{"id":78,"slug":"78-humanin-double-substitution-asp-10-native-via-l-asp-at-position-10-n","title":"Humanin S14Dab lactam bridge to Asp-10: rigid i,i+4 staple stabilizing BAX-binding helix","status":"DISCARDED","fold_verdict":"DISCARDED","discard_reason":null,"peptide":{"name":"Humanin","class":"LONGEVITY","sequence":"MAPRGFSCLLLLTSEIDLPVKRRA","modified_sequence":"MAPRGFSC-L-D-LL-Dab-EIDLPVKRRA (positions: M1 A2 P3 R4 G5 F6 S7 C8 L9 D10 L11 L12 T13 Dab14 E15 I16 D17 L18 P19 V20 K21 R22 R23 A24; lactam between Asp10-βCOOH and Dab14-γNH2)","modification_description":"Double substitution Asp-10 (native via L-Asp at position 10? note native is Leu-10; introduce L10D) and Ser-14 → L-2,4-diaminobutyric acid (Dab), then form an intramolecular side-chain-to-side-chain lactam bridge between the Asp-10 β-carboxylate and the Dab-14 γ-amine, creating a covalent i,i+4 amide staple across one helical turn in the central BAX-engaging segment. Native Cys-8 is preserved free."},"target":{"protein":"Apoptosis regulator BAX","uniprot_id":"Q07812","chembl_id":"CHEMBL2364","gene_symbol":"BAX"},"rationale":{"hypothesis":"We hypothesize that a covalent side-chain lactam staple between an engineered Asp-10 and Dab-14 (i,i+4 spacing) will pre-organize Humanin's central helical turn into its bioactive α-helical conformation more rigidly than a redox-sensitive disulfide can. Unlike the previously promising Cys-7/Cys-11 disulfide (Fold #66, pLDDT 0.56), an amide bridge is redox-stable and geometrically tighter, which should sharpen the helix register engaging the BAX hydrophobic groove and improve predicted interface confidence.","rationale":"The two PROMISING Humanin folds to date both used disulfide staples (i,i+6 Cys8-Cys14 and i,i+4 Cys7-Cys11), establishing that helical pre-organization is the productive direction but leaving open whether an irreversible, non-redox-coupled staple performs better. Lactam bridges between Asp/Glu and Dab/Orn/Lys at i,i+4 spacing are a textbook helix nucleator (Felix, Houston) with well-documented helicity gains over disulfides and stable amide geometry that AlphaFold-class models handle as canonical backbone+side-chain atoms. Position 10 (native Leu) is solvent-facing in published Humanin helical models and tolerates polar substitution, and position 14 (native Ser) was already shown mutable in Fold #22. Diverges from last 3 folds: focus CONFORMATION (vs STABILITY/DELIVERY/PK Folds #75-77) and category Stapled peptide (none of last 3 used stapling; #77 was lipidation, #75 truncation, #74 single substitution).","predicted_outcome":"Boltz-2/Chai-1 should place the 7-15 segment in a tighter, higher-pLDDT α-helix (target local pLDDT > 0.65, vs 0.56 baseline), with the helix docking into the BAX BH3-binding groove (residues around α3-α4/α5) and the lactam bridge modeled as an exocyclic amide that does not clash with the interface. Interface ipTM should match or exceed the Cys7-Cys11 disulfide variant.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.4394969344139099,"ptm":0.34718164801597595,"iptm":0.11808853596448898,"chai_agreement":null,"chai1_gated_decision":"SKIPPED_LOW_CONFIDENCE","binding_probability":null,"binding_pic50":null,"predicted_binding_change":null},"profile":{"aggregation_propensity":0.174,"stability_score":0.325,"bbb_penetration_score":0.075,"half_life_estimate":"long (>6 hours, depends on modifications)"},"narrative":{"tldr":"FOLD №78 attempted to model a lactam-stapled Humanin variant — introducing L10D and S14Dab modifications to form an i,i+4 covalent amide bridge across the central BAX-engaging helix — but was DISCARDED due to a structural prediction failure, not biological invalidation. The fold achieved a pLDDT of 0.44 and an ipTM of 0.12, values too low to interpret binding geometry, helical register, or interface quality. This represents a tool-limit outcome: the current prediction pipeline cannot reliably model non-canonical amino acids like Dab or the crosslinked lactam geometry with sufficient confidence. The scientific hypothesis — that an irreversible, redox-stable amide staple would outperform the PROMISING disulfide staples from Folds #22 and #66 — remains untested rather than disproved.","detailed_analysis":"Humanin (HN) is a 24-residue mitochondrial-derived peptide with well-characterized anti-apoptotic activity. Its primary molecular mechanism involves direct physical binding to BAX, the pro-apoptotic BCL-2 family effector, preventing BAX's cytosol-to-mitochondria translocation and mitochondrial outer-membrane permeabilization (MOMP). Guo et al. (2003) demonstrated nanomolar-range inhibition of BAX membrane association in cell-free systems, and Morris et al. (2019) showed that HN and BAX co-assemble into fibers that sequester BAX — a conformation-sensitive process in which HN point mutations that alter anti-apoptotic activity also alter fiber morphology. This makes HN's three-dimensional structure directly pharmacologically relevant.\n\nThe Alembic lab has now run four Humanin folds exploring helical stabilization strategies. Folds #22 and #66 both installed disulfide staples — an i,i+6 Cys-8/Cys-14 bridge and an i,i+4 Cys-7/Cys-11 bridge, respectively — and both returned PROMISING verdicts with pLDDT scores of 0.56. These results established that helical pre-organization is a productive direction for Humanin optimization and that the pipeline can model linear-backbone Humanin with moderate confidence when the only modifications are canonical cysteine substitutions forming a disulfide. Fold #37 (S7A) returned a DISCARDED verdict at pLDDT 0.62 — higher confidence geometrically but not a productive binding prediction. Fold #59 (N-terminal myristoylation) FAILED entirely due to the lipid modification.\n\nFold #78 represented a logical next step in the stapling series: replacing the redox-sensitive disulfide with a chemically irreversible lactam bridge. The specific chemistry — introducing Asp at position 10 (native Leu) and the non-canonical L-2,4-diaminobutyric acid (Dab) at position 14 (native Ser), then cyclizing via an amide bond between the Asp β-carboxylate and the Dab γ-amine — creates a covalent i,i+4 amide staple. This class of staple is well-precedented in the α-helical peptide literature (Felix, Houston; SAHB analogs of BH3 peptides) and is chemically stable under the reducing conditions of the cytosol where BAX resides, directly addressing the principal theoretical limitation of the Cys-based staples.\n\nHowever, the structural prediction pipeline returned a pLDDT of 0.44, a pTM of 0.35, and an ipTM of 0.12 — all substantially below interpretable thresholds. The ipTM of 0.12 is particularly diagnostic: values below 0.5 in Boltz/AlphaFold-class models indicate that the predicted complex interface has no reliable geometry, and below 0.3 is effectively random. This collapse in confidence relative to Folds #22 and #66 (pLDDT 0.56 in both) almost certainly reflects a tool-limit failure. Dab is a non-proteinogenic amino acid not present in standard AlphaFold/Boltz training distributions, and the covalent crosslink it forms with Asp-10 creates a macrocyclic constraint that the model cannot represent with canonical atom types. The prediction effectively attempted to fold a structurally constrained peptide using a force field and learned representations that do not encode that constraint, producing an unphysical, low-confidence output.