Identification of the proteins interacting with neuroprotective peptide humanin in a yeast two-hybrid system

Humanine is a human neuroprotective peptide with a wide action spectrum. To analyze molecular mechanisms of humanin functioning, a search for proteins interacting with this peptide was conducted using yeast two-hybrid system. Screening of human fetal brain cDNA library identified seven proteins with different functions that specifically interacted with humanin.     

Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses

The formyl peptide receptor (FPR) family is involved in host defence against pathogens, but also in sensing internal molecules that may constitute signals of cellular dysfunction. It includes three subtypes in human and other primates. FPR responds to formyl peptides derived from bacterial and mitochondrial proteins. FPRL1 displays a large array of exogenous and endogenous ligands, including the chemokine variant sCKβ8-1, the neuroprotective peptide humanin, and lipoxin A4. Two high affinity agonists (F2L and humanin) were recently described for FPRL2. In mouse, eight FPR-related receptors have been described. Fpr1 is the ortholog of human FPR, while fpr2 appears to share many ligands with human FPRL1. Altogether, the physiological role of the FPR family is still incompletely understood, due in part to the large variety of ligands, the redundancy with other chemoattractant agents, and the lack of clear orthologs between human and mouse receptors. Newly developed tools will allow to study further this family of receptors.     

IGF‐I regulates the age‐dependent signaling peptide humanin

Aging is influenced by endocrine pathways including the growth hormone/insulin‐like growth factor‐1 (GH/IGF) axis. Mitochondrial function has also been linked to the aging process, but the relevant mitochondrial signals mediating the effects of mitochondria are poorly understood. Humanin is a novel signaling peptide that acts as a potent regulator of cellular stress responses and protects from a variety of in vitro and in vivo toxic and metabolic insults. The circulating levels of humanin decline with age in mice and humans. Here, we demonstrate a negative correlation between the activity of the GH‐IGF axis and the levels of humanin, as well as a positive correlation between humanin and lifespan in mouse models with altered GH/IGF‐I axis. Long‐lived, GH‐deficient Ames mice displayed elevated humanin levels, while short‐lived GH‐transgenic mice have reduced humanin levels. Furthermore, treatment with GH or IGF‐I reduced circulating humanin levels in both mice and human subjects. Our results indicate that GH and IGF are potent regulators of humanin levels and that humanin levels correlate with lifespan in mice. This suggests that humanin represents a circulating mitochondrial signal that participates in modulating the aging process, adding a coordinated mitochondrial element to the endocrine regulation of aging.     

Zinc(II) binds to the neuroprotective peptide humanin

The abnormal accumulation of the peptide amyloid-beta in the form of senile (or amyloid) plaques is one of the hallmarks of Alzheimer's disease (AD). Zinc ions have been implicated in AD and plaques formation. Recently, the peptide humanin has been discovered. Humanin showed neuroprotective activity against amyloid-beta insults. Here the question investigated is if humanin could interact directly with Zn(II). It is shown that Zn(II) and its substitutes Cd(II)/Co(II) bind to humanin via a thiolate bond from the side chain of the single cysteine at position 8. The low intensity of the d-d bands of Co(II)-humanin indicated an octahedral coordination geometry. Titration experiments suggest that Zn(II) binds to humanin with an apparent affinity in the low muM range. This apparent Zn-binding affinity is in the same order as for amyloid-beta and glutathione and could thus be of physiological relevance.     

High-yield, solid-phase synthesis of humanin, an Alzheimer's disease associated, novel 24-mer peptide which contains a difficult sequence.

Humanin is a novel, 24-mer residue bioactive peptide, which antagonizes Alzheimer's disease (AD) related neurotoxicity and offers a hope for developing new therapeutics against AD. Access to adequate amounts of pure humanin is a prerequisite for further, thorough, investigation of the pharmacological properties and therapeutic potency of the peptide. Until now, humanin has been obtained mainly by molecular biology techniques. In this work the Fmoc solid-phase synthesis of humanin on an in-house prepared 2-Cl-tritylamidomethyl polystyrene resin is described fully. Special precautions, i.e. prolonged deprotection steps, should be taken to achieve a high overall yield, since humanin seems to contain a 'difficult sequence' (R4G5F6S7C8L9) near its highly lipophilic, biologically important region L9L10L11L12.     

