Humanin 10mg Peptide

Humanin is a short peptide encoded inside the mitochondrial genome, within the 16S ribosomal RNA gene MT-RNR2. It was found in 2001 by Hashimoto and colleagues at Keio University, who transfected a cDNA library built from the occipital cortex of an Alzheimer's patient into neuronal cells that were being killed by a familial Alzheimer's mutant gene, then kept whichever clones survived. Four rounds of that death-trap selection returned 250 clones, which sorted into 36 groups, one of which encoded a 75-base open reading frame for a 24-residue peptide they named Humanin. Its identification did something larger than add another neuroprotective factor: it was the first demonstration that the mitochondrial genome, long treated as a set of respiratory-chain parts and two rRNAs, also encodes signalling peptides.

Two forms exist and the difference is not a manufacturing variant. The same open reading frame gives a 24-residue peptide when read by cytoplasmic ribosomes under the standard genetic code, and a 21-residue peptide when read inside the mitochondrion, because codon 22 is AGA, which specifies arginine in the standard code and terminates translation in the vertebrate mitochondrial code. The 24-residue form, MAPRGFSCLLLLTSEIDLPVKRRA, is the one with a CAS registry number, a PubChem entry and a catalogue mass of 2,687.2 g/mol. It is the form supplied here.

Almost everything sold or discussed as humanin research data was generated with S14G-humanin, usually written HNG, in which the serine at position 14 is replaced by glycine. In the original characterisation the native peptide gave complete neuronal protection at 10 micromolar while HNG gave complete protection at 10 nanomolar, a roughly thousand-fold separation that has been repeated in enough assays to become the standard shorthand for the analogue. Native humanin and HNG are different molecules with different potency and different pharmacokinetics, and a figure quoted for one does not transfer to the other.

Humanin acts on two sides of the plasma membrane, and the two accounts were built by different laboratories working on different problems. Outside the cell, secreted humanin engages a three-subunit cytokine receptor made of CNTFR alpha, WSX-1 and gp130, and signals through JAK/STAT. Inside the cell, humanin binds the pro-apoptotic protein Bax and prevents it moving from cytosol to mitochondrial membrane, and it binds insulin-like growth factor binding protein 3. Neither branch subsumes the other, and the receptor literature contains a genuine unresolved disagreement rather than a tidy consensus.

Hashimoto and colleagues established the trimeric receptor in Molecular Biology of the Cell in 2009 using 10 micromolar humanin or 100 nanomolar HNG on F11 neurohybrid cells, SH-SY5Y neuroblastoma and primary cortical neurons. A dominant-negative gp130 construct abolished humanin-mediated protection completely; siRNA against either CNTFR or WSX-1 removed the response; neutralising antibodies against CNTFR or WSX-1 blocked it in human neuroblastoma. Phosphorylation of STAT3 at Tyr705 tracked the protection and was required for it. The same paper argued against the earlier receptor assignment: siRNA knockdown of FPR2, the mouse counterpart of FPRL1, did not reduce humanin activity in their hands.

The FPRL1 account came from Ying and colleagues in the Journal of Immunology in 2004, working on the inflammatory rather than the neuronal side. They showed that humanin induces chemotaxis of mononuclear phagocytes through human FPRL1 and mouse FPR2, the same G protein coupled receptor that amyloid-beta 42 uses to recruit and activate phagocytes. Humanin reduced amyloid-beta aggregation and fibril formation on those cells, and in neuroblasts both peptides activated FPRL1 but only amyloid-beta killed the cells. Their reading was competitive occupancy: humanin blocks amyloid-beta from reaching FPRL1. The most defensible current position is that humanin has at least two receptor systems serving different cell types, and that which one dominates depends on the assay.

The intracellular arm is the most structurally specific part of the pharmacology. Guo and colleagues reported in Nature in 2003 that humanin binds Bax and prevents its translocation from cytosol to mitochondria; silencing humanin with siRNA sensitised cells to Bax and increased Bax movement to membranes; humanin peptides blocked Bax association with isolated mitochondria and suppressed cytochrome c release in a cell-free system. Separately, Ikonen and colleagues pulled humanin out of a yeast two-hybrid screen for IGFBP-3 binding partners, mapped the binding site to the 18-residue heparin-binding domain of IGFBP-3, and showed by alanine scanning that the F6A substitution abolishes binding without abolishing the peptide's other activities. That single-residue separation is what made HNGF6A, a potent analogue that does not bind IGFBP-3, a usable tool for asking which effects run through the IGF axis.