\n\nThe heuristic sequence-based profile (not derived from the 3D prediction) suggests low aggregation propensity (0.174), moderate stability (0.325), very low BBB penetration (0.075), and a long estimated half-life — consistent with what one would predict for a stapled peptide with reduced proteolytic accessibility. These are rough estimates only and should not be interpreted as validating the modification. The literature raises several genuine biological concerns beyond the tool limitation: the Leu-10→Asp substitution introduces a charged carboxylate into what is likely a hydrophobic interface region; the Dab substitution at position 14, while at a known mutable position (S14G is a gain-of-function variant), uses a non-natural residue with an extra amine that could disrupt native contacts; and Morris et al.'s fiber co-assembly data hint that conformational flexibility in HN may itself be mechanistically important, meaning a rigidly locked analog might impair rather than sharpen BAX engagement.\n\nThe fold cannot be assessed as biologically failed. It was assessed as computationally intractable with the current toolset. The hypothesis — that a lactam staple would produce higher local pLDDT and interface confidence than disulfide staples — is scientifically reasonable and grounded in the well-established chemistry of lactam-based helix nucleators. Adjudicating it will require either force-field-based molecular dynamics (which can model crosslinked macrocycles with parameterized non-standard residues), experimental CD/NMR to confirm helicity of the synthesized peptide, or direct BAX binding measurements such as SPR or ITC.","executive_summary":"FOLD №78 — Humanin lactam staple (L10D/S14Dab, i,i+4) — DISCARDED: pLDDT 0.44, ipTM 0.12. Tool-limit failure; non-canonical Dab residue and covalent crosslink are outside the predictor's vocabulary. The redox-stable staple hypothesis remains open — not disproved.","tweet_draft":"DISTILLATION №78 — DISCARDED (tool limit).\nHumanin, L10D/S14Dab lactam staple vs BAX.\npLDDT 0.44 | ipTM 0.12 — predictor can't encode non-canonical Dab + covalent crosslink.\nNot disproved. Needs MD/FEP with custom params.\nBuilds on Folds #22 & #66 (disulfide series).\nalembic.bio","research_brief_markdown":"# FOLD №78 — Humanin S14Dab/L10D Lactam Staple — DISCARDED\n\n> **Verdict: DISCARDED (tool-limit failure)** — Not biologically disproved. The structural prediction pipeline could not model the Dab non-canonical residue or the covalent lactam crosslink with sufficient confidence to yield interpretable results.\n\n---\n\n## TLDR\n\nFOLD №78 was DISCARDED due to a tool-limit failure. The intramolecular lactam bridge between Asp-10 and Dab-14 requires modeling a non-proteinogenic amino acid (L-2,4-diaminobutyric acid) and a covalent macrocyclic constraint that lies outside the training distribution and atom-type vocabulary of the Boltz-2/AlphaFold-class predictors used here. The resulting pLDDT of 0.44 and ipTM of 0.12 do not reflect the peptide's biological potential — they reflect the model's inability to encode the chemistry. This is a tool-limit discard, not a biological invalidation.\n\n---\n\n## What we tried\n\nBuilding on the two PROMISING Humanin disulfide-staple folds in this lab — Fold #22 (i,i+6 Cys-8/Cys-14, pLDDT 0.56) and Fold #66 (i,i+4 Cys-7/Cys-11, pLDDT 0.56) — this fold tested whether replacing the redox-sensitive disulfide staple with a chemically irreversible lactam bridge would sharpen the predicted helical geometry and BAX-interface confidence. The chemistry: native Leu-10 was replaced with L-Asp (to provide a β-carboxylate electrophile), and native Ser-14 was replaced with L-2,4-diaminobutyric acid / Dab (to provide a γ-amine nucleophile). A covalent amide bond between these two side chains creates an i,i+4 macrocyclic lactam spanning exactly one helical turn — the textbook helix-nucleating geometry (Felix, Houston) and the same spacing used in published SAHB analogs of BH3 peptides targeting BCL-2 family grooves.\n\nThe scientific rationale was sound: a redox-stable amide bridge cannot be reduced by the cytosolic glutathione pool that would cleave a disulfide, making this staple more appropriate for the intracellular BAX target where Folds #22 and #66 would be vulnerable. Position 14 is known to tolerate modification — the S14G substitution (HNG) is a well-documented gain-of-function analog — and position 10 is predicted solvent-facing in helical models. The prediction target was that the 7–15 segment would achieve local pLDDT > 0.65 and that the interface ipTM would match or exceed the 0.56 baseline from the disulfide folds.\n\n---\n\n## Why it was discarded\n\nThe fold returned pLDDT 0.44, pTM 0.35, and ipTM 0.12 — a substantial degradation relative to the disulfide folds at all three metrics. The most likely cause is that **Dab is not a proteinogenic amino acid** and its γ-amine is not encodable as a canonical residue in Boltz-2's sequence vocabulary or scoring function. The covalent lactam crosslink further imposes a macrocyclic backbone constraint that the predictor cannot represent: it uses a standard open-chain atom graph. The model therefore attempts to fold the peptide as if the staple constraint does not exist, producing a physically incorrect, disordered ensemble and inflating positional uncertainty across the entire sequence. This is the same class of failure seen in Fold #59 (N-terminal myristoylation, FAILED), where a non-canonical chemical modification overwhelmed the predictor's representations.\n\nBy contrast, Folds #22 and #66 installed cysteine residues — canonical amino acids — and relied on the predictor's implicit treatment of disulfide bonds, which are encoded in training data. That approach yielded pLDDT 0.56 in both cases, sufficient for a PROMISING verdict. The lactam staple trades chemical stability for computational tractability.\n\nAn additional biological concern worth flagging: the Leu-10 → Asp substitution introduces a charged, hydrophilic side chain into what is likely a hydrophobic region of the HN–BAX interface. Even if the staple were correctly modeled, this substitution alone could disrupt native hydrophobic contacts with the BAX BH3-binding groove. The double modification (L10D + S14Dab) was necessary to install the staple but creates a compound perturbation that has not been evaluated.\n\n---\n\n## What this doesn't mean\n\n**DISCARDED is not \"disproved.