Solution structure of humanin, a peptide against Alzheimer's disease-related neurotoxicity

Humanin is a newly identified 24-residue peptide that suppresses neuronal cell death caused by a wide spectrum of familial Alzheimer's disease genes and the beta-amyloid peptide. In this study, NMR and circular dichroism studies of synthetic humanin in aqueous and 30% 2,2,2-trifluoroethanol (TFE) solutions are reported. In aqueous solution, humanin exists predominantly in an unstructured conformation in equilibrium with turn-like structures involving residues Gly5 to Leu10 and Glu15 to Leu18, providing indication of nascent helix. In the less polar environment of 30% TFE, humanin readily adopts helical structure with long-range order spanning residues Gly5 to Leu18. Comparative 3D modeling studies and topology predictions are in qualitative agreement with the experimental findings in both environments. Our studies reveal a flexible peptide in aqueous environment, which is free to interact with possible receptors that mediate its action, but may also acquire a helical conformation necessary for specific interactions and/or passage through membranes.     

Cardio-protective role of Humanin in myocardial ischemia-reperfusion

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.     

The role of humanin in the regulation of reproduction

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     

Circulating humanin is lower in coronary artery disease and is a prognostic biomarker for major cardiac events in humans

BackgroundHumanin is an endogenous mitochondria-derived peptide that plays critical roles in oxidative stress, inflammation and CAD. In this study, we measured the levels of circulating humanin, markers of oxidative stress and inflammation in patients with unstable angina and MI and studied the relationship between these parameters and major adverse cardiac events (MACE).MethodsA total of 327 subjects were recruited from the inpatient department at First Hospital of Jilin University and divided into 3 groups [control, angina and myocardial infarction (MI)] based on the clinical data and the results of the angiography. Serum humanin and thiobarbituric acid reactive substances (TBARS) were measured at the time of initial admission. The hospitalization data and MACE of all patients were collected.ResultsCirculating humanin levels were lower in the angina group compared to controls [124.22 ± 63.02 vs. 157.77 ± 99.93 pg/ml, p < 0.05] and even lower in MI patients [67.17 ± 24.35 pg/ml, p < 0.05 vs controls] and oxidative stress marker were higher in MI patients compared to the control and angina groups [12.94 ± 4.55 vs. 8.26 ± 1.66 vs. 9.06 ± 2.47 umol/ml, p < 0.05]. Lower circulating humanin levels was an independent risk factor of MI patients. Circulating humanin levels could be used to predict MACE in angina group.ConclusionsLower circulating humanin levels was an independent risk factor for CAD, and a potential prognostic marker for mild CAD.General significanceHumanin may become a new index for the diagnosis and treatment of CAD.     

妊娠期糖尿病孕妇血清CST、humanin、VAP-1与糖脂代谢及胰岛素抵抗的关系研究

目的:探讨妊娠期糖尿病(GDM)孕妇血清皮质醇激素抑制素(CST)、humanin、血管黏附蛋白-1(VAP-1)与糖脂代谢及胰岛素抵抗的关系。方法:选择2017年1月至2019年10月我院妇产科门诊收治的79例GDM患者(GDM组),另选取同期到我院行产检的52例正常妊娠孕妇(NGT组)。检测并比较血清CST、humanin、VAP-1水平,分析CST、humanin、VAP-1与GDM患者糖脂代谢、胰岛素抵抗的相关性,并作二元Logistic回归分析探讨GDM发病的危险因素。结果:GDM组血清CST、humanin、空腹C肽(FC-P)、C-P峰值/FC-P、胰岛素β细胞功能指数[HOMA-β(C-P)]水平低于NGT组(P<0.05),VAP-1、空腹血糖(FPG)、胰岛素抵抗指数[HOMA-IR(C-P)]、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)水平高于NGT组(P<0.05)。Pearson相关性分析结果显示血清CST水平与FPG、HOMA-IR(C-P)呈负相关(P<0.05),与FC-P、C-P峰值/FC-P、HOMA-β(C-P)呈正相关(P<0.05)。humanin水平与TG、FPG、HOMA-IR(C-P)呈负相关,与FC-P、C-P峰值/FC-P、HOMA-β(C-P)呈正相关(P<0.05)。VAP-1与TG、FPG、HOMA-IR(C-P)呈正相关(P<0.05),与FC-P、C-P峰值/FC-P、HOMA-β(C-P)呈负相关(P<0.05)。二元Logistic回归分析结果显示CST、humanin、HOMA-β(C-P)水平降低,年龄、BMI、LDL-C、VAP-1、HOMA-IR(C-P)水平升高是GDM发病的危险因素(P<0.05)。结论:GDM患者血清CST、humanin水平降低,VAP-1水平升高,三者均参与GDM发病和胰岛素抵抗。CST与糖代谢紊乱有关,humanin、VAP-1与糖脂代谢异常有关。     

