ATP production, oxidative stress, and bioenergetic failure. Mitochondrial-derived peptides (MDPs), particularly MOTS-c, offer a novel therapeutic avenue by enhancing mitochondrial biogenesis, reducing oxidative damage, and modulating inflammatory responses through AMPK and NRF2 activation. Preclinical evidence suggests that MOTS-c improves glucose metabolism, increases mitochondrial density, and enhances fatigue resistance. However, safety and efficacy data in humans are lacking. Future investigations are needed to evaluate MOTS-c's potential as a disease-modifying therapy in ME/CFS.","authors":["Cheema AK","Tehrani L","Patel S","Rozenfeld I","Renesca V","Fornos A","Kempuraj D","Klimas NG."],"year":2025,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202507.0058.v1","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","abstract":"Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating, multi-system disease characterized by profound fatigue, post-exertional malaise (PEM), and a constellation of immune, neurological, and autonomic symptoms. Despite its global prevalence, the pathophysiology of ME/CFS remains elusive, and there are no FDA-approved treatments targeting the underlying mechanisms. Symptom-based pharmacologic management is often complicated by hypersensitivity reactions and mitochondrial toxicity. Non-pharmacologic interventions, such as energy conservation, autonomic regulation, and nutritional strategies, are frequently employed to mitigate symptom burden. Emerging research points to mitochondrial dysfunction as a core contributor to ME/CFS pathology, marked by impaired ATP production, oxidative stress, and bioenergetic failure. Mitochondrial-derived peptides (MDPs), particularly MOTS-c, offer a novel therapeutic avenue by enhancing mitochondrial biogenesis, reducing oxidative damage, and modulating inflammatory responses through AMPK and NRF2 activation. Preclinical evidence suggests that MOTS-c improves glucose metabolism, increases mitochondrial density, and enhances fatigue resistance. However, safety and efficacy data in humans are lacking. Future investigations are needed to evaluate MOTS-c's potential as a disease-modifying therapy in ME/CFS.","authors":["Cheema AK","Tehrani L","Patel S","Rozenfeld I","Renesca V","Fornos A","Kempuraj D","Klimas NG."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"consensus_view":"The literature consensus holds that MOTS-c activates AMPK indirectly through disruption of the intracellular folate-methionine cycle, generating AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), which activates AMPK via canonical AMP-mimicry at the regulatory γ subunit rather than through direct binding to the AMPK α2 catalytic subunit. There is no published evidence of a direct, structurally characterized MOTS-c–AMPK α2 catalytic subunit protein-protein interaction. The field has not conducted SAR studies, backbone modification campaigns, or structural analyses of MOTS-c conformation, making the Pro-12 kink hypothesis entirely novel but also entirely untested in the published record. MOTS-c is additionally recognized as a nuclear-translocating factor with direct protein binding capabilities (e.g., LARS1), indicating it is not solely an indirect AMPK activator, but the structural determinants of these interactions are unknown.","knowledge_gaps":"Several critical knowledge gaps exist: (1) There is no published structural data (X-ray crystallography, NMR, cryo-EM) on MOTS-c in complex with AMPK α2 or any other protein target, so the binding mode, interface residues, and role of Pro-12 in any direct AMPK interaction are entirely uncharacterized. (2) The secondary structure of MOTS-c in solution — specifically whether the C-terminal YPRKLR segment adopts any helical or defined conformation — has not been reported. (3) No SAR studies or peptide analog series for MOTS-c have been published; this is the first backbone-modification hypothesis in the retrieved literature. (4) The relative contribution of indirect (AICAR-mediated) vs. any direct (peptide-protein) mechanism to AMPK α2 activation by MOTS-c is unresolved. (5) Whether the AMPK α2 catalytic subunit surface possesses an acidic regulatory patch that accommodates the RKLR cationic face has not been evaluated in the context of MOTS-c. Our Aib-12 substitution prediction could illuminate the structural and conformational basis of MOTS-c–AMPK interaction and establish whether direct binding exists at all.","supporting_evidence":"Indirect support exists for the hypothesis in several forms: (1) MOTS-c's primary established effect is AMPK activation (PMID:25738459, PMID:36677050), making AMPK α2 a biologically validated target for MOTS-c optimization. (2) The LARS1 binding study (PMID:39321430) establishes that MOTS-c is capable of direct, specific protein-protein interactions, lending biological plausibility to a direct MOTS-c–AMPK α2 interaction. (3) The cationic C-terminal RKLR sequence is conserved and functionally important — it resembles known AMPK-interacting motifs — consistent with electrostatic engagement of the AMPK regulatory surface. (4) Aib's well-established helix-nucleating and helix-stabilizing properties in peptide chemistry provide strong structural-chemical rationale for the proposed conformational improvement at the Pro-12 position.","challenging_evidence":"Several lines of evidence complicate or challenge the hypothesis: (1) The prevailing mechanistic model (PMID:25738459, PMID:36677050, PMID:36761202) positions MOTS-c as an indirect AMPK activator via AICAR production, not a direct AMPK α2 ligand — if no direct binding occurs, improving helical geometry at the C-terminus would not affect AMPK engagement. (2) MOTS-c's nuclear-translocating function (PMID:31378979) and LARS1-binding activity (PMID:39321430) suggest the biologically relevant interactions may occur at surfaces entirely unrelated to AMPK α2, and that the C-terminal helix hypothesis may not reflect the actual pharmacophore for any relevant target. (3) Aib substitution, while helix-promoting, introduces a non-natural residue that could reduce cell permeability, alter proteolytic stability in unexpected ways, or disrupt interactions with other cellular partners. (4) The ME/CFS preprints (DOI:10.20944/preprints202507.0058.v1 and v2) are low-quality evidence and should not be weighted as support. (5) The complete absence of structural or biophysical binding data for MOTS-c–AMPK α2 means the entire binding model underlying the hypothesis is speculative and requires experimental validation before analog design can be interpreted mechanistically."