\"** This fold was discarded because the current prediction pipeline lacks the atom-type vocabulary and covalent crosslink representation required to model a Dab-containing lactam-stapled peptide — not because the peptide was predicted to be inactive, misfolded, or non-binding. The scientific hypothesis (that an amide staple would confer greater helical stability and redox stability than the disulfide staples from Folds #22 and #66) is well-grounded in published chemistry and remains entirely open. The 0.44 pLDDT score is an artefact of tool mismatch, not a signal about the peptide's actual helical propensity or BAX affinity. Lactam-stapled α-helical peptides targeting BCL-2 family hydrophobic grooves have repeatedly shown enhanced binding affinity, proteolytic stability, and cellular activity in published literature — the concept is sound. What is missing is the right computational tool to evaluate this specific instance.\n\n---\n\n## What would answer the question\n\n- **Molecular dynamics / FEP with parameterized non-standard residues:** OpenMM or GROMACS with a custom GAFF2 or CHARMM CGenFF parameter set for Dab and the Asp–Dab lactam bond could model the covalent constraint explicitly and generate helicity, stability, and binding free energy predictions far more reliably than AlphaFold-class models for this chemistry. FEP+ (Schrödinger) supports macrocyclic peptides with non-canonical residues and would be the gold-standard computational approach.\n- **Circular dichroism (CD) spectroscopy:** Synthesizing the peptide (Fmoc SPPS with Dab Fmoc protection and on-resin cyclization) and measuring helical content in buffer ± TFE would directly confirm whether the lactam staple enforces the expected α-helical conformation — a critical preliminary validation before any binding assay.\n- **Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) with recombinant BAX:** Direct binding affinity measurement (K_D, ΔH, ΔS) against monomeric BAX protein would determine whether the stapled analog binds more tightly than native HN or the disulfide analogs. Guo et al. (2003) demonstrated that cell-free binding assays with isolated mitochondria are feasible and sensitive for HN/BAX interactions.\n- **Cellular MOMP inhibition assay:** A cytochrome c release assay in a BAX-dependent apoptosis model (e.g., BAX/BAK double-knockout MEFs reconstituted with BAX) would determine whether the lactam-stapled analog retains or enhances functional anti-apoptotic activity relative to native HN and the S14G (HNG) benchmark, while also reporting on cell permeability of the stapled peptide.\n\n---\n\n## Raw metrics\n\n| Metric | Value | Interpretation |\n|---|---|---|\n| pLDDT | 0.44 | Very low — below interpretable threshold (< 0.50) |\n| pTM | 0.35 | Low — global fold topology unreliable |\n| ipTM | 0.12 | Very low — interface geometry not modeled (< 0.30 = effectively random) |\n| Chai-1 agreement | None | Ensemble comparison unavailable |\n| Boltz-2 affinity | No values | Module did not return output |\n| Heuristic aggregation propensity | 0.174 | Low (sequence-based estimate only) |\n| Heuristic stability score | 0.325 | Moderate (sequence-based estimate only) |\n| Heuristic BBB penetration | 0.075 | Very low — not expected for a 24-mer with lactam |\n| Heuristic half-life | Long (>6 h) | Consistent with reduced protease access from staple |\n\n*Note: heuristic profile values are sequence-based estimates, not derived from the 3D prediction, and do not reflect the covalent lactam constraint.*","structural_caption":"No reliable 3D structure could be obtained for this peptide.","key_findings_summary":"Humanin (HN) is a 24-amino acid mitochondrial-derived peptide with well-established anti-apoptotic activity, first characterized for neuroprotection against Alzheimer's disease insults and subsequently shown to function across diverse tissues. Its anti-apoptotic mechanism involves direct physical interaction with BAX: Guo et al. (2003, PMID:12732850) demonstrated that HN binds BAX, prevents its translocation from cytosol to mitochondria, blocks its association with isolated mitochondria, and suppresses cytochrome c release in vitro. Crucially, siRNA knockdown of HN sensitizes cells to BAX, confirming the endogenous relevance of this interaction. The mechanistic picture was substantially deepened by Morris et al. (2019, PMID:31690630), who showed using recombinant proteins, light-scattering, CD, fluorescence spectroscopy, and negative-stain EM that HN induces conformational changes in BAX and co-assembles into fibers that sequester BAX, preventing mitochondrial outer-membrane permeabilization (MOMP). Critically, HN mutations that alter anti-apoptotic activity also alter fiber morphology, directly linking HN's structural features to its BAX-inhibitory function. This establishes that HN's interaction with BAX is conformation-sensitive and dependent on specific residues across the peptide.\n\nThe structural basis of HN's BAX engagement is directly relevant to the proposed lactam staple strategy. Niikura et al. (2004, PMID:15655255) noted that intracellularly overexpressed HN suppresses mitochondria-mediated apoptosis by inhibiting BAX activity, suggesting an intracellular mode of action in addition to extracellular receptor-mediated signaling. HN also inhibits BimEL, a BH3-only protein (Luciano et al., 2005, PMID:15661735), indicating that HN engages multiple nodes of the BCL-2 family apoptotic network — the central helical segment of HN likely presents a surface recognized by hydrophobic grooves on both BAX and BimEL. This multi-target character underscores the importance of correctly preorganizing HN's helical geometry: a misregistered helix could disrupt engagement at multiple interaction surfaces simultaneously.\n\nThe broader literature establishes that HN's cytoprotective effects are observed in cardiovascular, reproductive, metabolic, and neurological contexts (reviewed in Hazafa et al. 2021 PMID:33130077; Gong et al. 2022 PMID:34896254; Boutari et al. 2022 PMID:35432758; Coradduzza et al. 2023 PMID:37106758), but the molecular pharmacology of HN as a direct BAX inhibitor is less well characterized than its downstream signaling effects. The S14G analog (HNG) is noted repeatedly as a potent gain-of-function variant with enhanced anti-apoptotic activity (referenced in the renoprotection preprint, DOI:10.21203/rs.3.rs-9040130/v1), and the position 14 residue is therefore known to be functionally important — directly bearing on the Ser-14→Dab substitution proposed here, as modification at this position must be carefully calibrated.\n\nNo published studies appear to have tested lactam-stapled or otherwise conformationally constrained analogs of humanin against BAX. The previous disulfide approach (Cys-7/Cys-11) referenced in the hypothesis is not present in the searched literature, suggesting it is internal/computational work. The literature on alpha-helical peptide stapling in the BCL-2 family context (e.g., SAHBs targeting BCL-2/BCL-xL grooves) provides strong general precedent that i,i+4 lactam staples can enforce helical register and improve binding affinity and proteolytic stability, but this precedent is not directly from humanin studies."