Mitochondrial Peptides: MOTS-c, Humanin and SS-31

"Mitochondrial peptide" covers two quite different ideas. MOTS-c and humanin are mitochondrial-derived peptides: short sequences encoded within the mitochondrial genome itself, studied as signals between mitochondria and the rest of the cell. SS-31 is a synthetic tetrapeptide designed to concentrate at the inner mitochondrial membrane. The three are often grouped together, but their origins, mechanisms and evidence bases differ substantially.

Origin and discovery

Humanin was the first mitochondrial-derived peptide described. In 2001, Hashimoto and colleagues in the laboratory of Ikuo Nishimoto screened a cDNA library from the relatively spared occipital cortex of a person with Alzheimer's disease, looking for sequences that protected neuronal cells from death induced by familial Alzheimer's disease genes and amyloid-β. The protective clone encoded a 24-residue peptide. Its open reading frame lies within the mitochondrial 16S ribosomal RNA gene (MT-RNR2). Independent groups soon reported humanin binding to the pro-apoptotic protein BAX (Guo et al., 2003) and to IGFBP-3 (Ikonen et al., 2003).

MOTS-c (mitochondrial open reading frame of the 12S rRNA type-c) was identified by Changhan Lee, Pinchas Cohen and colleagues, who reported in 2015 that a short open reading frame within the mitochondrial 12S rRNA gene encoded a 16-residue peptide with metabolic activity.

SS-31 comes from a different tradition. Hazel Szeto and Peter Schiller developed a series of aromatic-cationic tetrapeptides, the Szeto–Schiller peptides, while working on opioid peptide analogues. They found that certain members, including SS-31, were cell-permeable and accumulated at the inner mitochondrial membrane without opioid activity. SS-31 was later developed as elamipretide by Stealth BioTherapeutics.

Structure and chemistry

	MOTS-c	Humanin	SS-31
Sequence	MRWQEMGYIFYPRKLR	MAPRGFSCLLLLTSEIDLPVKRRA	D-Arg-Dmt-Lys-Phe-NH2
Length	16 residues	24 residues	4 residues
Approx. mass	~2,175 Da	~2,687 Da	~640 Da
Notable features	Encoded in 12S rRNA region	Encoded in 16S rRNA region; S14G analogue (HNG) widely studied	D-arginine and 2′,6′-dimethyltyrosine (Dmt); alternating aromatic and cationic residues

Humanin can be translated either by the mitochondrial or the cytoplasmic genetic code, which yields peptides of slightly different length, and nuclear-encoded humanin-like sequences complicate attribution. SS-31's non-natural residues resist proteolysis, and its alternating cationic and aromatic residues let it cross membranes and associate with anionic phospholipids.

Mechanism studied

MOTS-c

Lee et al. (2015) reported that MOTS-c inhibited the folate–methionine pathway, causing accumulation of the purine intermediate AICAR and activation of AMP-activated protein kinase (AMPK) in skeletal muscle. Kim et al. (2018) showed that under metabolic stress MOTS-c translocated to the nucleus in an AMPK-dependent manner and regulated antioxidant-response genes in cooperation with NRF2. This dual cytoplasmic and nuclear role is studied as a form of mitochondrial-to-nuclear signalling.

Humanin

Humanin acts both outside and inside cells. Extracellularly, it has been reported to bind formyl peptide receptor-like 1 and a trimeric receptor composed of CNTFR, WSX-1 and gp130, activating JAK2–STAT3 signalling. Intracellularly, it binds BAX and IGFBP-3, interfering with apoptosis. Central humanin signalling improved hepatic insulin sensitivity in rodent studies (Muzumdar et al., 2009).

SS-31

SS-31 binds cardiolipin, a phospholipid found almost exclusively in the inner mitochondrial membrane, where it organises electron transport chain supercomplexes and anchors cytochrome c. By associating with cardiolipin, SS-31 is reported to limit cardiolipin peroxidation, preserve cristae structure and improve electron transfer (Birk et al., 2013). It is described as a membrane-targeted stabiliser rather than a classic radical scavenger.

Research landscape by model system

In vitro

Cell work includes MOTS-c in myoblasts and adipocytes (glucose uptake, AMPK signalling), humanin in neuronal lines exposed to amyloid-β or oxidative stress, and SS-31 in isolated mitochondria and oxidative-injury models.