The cysteine at position 8 is not incidental. Substituting it with alanine abolished protection against V642I-APP toxicity even at 100 micromolar, whereas replacing the C-terminal KRRA with AAAA barely changed activity. Humanin therefore carries exactly one cysteine and one methionine, both oxidation-sensitive, in a molecule whose activity depends on one of them.

The 2001 screen that found humanin was not looking for a mitochondrial peptide. Hashimoto and colleagues transfected F11 neurohybrid cells, a fusion of embryonic day 13 rat primary neurons with mouse neuroblastoma, with a cDNA library from the occipital cortex of an Alzheimer's brain, then applied a familial Alzheimer's mutant gene as the selective pressure and kept the survivors. Four rounds returned 250 clones in 36 groups. One encoded the 75-base open reading frame. That the same reading frame also existed in mitochondrial DNA was noticed later, and Guo and colleagues remarked on it in 2003 when they found the mitochondrial version could also bind and suppress Bax.

Three laboratories arrived at humanin from three directions within two years, which is unusual and is part of why the mechanism literature reads as three parallel stories. The Nishimoto group came through neuroprotection against amyloid-beta. The Reed group came through Bax and apoptosis. The Cohen group came through a yeast two-hybrid screen for IGFBP-3 partners. None of the three was studying mitochondrial signalling when they started.

Humanin is the founding member of what is now a small class. Cobb and colleagues ran an in silico search of the same 16S rRNA region in 2016 and found six further peptides, named small humanin-like peptides, SHLP1 through SHLP6. SHLP2 and SHLP3 reduced apoptosis and reactive oxygen species production and improved mitochondrial metabolism in vitro, while SHLP6 did the opposite and increased apoptosis in both cell lines tested. MOTS-c, reported by Lee and colleagues in Cell Metabolism in 2015, comes from the other ribosomal RNA gene, the 12S. The pattern that emerged is that the mitochondrial genome encodes a set of peptides with different and sometimes opposing effects, not a single protective factor.

The mitochondrial genetic code is not the standard genetic code. In vertebrate mitochondria, AGA and AGG terminate translation instead of specifying arginine. Codon 22 of the humanin reading frame is AGA. Translated on a cytoplasmic ribosome under the standard code, the peptide runs through Lys21-Arg22-Arg23-Ala24 and is 24 residues long, MAPRGFSCLLLLTSEIDLPVKRRA, at 2,687.2 g/mol. Translated inside the mitochondrion, it stops after Lys21 and is 21 residues long, MAPRGFSCLLLLTSEIDLPVK, calculating to 2,303.8 g/mol. Both are biologically active.

A 2023 analysis of selection across vertebrates found the humanin start codon conserved in 79 percent of species examined, with codon-level conservation strongest at the codons for Cys8, Leu9, Glu15 and Ile16. Codon 22 carried a synonymous-fraction value of 0.86 for the stop codon reading, which is the kind of conservation you would expect if the mitochondrial translation product is under selection in its own right rather than being an artefact of where the reading frame happens to sit. The synonymous codon bias across the reading frame reached p less than 0.00063, consistent with purifying selection on a protein-coding element rather than on rRNA structure alone.

There is a second complication that most sources skip entirely. Thirteen humanin-like sequences sit in the nuclear genome as nuclear insertions of mitochondrial origin, the MTRNR2L family, and ten of them are transcribed across human tissues. This matters for interpreting the human data: when a study reports that circulating humanin falls with age, the immunoassay is measuring an epitope that several nuclear paralogues could also present. It is a real limitation of the human biomarker literature, and it is why the age-decline claim rests better on the direction and reproducibility of the finding than on any single absolute concentration.

S14G-humanin, HNG, replaces the serine at position 14 with glycine. In the original characterisation, native synthetic humanin at 10 nanomolar had minimal effect and needed 10 micromolar for complete protection of V642I-APP transfected F11 cells, while HNG achieved complete protection at 10 nanomolar. That thousand-fold figure is the number quoted everywhere, but it is an assay-specific ratio, not a universal constant, and the underlying reason for the potency gain has never been fully resolved.