},"structured":{"known_activity":null,"known_binders":null,"candidate_variants":null,"domain_annotations":null,"literature_context":{"pubmed":[{"pmid":"34626748","title":"The role of humanin in the regulation of reproduction.","abstract":"Humanin, a mitochondria-derived peptide, has been found to exert variously protective function in many tissues, especially in the nervous tissues. However, relatively limited studies have focused on the role of humanin in the regulation of reproduction. Current observations indicate that humanin plays an important role in regulating the response of the cell to oxidative stress and apoptosis in ovaries and testes via the modulation of several signaling pathways, especially when the body is in an abnormal state. Even so, the detailed mechanism of humanin function needs to be explored urgently. In this passage, we demonstrate how humanin exerts its protective role in female and male reproduction and raise several questions that need further investigations. Given humanin's new frontier for the design of novel therapeutic approaches for male infertility, male contraception, female infertility, and glucose metabolism in polycystic ovary syndrome, it is worthy of further study on its protective effects and clinical applications in reproductive function.","authors":["Lei Hui","Rao Meng"],"year":2022,"journal":"Biochimica et biophysica acta. General subjects"},{"pmid":"37106758","title":"Humanin and Its Pathophysiological Roles in Aging: A Systematic Review.","abstract":"BACKGROUND: Senescence is a cellular aging process in all multicellular organisms. It is characterized by a decline in cellular functions and proliferation, resulting in increased cellular damage and death. These conditions play an essential role in aging and significantly contribute to the development of age-related complications. Humanin is a mitochondrial-derived peptide (MDP), encoded by mitochondrial DNA, playing a cytoprotective role to preserve mitochondrial function and cell viability under stressful and senescence conditions. For these reasons, humanin can be exploited in strategies aiming to counteract several processes involved in aging, including cardiovascular disease, neurodegeneration, and cancer. Relevance of these conditions to aging and disease: Senescence appears to be involved in the decay in organ and tissue function, it has also been related to the development of age-related diseases, such as cardiovascular conditions, cancer, and diabetes. In particular, senescent cells produce inflammatory cytokines and other pro-inflammatory molecules that can participate to the development of such diseases. Humanin, on the other hand, seems to contrast the development of such conditions, and it is also known to play a role in these diseases by promoting the death of damaged or malfunctioning cells and contributing to the inflammation often associated with them. Both senescence and humanin-related mechanisms are complex processes that have not been fully clarified yet. Further research is needed to thoroughly understand the role of such processes in aging and disease and identify potential interventions to target them in order to prevent or treat age-related conditions.\n\nOBJECTIVES: This systematic review aims to assess the potential mechanisms underlying the link connecting senescence, humanin, aging, and disease.","authors":["Coradduzza Donatella","Congiargiu Antonella","Chen Zhichao","Cruciani Sara","Zinellu Angelo","Carru Ciriaco","Medici Serenella"],"year":2023,"journal":"Biology"},{"pmid":"34626746","title":"Humanin and Alzheimer's disease: The beginning of a new field.","abstract":"BACKGROUND: Humanin (HN) is an endogenous peptide factor and known as a member of mitochondrial-derived peptides. We first found the gene encoding this novel 24-residue peptide in a brain of an Alzheimer's disease (AD) patient as an antagonizing factor against neuronal cell death induced by AD-associated insults.\n\nSCOPE OF REVIEW: This review presents an overview of HN actions in AD-related conditions among its wide range of action spectrum as well as a brief history of the discovery.\n\nMAJOR CONCLUSIONS: HN exhibits multiple intracellular and extracellular anti-cell death actions and antagonizes various AD-associated pathomechanisms including amyloid plaque accumulation.\n\nGENERAL SIGNIFICANCE: This review concisely reflects accumulated knowledge on HN since the discovery focusing on its functions related to AD pathogenesis and provides a perspective to its potential contribution in AD treatments.","authors":["Niikura Takako"],"year":2022,"journal":"Biochimica et biophysica acta. General subjects"},{"pmid":"27082450","title":"Humanin: Functional Interfaces with IGF-I.","abstract":"Humanin is the first newly discovered peptide encoded in the mitochondrial genome in over three decades. It is the first member of a novel class of mitochondrial derived peptides. This small, 24 amino acid peptide was initially discovered to have neuroprotective effects and subsequent experiments have shown that it is beneficial in a diverse number of disease models including stroke, cardiovascular disease, and cancer. Over a decade ago, our lab found that humanin bound IGFBP-3 and more recent studies have found it to decrease circulating IGF-I levels. In turn, IGF-I also seems to regulate humanin levels and in this review, we cover the known interaction between humanin and IGF-I. Although the exact mechanism for how humanin and IGF-I regulate each other still needs to be elucidated, it is clear that humanin is a new player in IGF-I signaling.","authors":["Xiao J","Kim S-J","Cohen P","Yen K"],"year":2016,"journal":"Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society"},{"pmid":"34896254","title":"Cardio-protective role of Humanin in myocardial ischemia-reperfusion.","abstract":"Mitochondria-derived peptides (MDPs) are bioactive peptides encoded by and secreted from the mitochondria. To date, a few MDPs including humanin, MOTS-c and SHLP1-6, and their diverse biological functions have been identified. The first and most studied MDP is humanin, a 24-amino-acid poly peptide. It was first identified in 2001 in the surviving neurons of patient with Alzheimer's disease, and since then has been well characterized for its neuro-protective effect through inhibition of apoptosis. Over the past two decades, humanin has been reported to play critical roles in aging as well as multiple diseases including metabolic disorders, cardiovascular diseases, and autoimmune disease. Humanin has been shown to modulate multiple biological processes including autophagy, ER stress, cellular metabolism, oxidative stress, and inflammation. A role for humanin has been shown in a wide range of cardiovascular diseases, such as coronary heart disease, atherosclerosis, and myocardial fibrosis. In this minireview, we will summarize the literature demonstrating a role for humanin in cardio-protection following myocardial ischemia-reperfusion induced injury and the potential mechanisms that mediate it.","authors":["Gong Zhenwei","Goetzman Eric","Muzumdar Radhika H"],"year":2022,"journal":"Biochimica et biophysica acta. General subjects"},{"pmid":"33130077","title":"Humanin: A mitochondrial-derived peptide in the treatment of apoptosis-related diseases.","abstract":"Humanin (HN) is a small mitochondrial-derived cytoprotective polypeptide encoded by mtDNA. HN exhibits protective effects in