Animal models

• MOTS-c: reduced diet-induced obesity and insulin resistance in mice (Lee et al., 2015); improved physical capacity in young, middle-aged and old mice and was induced by exercise (Reynolds et al., 2021).
• Humanin: neuroprotection in rodent Alzheimer's and stroke models, improved insulin sensitivity, and cardioprotection in ischaemia–reperfusion. Circulating levels were reported to decline with age in rodents.
• SS-31: reduced ischaemia–reperfusion injury in heart and kidney, and improved mitochondrial function in aged mouse skeletal muscle and heart.

Human trials

• MOTS-c: an analogue, CB4211, entered early-stage clinical testing in obesity and fatty liver disease before the sponsor discontinued development. Observational studies have measured circulating MOTS-c in various populations.
• Humanin: no interventional trials have been published. Observational work has examined circulating humanin in relation to age and disease.
• SS-31 (elamipretide): evaluated in phase 2 and phase 3 trials in primary mitochondrial myopathy, heart failure, Barth syndrome and dry age-related macular degeneration, with mixed results. In 2025 the FDA granted elamipretide accelerated approval for Barth syndrome, a rare X-linked disorder of cardiolipin metabolism.

Evidence quality and open questions

SS-31 has the strongest evidence of the three, with independent preclinical replication and a regulatory approval in one rare disease, although several larger trials in broader indications missed their primary endpoints.

MOTS-c and humanin have credible preclinical foundations from leading laboratories, but evidence is low. Open questions include:

1. Endogenous relevance. How much MOTS-c and humanin are produced from the mitochondrial genome, versus nuclear pseudogene copies, is debated.
2. Assay reliability. Immunoassays for circulating mitochondrial-derived peptides vary, and mass-spectrometry confirmation is limited.
3. Receptors. MOTS-c lacks a defined receptor, and humanin's multiple binding partners make its primary mechanism hard to isolate.
4. Translation. Whether rodent metabolic findings extend to humans remains untested in controlled trials.

Regulatory and anti-doping status

MOTS-c and humanin are not approved by the FDA or EMA. Elamipretide has FDA accelerated approval for Barth syndrome and is not approved by the EMA; synthetic SS-31 from research suppliers is not the approved product. AMPK activators such as AICAR are prohibited under WADA's metabolic modulator class, and MOTS-c may fall within that wording; researchers should check the current list for all three compounds.

Summary table

Attribute	MOTS-c	Humanin	SS-31 (elamipretide)
Origin	Mitochondrial 12S rRNA ORF	Mitochondrial 16S rRNA ORF	Synthetic (Szeto–Schiller series)
Core mechanism	Folate–methionine pathway inhibition, AICAR accumulation, AMPK activation; nuclear gene regulation	Anti-apoptotic binding (BAX, IGFBP-3); receptor-mediated STAT3 signalling	Cardiolipin binding and stabilisation
Main model systems	Mouse metabolic and exercise models	Neuronal cell and rodent neuroprotection models	Isolated mitochondria; rodent ischaemia and ageing models
Human data	Early trials of an analogue; observational	Observational only	Phase 2–3 trials
Evidence level	Low	Low	Moderate
FDA	Not approved	Not approved	Accelerated approval, Barth syndrome
WADA	Check current list	Check current list	Check current list

References

• Hashimoto Y, Niikura T, Tajima H, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ. Proc Natl Acad Sci USA. 2001;98(11):6336–6341.
• Guo B, Zhai D, Cabezas E, et al. Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature. 2003;423:456–461.
• Ikonen M, Liu B, Hashimoto Y, et al. Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis. Proc Natl Acad Sci USA. 2003;100(22):13042–13047.
• Muzumdar RH, Huffman DM, Atzmon G, et al. Humanin: a novel central regulator of peripheral insulin action. PLoS One. 2009;4(7):e6334.
• Lee C, Zeng J, Drew BG, et al. The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance. Cell Metab. 2015;21(3):443–454.
• Kim KH, Son JM, Benayoun BA, Lee C. The mitochondrial-encoded peptide MOTS-c translocates to the nucleus to regulate nuclear gene expression in response to metabolic stress. Cell Metab. 2018;28(3):516–524.
• Reynolds JC, Lai RW, Woodhead JST, et al. MOTS-c is an exercise-induced mitochondrial-encoded regulator of age-dependent physical decline and muscle homeostasis. Nat Commun. 2021;12:470.
• Zhao K, Zhao GM, Wu D, et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem. 2004;279(33):34682–34690.
• Birk AV, Liu S, Soong Y, et al. The mitochondria-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin. J Am Soc Nephrol. 2013;24(8):1250–1261.
• Reid Thompson W, Hornby B, Manuel R, et al. A phase 2/3 randomized clinical trial followed by an open-label extension to evaluate the effectiveness of elamipretide in Barth syndrome. Genet Med. 2021;23(3):471–478.