The practical consequence is that the great majority of in vivo humanin work uses HNG or a further-modified analogue, not the native sequence. The myocardial ischaemia work used HNG at up to 2 mg/kg intraperitoneally in mice. The stroke work used HNG at 0.1 microgram intracerebroventricularly or 1 microgram intraperitoneally in mice. The healthspan work in middle-aged mice used HNG. The insulin work used HNGF6A, an analogue combining the S14G potency change with the F6A substitution that abolishes IGFBP-3 binding, which was the point: it let the investigators ask whether the metabolic effect needed the IGF axis. A figure taken from any of those papers describes the analogue that was injected, not the native 24-mer.

Chin and colleagues measured this directly in Endocrinology in 2013. Wild-type mice given HNGF6A and IGFBP-3 knockout mice given HNG both showed longer humanin half-lives than wild-type mice given HNG, which places IGFBP-3 binding squarely in the clearance pathway. Half-life was longer in rats than in mice. In Sprague-Dawley rats given HNG intraperitoneally, humanin was highest in plasma, present in liver, and undetectable in brain or heart. Circulating IGF-I and IGFBP-3 both fell over time after HNG injection. Any single half-life number quoted without naming the analogue, the species and the route is not a fact about humanin.

The observation driving most interest in humanin is that it declines with age. Muzumdar and colleagues reported in 2009 that detectable humanin fell with age in rodent hypothalamus, skeletal muscle and cortex, and that circulating levels fell with age in both humans and mice. Cobb and colleagues found the same age-related decline for circulating SHLP2, suggesting the pattern is not unique to humanin among the peptides from that region.

Yen and colleagues extended this in 2020 with the comparisons that make the association interesting rather than merely descriptive. Humanin levels declined with age in multiple species but were stable across the lifespan of the naked mole-rat, an animal used as a model of negligible senescence. In children of centenarians, a group with an elevated likelihood of reaching extreme age themselves, circulating humanin was substantially higher than in age-matched controls. Levels were reduced in Alzheimer's disease and in MELAS, a mitochondrial encephalomyopathy. The same study showed that overexpression in Caenorhabditis elegans increased lifespan in a daf-16 dependent manner, which connects the correlational human data to a causal handle in a genetic model.

Exercise moves humanin acutely in people. Woodhead and colleagues took vastus lateralis biopsies and plasma from ten untrained healthy young men, mean age 24.5 plus or minus 3.7 years, before, immediately after and four hours after ten 60-second cycle ergometer bouts at peak oxygen uptake power output. Humanin protein rose in both muscle and plasma after the acute bout. SHLP6 behaved the same way and SHLP2 did not, which points to selective regulation of peptides from the same MT-RNR2 gene. Notably, the muscle rise was not accompanied by a rise in MT-RNR2 or humanin mRNA, which actually fell, so the acute response is not transcriptional. Contracting isolated mouse extensor digitorum longus muscle raised humanin roughly fourfold. Six sessions of high-intensity interval training over two weeks trended toward lowering resting plasma humanin, p equals 0.063, without changing the acute response.

Both are mitochondrial-derived peptides and both are in this catalogue, which makes the comparison worth doing properly rather than treating them as interchangeable siblings. They come from different genes: humanin from MT-RNR2, the 16S rRNA gene, and MOTS-c from MT-RNR1, the 12S rRNA gene. They differ in size and mass, humanin at 24 residues and 2,687.2 g/mol against MOTS-c at 16 residues and 2,174.6 g/mol. They were reported fourteen years apart, humanin in 2001 and MOTS-c in 2015.

The mechanisms do not overlap. Humanin works through a cell-surface cytokine receptor complex of CNTFR alpha, WSX-1 and gp130 with STAT3 downstream, plus intracellular binding to Bax and IGFBP-3. MOTS-c has no identified cell-surface receptor. Its reported cellular action is inhibition of the folate cycle and the de novo purine biosynthesis tethered to it, which raises AICAR and activates AMPK, and under metabolic stress it translocates to the nucleus and acts on stress-response gene transcription. Nothing in the literature places the two peptides on a shared receptor or a shared immediate signalling step.