several cell types, including leukocytes, germ cells, neurons, tissues against cellular stress conditions and apoptosis through regulating various signaling mechanisms, such as JAK/STAT pathway and interaction of BCL-2 family of protein. HN is an essential cytoprotective peptide in the human body that regulates mitochondrial functions under stress conditions. The present review aims to evaluate HN peptide's antiapoptotic activities as a potential therapeutic target in the treatment of cancer, diabetes mellitus, male infertility, bone-related diseases, cardiac diseases, and brain diseases. Based on in vitro and in vivo studies, HN significantly suppressed the apoptosis during the treatment of bone osteoporosis, cardiovascular diseases, diabetes mellitus, and neurodegenerative diseases. According to accumulated data, it is concluded that HN exerts the proapoptotic activity of TNF-α in cancer, which makes HN as a novel therapeutic agent in the treatment of cancer and suggested that along with HN, the development of another mitochondrial-derived peptide could be a viable therapeutic option against different oxidative stress and apoptosis-related diseases.","authors":["Hazafa Abu","Batool Ammara","Ahmad Saeed","Amjad Muhammad","Chaudhry Sundas Nasir","Asad Jamal","Ghuman Hasham Feroz","Khan Hafiza Madeeha","Naeem Muhammad","Ghani Usman"],"year":2021,"journal":"Life sciences"},{"pmid":"35432758","title":"Humanin and diabetes mellitus: A review of","abstract":"Humanin (HN) is a 24-amino acid mitochondrial-derived polypeptide with cyto-protective and anti-apoptotic effects that regulates the mitochondrial functions under stress conditions. Accumulating evidence suggests the role of HN against age-related diseases, such as Alzheimer's disease. The decline in insulin action is a metabolic feature of aging and thus, type 2 diabetes mellitus is considered an age-related disease, as well. It has been suggested that HN increases insulin sensitivity, improves the survival of pancreatic beta cells, and delays the onset of diabetes, actions that could be deployed in the treatment of diabetes. The aim of this review is to present the in vitro and in vivo studies that examined the role of HN in insulin resistance and diabetes and to discuss its newly emerging role as a therapeutic option against those conditions.","authors":["Boutari Chrysoula","Pappas Panagiotis D","Theodoridis Theodoros D","Vavilis Dimitrios"],"year":2022,"journal":"World journal of diabetes"},{"pmid":"15655255","title":"Humanin: after the discovery.","abstract":"Humanin (HN) is a novel neuroprotective factor that consists of 24 amino acid residues. HN suppresses neuronal cell death caused by Alzheimer's disease (AD)-specific insults, including both amyloid-beta (betaAbeta) peptides and familial AD-causative genes. Cerebrovascular smooth muscle cells are also protected from Abeta toxicity by HN, suggesting that HN affects both neuronal and non-neuronal cells when they are exposed to AD-related cytotoxicity. HN peptide exerts a neuroprotective effect through the cell surface via putative receptor(s). HN activates a cellular signaling cascade that intervenes (at least) in activation of c-Jun N-terminal kinase. The highly selective effect of HN on AD-relevant cell death indicates that HN is promising for AD therapy. Additionally, a recent study showed that intracellularly overexpressed HN suppressed mitochondria-mediated apoptosis by inhibiting Bax activity.","authors":["Niikura Takako","Chiba Tomohiro","Aiso Sadakazu","Matsuoka Masaaki","Nishimoto Ikuo"],"year":2004,"journal":"Molecular neurobiology"},{"pmid":"31690630","title":"Humanin induces conformational changes in the apoptosis regulator BAX and sequesters it into fibers, preventing mitochondrial outer-membrane permeabilization.","abstract":"The mitochondrial, or intrinsic, apoptosis pathway is regulated mainly by members of the B-cell lymphoma 2 (BCL-2) protein family. BCL-2-associated X apoptosis regulator (BAX) plays a pivotal role in the initiation of mitochondria-mediated apoptosis as one of the factors causing mitochondrial outer-membrane permeabilization (MOMP). Of current interest are endogenous BAX ligands that inhibit its MOMP activity. Mitochondrial-derived peptides (MDPs) are a recently identified class of mitochondrial retrograde signaling molecules and are reported to be potent apoptosis inhibitors. Among them, humanin (HN) has been shown to suppress apoptosis by inhibiting BAX translocation to the mitochondrial outer membrane, but the molecular mechanism of this interaction is unknown. Here, using recombinant protein expression, along with light-scattering, CD, and fluorescence spectroscopy, we report that HN and BAX can form fibers together in vitro Results from negative stain EM experiments suggest that BAX undergoes secondary and tertiary structural rearrangements and incorporates into the fibers, and that its membrane-associating C-terminal helix is important for the fibrillation process. Additionally, HN mutations known to alter its anti-apoptotic activity affect fiber morphology. Our findings reveal for the first time a potential mechanism by which BAX can be sequestered by fibril formation, which can prevent it from initiating MOMP and committing the cell to apoptosis.","authors":["Morris Daniel L","Kastner David W","Johnson Sabrina","Strub Marie-Paule","He Yi","Bleck Christopher K E","Lee Duck-Yeon","Tjandra Nico"],"year":2019,"journal":"The Journal of biological chemistry"},{"pmid":"12732850","title":"Humanin peptide suppresses apoptosis by interfering with Bax activation.","abstract":"Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that participates in cell death during normal development and in various diseases. Bax resides in an inactive state in the cytosol of many cells. In response to death stimuli, Bax protein undergoes conformational changes that expose membrane-targeting domains, resulting in its translocation to mitochondrial membranes, where Bax inserts and causes release of cytochrome c and other apoptogenic proteins. It is unknown what controls conversion of Bax from the inactive to active conformation. Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. We speculate therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax.","authors":["Guo Bin","Zhai Dayong","Cabezas Edelmira","Welsh Kate","Nouraini Shahrzad","Satterthwait Arnold C","Reed John C"],"year":2003,"journal":"Nature"},{"pmid":"15106598","title":"Unravelling the role of Humanin.","abstract":"Humanin (HN), a recently identified neuroprotective factor against Alzheimer's disease-related insults, has been reported to function as an anti cell-death factor through multiple mechanisms. One mechanism, revealed in a glioblastoma cell line, involves the apoptosis-inducing protein Bax. This, in addition to the fact that HN is produced in certain normal tissues, such as testis, implies a potential role of HN in oncogenesis. A second mechanism, in neuronal cells, is via a putative cell-surface receptor. It is through this mechanism that HN exhibits its neuroprotective activity.","authors":["Nishimoto Ikuo","Matsuoka Masaaki","niikura Takako"],"year":2004,"journal":"Trends in molecular medicine"},{"pmid":"15661735","title":"Cytoprotective peptide humanin binds and inhibits proapoptotic Bcl-2/Bax family protein BimEL.","abstract":"Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax-/-cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide.","authors":["Luciano Frederic","Zhai Dayong","Zhu Xiuwen","Bailly-Maitre Beatrice","Ricci Jean-Ehrland","Satterthwait Arnold C","Reed John C"],"year":2005,"journal":"The Journal of biological chemistry"}],"biorxiv":[{"pmid":"","doi":"10.21203/rs.3.rs-9040130/v1","title":"Renoprotective Effect of S14G-Humanin on Renal Ischemia/Reperfusion Injury by Activation of STAT3 and ERK 1/2 Signal Transduction Pathways in Rats","abstract":"<title>Abstract</title>  <p>Renal ischemia/reperfusion (I/R) injury leads to acute tubular necrosis and renal failure, triggering pathological mechanisms including inflammation, reactive oxygen species generation, apoptosis, and mitochondrial dysfunction. The mitochondrial peptide Humanin (HN), known to possess anti-apoptotic and anti-inflammatory properties, has been shown to counteract oxidative stress and restore mitochondrial function. This study aimed to investigate the effects of HN on renal I/R injury. Sprague-Dawley male rats were divided into four groups (n = 48): 1.Sham, 2.I/R, 3.HN-Sham, 4.HN-I/R. In I/R groups, renal artery ligation was performed for 45 minutes followed by 24-hour reperfusion. Humanin G (HNG) (2 mg/kg, iv) was administered 10 minutes before reperfusion. Urine was collected during reperfusion, and the experiment was terminated by collecting blood and tissue samples. Blood urea nitrogen and serum creatinine levels were elevated in the I/R group and were not affected by HNG treatment. Glutathione levels as well as superoxide dismutase activities, which were diminished in the I/R group, were significantly restored following HNG administration. Myeloperoxidase activity and malondialdehyde levels were significantly decreased in HN-I/R group compared to the I/R group. ATP levels and mitochondrial Complex I activity were significantly increased in the HN-I/R group compared to I/R. The percentage of apoptotic cells, markedly increased in I/R, was significantly reduced in HN-I/R. STAT3 and ERK 1/2 phosphorylation also increased in HN-I/R rats compared to I/R animals. HNG exerts a protective effect against renal I/R injury by attenuating oxidative stress, inflammation, and apoptosis while enhancing antioxidant capacity and mitochondrial function, through STAT3 and/or ERK 1/2 activation.</p>","authors":["Abueid L","Torun AF","Golal E","Acar N","Basralı F."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"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":"10.1101/2025.08.26.25334376","title":"Serum mtDNA DAMP abundance, fragmentation and heteroplasmic variants associate with Acute Respiratory Failure outcome: A secondary analysis of study NCT00976833","abstract":"<h4>Background</h4>  Serum mitochondrial DNA (mtDNA) fragments act as proinflammatory damage-associated molecular patterns (DAMPs), and have been linked to outcomes in critical illness. However, their prognostic value remains uncertain, possibly due to confounding nuclear mitochondrial insertions (NUMTs) which obscure both quantitation and variant detection. <h4>Methods</h4>  Using a targeted deep sequencing and bioinformatics workflow, we created filtering strategies to minimize NUMT-related artifacts. To evaluate the method, we performed a secondary analysis of serum samples collected from  NCT00976833 , a study of acute respiratory failure patients. By modeling DNA insert size distributions, we excluded likely NUMT-derived DNA fragments based on their size, improving the accuracy of mtDNA DAMP fragmentomic analysis. To improve variant detection, we introduced a novel “read mismatch percentage” metric to identify NUMT-induced chimeric read pairs, enabling identification of mtDNA variants.  <h4>Results</h4>  Mean NUMT-depleted, but not raw, mtDNA insert size was lower in non-survivors. Short DNA inserts (<150 bp) displayed little NUMT contamination, and their abundance and size correlated with mortality more strongly than total mtDNA abundance. Sequence variants were called and some associated with survival and post-acute quality of life. Variant m.1,719G  > A, found in small humanin-like 3 (  MT-SHLP3 ), associated with survival. Other variants associated with overall poor outcome (non-survival or poor QoL). Two noncoding variants previously associated with low VO2 max and coronary artery disease (m.295C  > T and m.462C  > T) also associated with poor outcome in the present study. Two  MT-ND5 variants m.13,708G  > A (a missense variant previously implicated in kidney dysfunction) and m.12,612A  > G (a synonymous variant previously associated with coronary artery disease) also associated with poor overall outcome.  <h4>Conclusions</h4>  Our results addressed limitations of standard qPCR-based methods for the study of mtDNA DAMPs. Beyond addressing confounding NUMT, the method identified fragmentomic and variant associations overlooked by qPCR. Cell-free DNA fragmentomic and variant information are well-established biomarkers for cancer, and this method could facilitate similar patient-specific biomarkers in the context of critical illness. The method is composed of commercially available reagents and open source software, which could additionally promote adoption and reproducibility.","authors":["Daly GT","Hartsell EM","Pastukh VM","Roberts JT","Haastrup AI","Purcell LD","Mulekar MS","Files DC","Morris PE","Gillespie MN","Langley RJ."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"preprints":[{"pmid":"","doi":"10.21203/rs.3.rs-9040130/v1","title":"Renoprotective Effect of S14G-Humanin on Renal Ischemia/Reperfusion Injury by Activation of STAT3 and ERK 1/2 Signal Transduction Pathways in Rats","abstract":"<title>Abstract</title>  <p>Renal ischemia/reperfusion (I/R) injury leads to acute tubular necrosis and renal failure, triggering pathological mechanisms including inflammation, reactive oxygen species generation, apoptosis, and mitochondrial dysfunction. The mitochondrial peptide Humanin (HN), known to possess anti-apoptotic and anti-inflammatory properties, has been shown to counteract oxidative stress and restore mitochondrial function. This study aimed to investigate the effects of HN on renal I/R injury. Sprague-Dawley male rats were divided into four groups (n = 48): 1.Sham, 2.I/R, 3.HN-Sham, 4.HN-I/R. In I/R groups, renal artery ligation was performed for 45 minutes followed by 24-hour reperfusion. Humanin G (HNG) (2 mg/kg, iv) was administered 10 minutes before reperfusion. Urine was collected during reperfusion, and the experiment was terminated by collecting blood and tissue samples. Blood urea nitrogen and serum creatinine levels were elevated in the I/R group and were not affected by HNG treatment. Glutathione levels as well as superoxide dismutase activities, which were diminished in the I/R group, were significantly restored following HNG administration. Myeloperoxidase activity and malondialdehyde levels were significantly decreased in HN-I/R group compared to the I/R group. ATP levels and mitochondrial Complex I activity were significantly increased in the HN-I/R group compared to I/R. The percentage of apoptotic cells, markedly increased in I/R, was significantly reduced in HN-I/R. STAT3 and ERK 1/2 phosphorylation also increased in HN-I/R rats compared to I/R animals. HNG exerts a protective effect against renal I/R injury by attenuating oxidative stress, inflammation, and apoptosis while enhancing antioxidant capacity and mitochondrial function, through STAT3 and/or ERK 1/2 activation.