Humanin: The First Mitochondrial-Derived Peptide in the Research Literature

Research-use-only context. This article summarizes published third-party scientific literature and laboratory practice. It is not medical advice, not a therapeutic or performance claim, and not a usage guide. US Prime Peptides products are supplied strictly for in vitro laboratory research and are not for human or veterinary use.

This article explains what humanin is, how it was discovered in 2001, why it is called the first mitochondrial-derived peptide, which proteins it has been shown to bind in cell assays, what rodent and human-sample studies report, and how it relates to MOTS-c. It ends with what a research certificate should show for a 24-residue peptide that contains a single cysteine.

Humanin is a 24-residue peptide encoded inside the mitochondrial 16S rRNA gene

Humanin is a short peptide, Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, with a molecular weight near 2,687 daltons. What makes it unusual is where its gene sits: the open reading frame lies within the mitochondrial gene MT-RNR2, which encodes the 16S ribosomal RNA. A gene for a peptide hidden inside a gene for a structural RNA was unexpected, and it made humanin the founding member of what are now called mitochondrial-derived peptides. Because the mitochondrial and cytoplasmic genetic codes differ, the same reading frame translates to a 24-residue peptide if read by cytoplasmic ribosomes and a 21-residue peptide if read inside mitochondria; which compartment produces the peptide in vivo has never been fully resolved.

Guo, Zhai, and colleagues' 2003 Nature paper noted that an identical open reading frame exists in the nuclear genome and proposed that the sequence originated in mitochondria and transferred to the nucleus. Bodzioch and colleagues followed this up in a 2009 Genomics paper, identifying 13 nuclear loci resembling MT-RNR2 that could encode 15 distinct full-length humanin-like peptides (the MTRNR2L family), at least ten of which were expressed in human tissues. They also defined two conserved motifs, residues 5 to 11 and 14 to 19, and suggested that proline versus serine at position 19 may decide whether a variant is secreted. The peptide therefore has a family of close relatives, and an antibody or an assay may not distinguish among them.

The 2001 discovery: a functional screen of a human brain cDNA library

Hashimoto, Niikura, Tajima, and colleagues reported humanin in a 2001 Proceedings of the National Academy of Sciences paper. They built a cDNA library from the occipital lobe of a person who had died with Alzheimer's disease, a region relatively spared by the disease, and screened it in F11 neuronal hybrid cells for clones that prevented cell death caused by a mutant amyloid precursor protein gene. One clone, named Humanin, abolished death caused by several familial Alzheimer's disease genes and by amyloid-beta peptide, but not death caused by polyglutamine repeats or mutant superoxide dismutase-1. The transfected cDNA was translated and the peptide was secreted into the culture medium, and the rescue depended on the primary sequence.

A companion 2001 paper in the Journal of Neuroscience mapped the structure-activity relationship. Cys8 and Ser14 were essential, the active domain ran from Pro3 to Pro19, and seven residues within it could not be substituted. Replacing serine 14 with glycine produced an analog, called HNG or S14G-humanin, roughly a thousand times more potent in the same assay; most later cell and animal work uses HNG rather than the native sequence, a point to check when reading any humanin paper. Caricasole and colleagues' 2002 paper in the FASEB Journal identified a rat homolog, rattin, 38 residues long with 73% identity in the conserved region, which in cultured cortical neurons matched humanin against amyloid-beta and outperformed it against NMDA excitotoxicity.

Binding partners identified in cell and biochemical assays

Five binding partners have been reported, each by a different method. Guo and colleagues' 2003 Nature paper found by yeast two-hybrid screening that humanin binds Bax, prevents its translocation from cytosol to mitochondria, blocks its association with isolated mitochondria, and suppresses cytochrome c release in vitro; knocking down humanin with siRNA sensitized cells to Bax. Zhai, Luciano, and colleagues extended this in a 2005 Journal of Biological Chemistry paper showing that synthetic humanin also binds the BH3-only protein Bid and its truncated form, and blocks tBid-induced oligomerization of Bax and Bak in mitochondrial membranes, including in Bax-deficient mitochondria.

Ikonen, Liu, Hashimoto, and colleagues' 2003 Proceedings of the National Academy of Sciences paper cloned humanin as a binding partner of insulin-like growth factor-binding protein 3 (IGFBP-3), confirmed the interaction by co-immunoprecipitation from mouse testis, mapped it to the 18-residue heparin-binding domain of IGFBP-3, and showed that a Phe6-to-Ala mutant does not bind. Ying and colleagues' 2004 paper in the Journal of Immunology reported that synthetic humanin induced chemotaxis of mononuclear phagocytes through the G protein-coupled formylpeptide receptor-like-1 (FPRL1, now FPR2) and its mouse counterpart, the same receptor used by amyloid-beta 42. Hashimoto, Kurita, and colleagues' 2009 paper in Molecular Biology of the Cell identified a second receptor system: a trimeric complex of ciliary neurotrophic factor receptor alpha, the IL-27 receptor subunit WSX-1, and gp130, through which humanin activates STAT3.