Their strongest evidence sits in different places too. Humanin's deepest dataset is cytoprotection: neuronal rescue from amyloid-beta and familial Alzheimer's mutants, infarct-size reduction in cardiac and cerebral ischaemia-reperfusion models, retinal pigment epithelium survival. MOTS-c's deepest dataset is metabolic: prevention of age-dependent and diet-induced insulin resistance and of diet-induced obesity in mice, with skeletal muscle as the apparent primary target organ. Humanin does have metabolic data, but that arm runs through the hypothalamus and STAT3 rather than through skeletal muscle AMPK, and it was demonstrated with analogues rather than the native peptide.

Humanin is a hydrophobic peptide with a tetraleucine stretch at positions 9 through 12 and a single free cysteine at position 8. The cysteine is not spare capacity: substituting it with alanine abolished protection against V642I-APP toxicity even at 100 micromolar, while replacing the C-terminal Lys-Arg-Arg-Ala with four alanines barely changed activity. A material that has oxidised or disulfide-dimerised at Cys8 has lost the residue that the activity depends on, which is why headspace, moisture and repeated freeze-thaw matter more for this sequence than for a peptide with no cysteine. The methionine at position 1 is a second oxidation target, and methionine sulfoxide formation shows as a plus 16 mass shift on LC-MS.

The mass a certificate reports depends on what was weighed. Free-base humanin is 2,687.2 g/mol against the formula C119H204N34O32S2. Peptides purified by reversed-phase HPLC with trifluoroacetic acid in the mobile phase are typically isolated as TFA salts, and a multiply-basic peptide like this one, carrying two arginines, a lysine and a further arginine in the C-terminal region, can pick up several TFA counterions. A mass in the 2,800 range with fluorine in the formula is the salt, not the peptide, and treating it as the peptide mass means every molar concentration calculated from it is wrong in the same direction. Ask what the number refers to before using it.

Two other numbers are routinely conflated. HPLC purity is the fraction of peptide-related material that is the target peptide. Net peptide content is the fraction of the vial mass that is peptide at all, the remainder being counterion, residual water and salt, and it is typically 70 to 85 percent for a TFA-salt peptide. A vial can be 99 percent pure by HPLC and still contain substantially less peptide by mass than its label weight suggests. Amino acid analysis or quantitative nitrogen determination is what settles net content; HPLC alone cannot.

The CAS number 330936-69-1 refers to the 24-residue sequence specifically. The 21-residue form and the S14G analogue are different substances and are not covered by it. A certificate of analysis that pairs that CAS number with a mass or a sequence for a different form has an internal inconsistency worth resolving before the material is used.

No interventional trial has administered humanin or a humanin analogue to humans. Searching ClinicalTrials.gov for humanin returns a small set of studies, and every one of them measures humanin as a biomarker rather than giving it. NCT03431844, completed, measured humanin isoforms in cardiac muscle and blood plasma against major complications after cardiac surgery. NCT06105229 examines plasma humanin in acute kidney injury. NCT07678073 compares general anaesthesia with combined spinal-epidural anaesthesia in renal transplantation and measures humanin and MOTS-c among its outcomes. Two further observational studies look at humanin in acute kidney injury after cardiac surgery and in IVF outcomes in polycystic ovary syndrome. The registry contains no phase designation for humanin as an agent because there is no trial of humanin as an agent.

There is also a finding in the literature that runs against the general direction of the field and deserves to be stated rather than omitted. Moreno Ayala and colleagues reported in Scientific Reports in 2020 that exogenous humanin protected triple-negative breast cancer cells from apoptotic stimuli, that shRNA silencing of humanin reduced their viability and increased chemosensitivity, and that systemic humanin in tumour-bearing mice reduced tumour apoptosis, blunted the antitumour and antimetastatic effect of chemotherapy, and accelerated tumour growth and spontaneous lung metastasis. Humanin and its receptors were expressed in breast cancer specimens in TCGA transcriptomic data and were upregulated in triple-negative biopsies relative to healthy mammary tissue. An anti-apoptotic peptide protects whatever cell it reaches, and that is the mechanism working as designed, not a paradox.

The open questions that a specific answer would move are narrow. Whether the trimeric receptor and FPRL1 accounts describe two receptors on different cell types or one pathway misassigned by one of the two groups has not been settled by a study designed to test it. Whether native 24-residue humanin, as opposed to HNG, produces any of the in vivo effects at attainable exposures is largely untested, because almost nobody has run the native peptide in vivo. And whether the human age-decline signal survives an assay that can distinguish mitochondrial humanin from the ten transcribed MTRNR2L paralogues remains open.