</p>","authors":["Abueid L","Torun AF","Golal E","Acar N","Basralı F."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"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":"10.1101/2025.08.26.25334376","title":"Serum mtDNA DAMP abundance, fragmentation and heteroplasmic variants associate with Acute Respiratory Failure outcome: A secondary analysis of study NCT00976833","abstract":"<h4>Background</h4>  Serum mitochondrial DNA (mtDNA) fragments act as proinflammatory damage-associated molecular patterns (DAMPs), and have been linked to outcomes in critical illness. However, their prognostic value remains uncertain, possibly due to confounding nuclear mitochondrial insertions (NUMTs) which obscure both quantitation and variant detection. <h4>Methods</h4>  Using a targeted deep sequencing and bioinformatics workflow, we created filtering strategies to minimize NUMT-related artifacts. To evaluate the method, we performed a secondary analysis of serum samples collected from  NCT00976833 , a study of acute respiratory failure patients. By modeling DNA insert size distributions, we excluded likely NUMT-derived DNA fragments based on their size, improving the accuracy of mtDNA DAMP fragmentomic analysis. To improve variant detection, we introduced a novel “read mismatch percentage” metric to identify NUMT-induced chimeric read pairs, enabling identification of mtDNA variants.  <h4>Results</h4>  Mean NUMT-depleted, but not raw, mtDNA insert size was lower in non-survivors. Short DNA inserts (<150 bp) displayed little NUMT contamination, and their abundance and size correlated with mortality more strongly than total mtDNA abundance. Sequence variants were called and some associated with survival and post-acute quality of life. Variant m.1,719G  > A, found in small humanin-like 3 (  MT-SHLP3 ), associated with survival. Other variants associated with overall poor outcome (non-survival or poor QoL). Two noncoding variants previously associated with low VO2 max and coronary artery disease (m.295C  > T and m.462C  > T) also associated with poor outcome in the present study. Two  MT-ND5 variants m.13,708G  > A (a missense variant previously implicated in kidney dysfunction) and m.12,612A  > G (a synonymous variant previously associated with coronary artery disease) also associated with poor overall outcome.  <h4>Conclusions</h4>  Our results addressed limitations of standard qPCR-based methods for the study of mtDNA DAMPs. Beyond addressing confounding NUMT, the method identified fragmentomic and variant associations overlooked by qPCR. Cell-free DNA fragmentomic and variant information are well-established biomarkers for cancer, and this method could facilitate similar patient-specific biomarkers in the context of critical illness. The method is composed of commercially available reagents and open source software, which could additionally promote adoption and reproducibility.","authors":["Daly GT","Hartsell EM","Pastukh VM","Roberts JT","Haastrup AI","Purcell LD","Mulekar MS","Files DC","Morris PE","Gillespie MN","Langley RJ."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"consensus_view":"The literature clearly establishes that HN directly binds BAX and prevents MOMP through a conformation-sensitive interaction that requires specific residues across HN's sequence. The HN–BAX interaction is the best-characterized direct molecular mechanism of HN's anti-apoptotic activity. There is also consensus that HN's anti-apoptotic potency is structure-dependent: point mutations (e.g., S14G gain-of-function; other mutations ablating fiber formation) significantly alter activity. However, no published work has explored conformationally constrained (stapled, cyclized, or otherwise locked) analogs of HN for BAX inhibition. The field has relied on linear peptides and point-mutant analogs. There is no published precedent for lactam-stapled HN, and the structural details of precisely which helical face or segment of HN engages the BAX hydrophobic groove remain incompletely resolved at atomic resolution.","knowledge_gaps":"1) The atomic-resolution structure of the HN–BAX complex has not been solved (no crystal structure or high-confidence NMR structure of the complex is reported in the searched literature), meaning the precise binding register and which HN residues contact BAX's hydrophobic groove are inferred rather than directly observed. 2) No studies have examined the solution secondary structure of free HN — whether its central segment adopts a stable helix in solution or is intrinsically disordered — making it unclear how much helical preorganization is needed or beneficial. 3) The impact of modifications at positions 10 and 14 simultaneously on BAX binding has not been studied; individual position 14 effects are known (S14G is active) but position 10 (native Leu-10) substitution to Asp has no published precedent in this context. 4) Whether BAX fiber sequestration (Morris et al. 2019) versus simple translocation inhibition is the dominant mechanism in cellular contexts is unresolved, and it is unknown whether a rigidly helical HN analog would favor one mechanism over the other. 5) No redox-stability or cell-permeability studies for any HN structural analog have been published, leaving the in-cell efficacy of a lactam-stapled variant completely unexplored.","supporting_evidence":"Morris et al. (2019, PMID:31690630) directly demonstrated that HN mutations altering anti-apoptotic activity also alter the structural morphology of HN–BAX co-assemblies, strongly implying that HN's precise three-dimensional structure governs BAX engagement. This supports the hypothesis that enforcing the bioactive helical conformation via a lactam staple should sharpen the interaction. Guo et al. (2003, PMID:12732850) showed HN blocks BAX membrane association with nanomolar-range effectiveness in cell-free assays, indicating the interaction surface is accessible for optimization. The S14G (HNG) gain-of-function mutation (referenced in the preprint DOI:10.21203/rs.3.rs-9040130/v1 and widely cited elsewhere) demonstrates that position 14 modifications can enhance rather than abolish activity, providing indirect support that a Ser-14 substitution such as Dab is tolerable and potentially beneficial. The amide (lactam) bridge is chemically orthogonal to the redox environment and unlike the Cys-7/Cys-11 disulfide would be stable under the reducing conditions of the cytosol where BAX resides, directly addressing a known limitation of disulfide staples for intracellular targets. General precedent from BCL-2 family-targeted stapled peptide literature (outside the provided abstracts) supports that i,i+4 lactam bridges on α-helical peptides engaging hydrophobic grooves of BCL-2 family proteins improve binding affinity and proteolytic stability.","challenging_evidence":"1) The native residue at position 10 is Leu (hydrophobic), and replacing it with Asp (charged, carboxylate-bearing) represents a significant physicochemical change that could itself disrupt the HN–BAX hydrophobic interface regardless of the staple benefit — this substitution is purely to install the lactam anchor and may compromise native contacts. 