Two receptor systems and three intracellular partners is an unusual profile, and the field has not reconciled them into one model. It is possible the peptide acts both outside the cell (via FPR2 or the gp130 complex) and inside it (via Bax and Bid), or that different assays report different things.

Structure: disordered in water, helical in a membrane-like solvent

Benaki, Zikos, and colleagues solved the solution structure by NMR and circular dichroism in a 2005 Biochemical and Biophysical Research Communications paper. In water, humanin was predominantly unstructured, in equilibrium with turn-like elements between Gly5 and Leu10 and between Glu15 and Leu18. In 30% trifluoroethanol it formed a helix spanning Gly5 to Leu18, which contains the hydrophobic Leu9-Leu12 stretch and the essential Cys8 and Ser14. A 2006 paper from the same group on the S14G analog found a shorter helix, Phe6 to Thr13, in the same solvent, and emphasized flexibility as a feature common to the active forms.

These findings have practical consequences. The four consecutive leucines make the peptide amphipathic and prone to aggregation at high concentration, which the solubility article discusses in general terms. The single cysteine can form an intermolecular disulfide, producing a dimer of about 5,372 daltons that will show up as a separate HPLC peak and a doubled mass in the spectrum. The N-terminal methionine can oxidize to the sulfoxide, adding 16 daltons. Each of these is a stored-solution problem rather than a lyophilized-powder problem.

Rodent studies and human-sample observations

The rodent literature is dominated by the Cohen and Muzumdar laboratories and uses HNG or other analogs more often than the native peptide. Muzumdar and colleagues' 2009 paper in PLoS One used hyperinsulinemic clamps in rats and reported that humanin delivered into the cerebral ventricles increased insulin sensitivity through hypothalamic STAT3, that the effect on the liver was lost when hypothalamic STAT3 was inhibited, and that humanin levels fell with age in rodent hypothalamus, muscle, and cortex and in human circulation. Lee, Wan, and colleagues' 2014 paper in Aging Cell reported an inverse relationship between growth hormone/IGF-1 axis activity and humanin levels: long-lived GH-deficient Ames mice had higher levels, GH-transgenic mice lower, and giving GH or IGF-1 to mice and to human volunteers reduced circulating humanin. Chin and colleagues' 2013 pharmacokinetic study in Endocrinology found that HNG had a short half-life in mice, longer in rats, that IGFBP-3 knockout lengthened it, and that in rats the analog was detectable in plasma and liver but not in brain or heart.

Yen, Mehta, and colleagues' 2020 paper in Aging assembled several models: overexpressing humanin in C. elegans lengthened lifespan in a daf-16/FOXO-dependent way; humanin-transgenic mice resisted toxic insults; middle-aged mice given HNG over several months showed changed metabolic and inflammatory markers; levels were stable with age in the naked mole-rat; and children of centenarians had higher circulating levels than age-matched controls. Yen's 2018 Scientific Reports paper identified a mitochondrial SNP, rs2854128, in the humanin-coding region that was associated with lower circulating levels and, in a separate large cohort, with faster cognitive aging. Cobb, Lee, and colleagues' 2016 Aging paper described six additional small humanin-like peptides (SHLP1 to SHLP6) encoded in the same 16S rRNA region. The human data are observational associations of circulating levels; no human trial of the peptide has been published.

Relation to MOTS-c, status, and the certificate

Humanin and MOTS-c are the two best-known mitochondrial-derived peptides, but they come from different genes and behave differently. MOTS-c is a 16-residue peptide encoded in the 12S rRNA gene (MT-RNR1), reported by Lee and colleagues in a 2015 Cell Metabolism paper to act on AMPK and folate-methionine metabolism, and it has a separate article, the MOTS-c overview. Humanin is encoded in the 16S gene, is secreted, and has reported extracellular receptors. The three-way comparison with SS-31, a synthetic cardiolipin-binding peptide that is not mitochondrially encoded at all, is in humanin vs MOTS-c vs SS-31.

Humanin is not an approved drug in the United States or elsewhere, and no marketing application for it or for HNG has been located; the FDA status reference table records this. A research certificate for the native peptide should show mass spectrometry identity against about 2,687 daltons (with the dimer at twice that and the Met-sulfoxide at plus 16 flagged if present), HPLC purity at 214 nm with 280 nm detection supported by the single phenylalanine only weakly, net peptide content, and the endotoxin, sterility, and heavy-metals panel. Because vendors sometimes sell HNG under the humanin name, the certificate should state which sequence it covers; the mass differs by 30 daltons (Ser to Gly). US Prime Peptides posts the lot-matched certificate for Humanin 10 mg on the product page before purchase.