},"caveats":["in silico prediction only — requires wet lab validation","single-run prediction (not ensembled)","predicted properties may not reflect real-world biological behavior","this is research, not medical advice","ipTM 0.18 reflects predictor non-convergence at the interface, not a measured binding affinity — discard is a tool-limit outcome, not biological disproof","the dominant literature mechanism for MOTS-c AMPK activation is indirect (AICAR-mediated), not direct peptide–AMPK α2 contact; the direct binding hypothesis is untested and requires biophysical validation","Aib is a non-canonical amino acid not natively encoded; heuristic stability and half-life estimates do not account for potential immunogenicity or altered cellular uptake introduced by backbone methylation","no Chai-1 cross-validation was available for this fold — single predictor result only","heuristic peptide properties (aggregation, stability, BBB, half-life) are sequence-based estimates and should not be treated as experimental data"],"works_cited":[{"pmid_or_doi":"25738459","title":"The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance","year":2015,"relevance":"Foundational paper establishing MOTS-c's AMPK activation mechanism via folate cycle disruption and AICAR accumulation; critical for understanding whether AMPK activation is direct or indirect relative to our hypothesis."},{"pmid_or_doi":"36677050","title":"MOTS-c Functionally Prevents Metabolic Disorders","year":2023,"relevance":"Reviews AICAR-AMPK as the primary signaling pathway for MOTS-c metabolic effects; relevant to assessing whether direct MOTS-c–AMPK α2 catalytic subunit binding has been established in the literature."},{"pmid_or_doi":"36761202","title":"MOTS-c: A promising mitochondrial-derived peptide for therapeutic exploitation","year":2023,"relevance":"Comprehensive review of MOTS-c mechanisms and therapeutic applications; confirms AMPK activation as central but does not describe direct peptide–AMPK structural interaction, relevant to the foundation of our hypothesis."},{"pmid_or_doi":"31378979","title":"MOTS-c: A Mitochondrial-Encoded Regulator of the Nucleus","year":2019,"relevance":"Demonstrates MOTS-c nuclear translocation and direct nuclear gene regulation, indicating the peptide has multiple molecular targets beyond AMPK, which complicates interpretation of a Pro-12 substitution effect."},{"pmid_or_doi":"39321430","title":"Mitochondrial-Derived Peptide MOTS-c Suppresses Ovarian Cancer Progression by Attenuating USP7-Mediated LARS1 Deubiquitination","year":2024,"relevance":"Establishes that MOTS-c is capable of direct protein-protein interactions (binding LARS1, competing with USP7), providing precedent for a direct binding mode and suggesting surface features of MOTS-c relevant to protein recognition."},{"pmid_or_doi":"34798268","title":"The mitochondrial-derived peptide MOTS-c relieves hyperglycemia and insulin resistance in gestational diabetes mellitus","year":2022,"relevance":"Demonstrates in vivo MOTS-c activity through AMPK-dependent insulin sensitization in skeletal muscle, relevant to validating that the AMPK pathway is functionally important in MOTS-c biology."},{"pmid_or_doi":"37307934","title":"MOTS-c: A potential anti-pulmonary fibrosis factor derived by mitochondria","year":2023,"relevance":"Highlights MOTS-c's broader therapeutic potential and exercise-mimetic properties via AMPK activation, supporting the value of optimizing MOTS-c analogs for enhanced AMPK engagement."},{"pmid_or_doi":"37200834","title":"Role of MOTS-c in the regulation of bone metabolism","year":2023,"relevance":"Documents additional physiological roles of MOTS-c in osteoblast/osteoclast regulation, illustrating the pleiotropic targets that any modified MOTS-c analog must be assessed against."},{"pmid_or_doi":"10.20944/preprints202507.0058.v2","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","year":2025,"relevance":"Preprint discussing MOTS-c's AMPK and NRF2 activation in a disease context; weak evidence quality but supports the therapeutic rationale for AMPK-optimized MOTS-c analogs."},{"pmid_or_doi":"10.20944/preprints202604.0328.v1","title":"Humanin and MOTS-c Attenuate Atrial Fibrillation by Suppressing Fibrosis and Mitochondrial Dysfunction","year":2026,"relevance":"Preprint with uninformative abstract content; provides minimal mechanistic data but suggests ongoing interest in MOTS-c's therapeutic profile in cardiovascular contexts."}]},"onchain":{"hash":"HZHtfzEXHLaHyjQRqchj1GRcNoZHkZUm1MW5Bv3vmPvqcdHELsM1zo9A7Wf4NmCVoPdKD9XUd8h9dv19t61brue","signature":"HZHtfzEXHLaHyjQRqchj1GRcNoZHkZUm1MW5Bv3vmPvqcdHELsM1zo9A7Wf4NmCVoPdKD9XUd8h9dv19t61brue","data_hash":"e28e15ac8cc315b34d87e93ecf68972793f158e1aa3f2f20fc3b646545a0d007","logged_at":"2026-05-05T07:09:36.181829+00:00","explorer_url":"https://solscan.io/tx/HZHtfzEXHLaHyjQRqchj1GRcNoZHkZUm1MW5Bv3vmPvqcdHELsM1zo9A7Wf4NmCVoPdKD9XUd8h9dv19t61brue"},"ipfs_hash":null,"created_at":"2026-05-05T06:34:49.924774+00:00","updated_at":"2026-05-05T07:09:36.188101+00:00"}