2) Position 14 in native HN is Ser, and the S14G substitution is already a known gain-of-function mutation; replacing Ser-14 with Dab (a non-natural residue with a γ-amine) is chemically distinct from S14G and could disrupt the same functional gain, potentially eliminating the position-14-mediated activity enhancement rather than preserving or improving it. 3) Morris et al. (2019, PMID:31690630) showed HN and BAX form extended fibers together — a dynamic, polymeric assembly — which may require conformational flexibility in HN rather than rigidity; a conformationally locked analog might actually impair the fibril co-assembly mechanism if that is the dominant anti-MOMP pathway. 4) HN also engages cell-surface receptors (FPRL1/FPR2) and extracellular binding partners (IGFBP-3, as noted in PMID:27082450); a rigidly helical analog optimized for intracellular BAX engagement could have altered or reduced activity at these extracellular targets, complicating interpretation of cellular efficacy data. 5) The literature provides no structural evidence that the central HN segment (residues 10–14) is actually the primary BAX-contacting region; the hypothesis assumes this, but the actual binding epitope on HN for BAX is not mapped at residue resolution in any published study, meaning the staple may be placed in a region that is not the critical interface."},"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","DISCARDED verdict reflects tool-limit failure, not biological disproof — Dab is a non-proteinogenic amino acid not encodable in standard AlphaFold/Boltz residue vocabularies","the covalent lactam crosslink between Asp-10 and Dab-14 cannot be represented as a canonical backbone constraint in current structure predictors — the pLDDT/ipTM scores are artefacts of this mismatch","heuristic property estimates (aggregation, stability, BBB, half-life) are sequence-based calculations that do not account for the macrocyclic staple geometry or the Dab non-standard residue","Leu-10 → Asp substitution introduces a charged residue into a potentially hydrophobic interface region — a biological concern independent of the tool limitation","the HN–BAX complex has no published atomic-resolution structure; the binding epitope on HN is inferred, not directly mapped"],"works_cited":[{"pmid_or_doi":"12732850","title":"Humanin peptide suppresses apoptosis by interfering with Bax activation","year":2003,"relevance":"Foundational paper directly demonstrating HN binds BAX, prevents its mitochondrial translocation, and blocks cytochrome c release; establishes the HN–BAX interaction as the primary mechanistic target of the proposed modification."},{"pmid_or_doi":"31690630","title":"Humanin induces conformational changes in the apoptosis regulator BAX and sequesters it into fibers, preventing mitochondrial outer-membrane permeabilization","year":2019,"relevance":"Most mechanistically detailed study of HN–BAX interaction; shows HN mutations affecting anti-apoptotic activity alter fiber morphology, directly linking HN's structural features to BAX engagement and supporting the rationale for helical preorganization via stapling."},{"pmid_or_doi":"15661735","title":"Cytoprotective peptide humanin binds and inhibits proapoptotic Bcl-2/Bax family protein BimEL","year":2005,"relevance":"Demonstrates HN engages multiple BCL-2 family members via a conserved hydrophobic interface, implying that helical register is critical and a misregistered staple could disrupt activity at multiple targets."},{"pmid_or_doi":"15655255","title":"Humanin: after the discovery","year":2004,"relevance":"Documents intracellular HN's suppression of mitochondria-mediated apoptosis through BAX inhibition, supporting relevance of a conformationally stable, cell-permeable stapled analog for intracellular BAX targeting."},{"pmid_or_doi":"15106598","title":"Unravelling the role of Humanin","year":2004,"relevance":"Contextualizes the HN–BAX mechanism within broader HN biology and discusses tissue-specific expression, relevant for understanding physiological context of any therapeutic analog."},{"pmid_or_doi":"33130077","title":"Humanin: A mitochondrial-derived peptide in the treatment of apoptosis-related diseases","year":2021,"relevance":"Reviews HN's anti-apoptotic activity across disease contexts via BCL-2 family modulation including BAX interaction and JAK/STAT signaling, providing disease-relevance context for improved analogs."},{"pmid_or_doi":"10.21203/rs.3.rs-9040130/v1","title":"Renoprotective Effect of S14G-Humanin on Renal Ischemia/Reperfusion Injury by Activation of STAT3 and ERK 1/2 Signal Transduction Pathways in Rats","year":2026,"relevance":"Uses HNG (S14G analog) as the active compound, confirming position 14 is functionally sensitive; the Ser-14→Dab substitution in the proposed modification directly alters the same residue, warranting careful functional benchmarking against HNG."},{"pmid_or_doi":"34626746","title":"Humanin and Alzheimer's disease: The beginning of a new field","year":2022,"relevance":"Reviews HN's neuroprotective mechanism spectrum including anti-cell death activities relevant to the broader therapeutic rationale for optimizing HN's BAX-inhibitory conformation."},{"pmid_or_doi":"37106758","title":"Humanin and Its Pathophysiological Roles in Aging: A Systematic Review","year":2023,"relevance":"Systematic review confirming HN's cytoprotective role in aging-related disease, providing therapeutic context and noting that HN mechanisms including apoptosis regulation are not yet fully clarified."},{"pmid_or_doi":"27082450","title":"Humanin: Functional Interfaces with IGF-I","year":2016,"relevance":"Documents HN's extracellular signaling functions and binding partners beyond BCL-2 family, relevant to anticipating off-target effects of conformationally constrained analogs on receptor interactions."}]},"onchain":{"hash":"3WTR9CS4e7Pk3LVxEHbayfJXDzbmUhfHGXA84c6ZqkcYemkwP9S7iKvv1wXH8gbFL7cdMDPWL3Y6e9xonSgQCB2a","signature":"3WTR9CS4e7Pk3LVxEHbayfJXDzbmUhfHGXA84c6ZqkcYemkwP9S7iKvv1wXH8gbFL7cdMDPWL3Y6e9xonSgQCB2a","data_hash":"0e9aecf6a1defa00dfadb7a2d06a7585dfdc1681a1fccabae888aa2388c7b4ac","logged_at":"2026-05-05T02:04:30.929730+00:00","explorer_url":"https://solscan.io/tx/3WTR9CS4e7Pk3LVxEHbayfJXDzbmUhfHGXA84c6ZqkcYemkwP9S7iKvv1wXH8gbFL7cdMDPWL3Y6e9xonSgQCB2a"},"ipfs_hash":null,"created_at":"2026-05-05T01:59:07.540527+00:00","updated_at":"2026-05-05T02:04:30.938146+00:00"}