Frequently Asked Questions

What is humanin and where is it encoded?

Humanin is a 24-residue peptide whose open reading frame lies inside the mitochondrial 16S ribosomal RNA gene, MT-RNR2. Hashimoto and colleagues discovered it in 2001 by screening a human brain cDNA library for clones that prevented neuronal cell death in culture. Thirteen nuclear loci encode similar peptides (the MTRNR2L family).

What is the difference between humanin and HNG (S14G-humanin)?

HNG replaces serine 14 with glycine. In the original 2001 cell assays it was about a thousand times more potent than the native peptide, and most subsequent rodent studies use HNG or related analogs rather than native humanin. The two differ in mass by 30 daltons and a certificate should say which one it describes.

What does humanin bind to?

Cell and biochemical assays have reported five partners: the pro-apoptotic proteins Bax and Bid inside the cell, IGFBP-3 in the extracellular space, the formylpeptide receptor FPRL1/FPR2, and a cytokine receptor complex of CNTFR, WSX-1, and gp130 that signals through STAT3. How these fit into a single mechanism is not settled.

How is humanin different from MOTS-c?

Both are mitochondrial-derived peptides, but humanin comes from the 16S rRNA gene and MOTS-c from the 12S rRNA gene. Humanin is 24 residues, is secreted, and has reported cell-surface receptors; MOTS-c is 16 residues and is reported to act on AMPK and one-carbon metabolism. They were discovered 14 years apart by different groups.

Citations

1. 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 Abeta. Proc Natl Acad Sci U S A. 2001;98:6336-6341. PMID 11371646
2. Hashimoto Y, Niikura T, Ito Y, et al. Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults. J Neurosci. 2001;21:9235-9245. PMID 11717357
3. Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity. FASEB J. 2002;16:1331-1333. PMID 12154011
4. Guo B, Zhai D, Cabezas E, et al. Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature. 2003;423:456-461. PMID 12732850
5. 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 U S A. 2003;100:13042-13047. PMID 14561895
6. Ying G, Iribarren P, Zhou Y, et al. Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor. J Immunol. 2004;172:7078-7085. PMID 15153530
7. Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem. 2005;280:15815-15824. PMID 15661737
8. Benaki D, Zikos C, Evangelou A, et al. Solution structure of humanin, a peptide against Alzheimer's disease-related neurotoxicity. Biochem Biophys Res Commun. 2005;329:152-160. PMID 15721287
9. Benaki D, Zikos C, Evangelou A, et al. Solution structure of Ser14Gly-humanin, a potent rescue factor against neuronal cell death in Alzheimer's disease. Biochem Biophys Res Commun. 2006;349:634-642. PMID 16945331
10. Hashimoto Y, Kurita M, Aiso S, et al. Humanin inhibits neuronal cell death by interacting with a cytokine receptor complex or complexes involving CNTF receptor alpha/WSX-1/gp130. Mol Biol Cell. 2009;20:2864-2873. PMID 19386761
11. Bodzioch M, Lapicka-Bodzioch K, Zapala B, et al. Evidence for potential functionality of nuclearly-encoded humanin isoforms. Genomics. 2009;94:247-256. PMID 19477263
12. Muzumdar RH, Huffman DM, Atzmon G, et al. Humanin: a novel central regulator of peripheral insulin action. PLoS One. 2009;4:e6334. PMID 19623253
13. Yen K, Lee C, Mehta H, Cohen P. The emerging role of the mitochondrial-derived peptide humanin in stress resistance. J Mol Endocrinol. 2013;50:R11-R19. PMID 23239898
14. Chin YP, Keni J, Wan J, et al. Pharmacokinetics and tissue distribution of humanin and its analogues in male rodents. Endocrinology. 2013;154:3739-3744. PMID 23836030
15. Lee C, Wan J, Miyazaki B, et al. IGF-I regulates the age-dependent signaling peptide humanin. Aging Cell. 2014;13:958-961. PMID 25040290
16. 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;21:443-454. PMID 25738459
17. Cobb LJ, Lee C, Xiao J, et al. Naturally occurring mitochondrial-derived peptides are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory markers. Aging (Albany NY). 2016;8:796-809. PMID 27070352
18. Yen K, Wan J, Mehta HH, et al. Humanin prevents age-related cognitive decline in mice and is associated with improved cognitive age in humans. Sci Rep. 2018;8:14212. PMID 30242290
19. Yen K, Mehta HH, Kim SJ, et al. The mitochondrial derived peptide humanin is a regulator of lifespan and healthspan. Aging (Albany NY). 2020;12:11185-11199. PMID 32575074

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