N-Formylated humanin activates both formyl peptide receptor-like 1 and 2

We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2). This discovery was based on our finding that HN suppressed forskolin-induced cAMP production in Chinese hamster ovary (CHO) cells expressing human FPRL1 (CHO-hFPRL1) or human FPRL2 (CHO-hFPRL2). In addition, we found that N-formylated HN (fHN) performed more potently as a ligand for FPRL1 than HN: in CHO-hFPRL1 cells, the effective concentration for the half-maximal response (EC(50)) value of HN was 3.5nM, while that of fHN was 0.012nM. We demonstrated by binding experiments using [(125)I]-W peptide that HN and fHN directly interacted with hFPRL1 on the membrane. In addition, we found that HN and fHN showed strong chemotactic activity for CHO-hFPRL1 and CHO-hFPRL2 cells. HN is known to have a protective effect against neuronal cell death. Our findings contribute to the understanding of the mechanism behind HN's function.     

Humanin-induced autophagy plays important roles in skeletal muscle function and lifespan extension

BackgroundAutophagy, a highly conserved homeostatic mechanism, is essential for cell survival. The decline of autophagy function has been implicated in various diseases as well as aging. Although mitochondria play a key role in the autophagy process, whether mitochondrial-derived peptides are involved in this process has not been explored.MethodsWe developed a high through put screening method to identify potential autophagy inducers among mitochondrial-derived peptides. We used three different cell lines, mice, c.elegans, and a human cohort to validate the observation.ResultsHumanin, a mitochondrial-derived peptide, increases autophagy and maintains autophagy flux in several cell types. Humanin administration increases the expression of autophagy-related genes and lowers accumulation of harmful misfolded proteins in mice skeletal muscle, suggesting that humanin-induced autophagy potentially contributes to the improved skeletal function. Moreover, autophagy is a critical role in humanin-induced lifespan extension in C. elegans.ConclusionsHumanin is an autophagy inducer.General significanceThis paper presents a significant, novel discovery regarding the role of the mitochondrial derived peptide humanin in autophagy regulation and as a possible therapeutic target for autophagy in various age-related diseases.     

Structural and dynamical studies of Humanin in water and TFE/water mixture: a molecular dynamics simulation

The structural and dynamical properties of Humanin, a small peptide with neuroprotective activity against the insults of the Alzheimer's disease-related genes and the neurotoxic amyloid peptide, are studied in two different environments by molecular dynamics simulation. In this study, we have performed comparative molecular dynamics simulations in the absence and in the presence of TFE. The resulting trajectories were analyzed in terms of structural and dynamical properties of peptide and compared to the available NMR data. In water humanin is observed to partly unfold. The peptide is readily stabilized in an ordered helical conformation in the TFE/water mixture. Our simulations show that the peptide is flexible with definite turn point in its structure in water environment. It is free to interact with receptors that mediate its action in polar environment. Humanin may also find an alpha helix structure necessary for passage through biomembranes and/or specific interactions.     

The role of humanin in natural stress tolerance: An underexplored therapeutic avenue

BackgroundThe discovery of humanin (HN/MTRNR2) 20 years ago blazed a trail to identifying mitochondrial derived peptides with biological function.ScopeHumanin is associated with pro-survival, cytoprotective, anti-inflammatory, and anti-oxidative properties and may play a role in reducing neurodegenerative and metabolic disease progression. Although the role of humanin in vitro and in vivo laboratory models is well characterized, the regulation of humanin in natural models that encounter lethal cytotoxic and oxidative insults, as part of their natural history, require immediate research. In this review, we discuss the conservation of humanin-homologues across champion hibernators, anoxia and freeze-tolerant vertebrates and postulate on the putative roles of humanin in non-model species.SignificanceWe hope characterization of humanin in animals that are naturally immune to cellular insults, that are otherwise lethal for non-tolerant species, will elucidate key biomarkers and cytoprotective pathways with therapeutic potential and help differentiate pro-survival mechanisms from cellular consequences of stress.     

The emerging role of mitochondrial derived peptide humanin in the testis

The discovery of mitochondrial derive peptides (MDPs) has spotlighted mitochondria as central hubs in control and regulation of cell viability and metabolism in the testis in response to intracellular and extracellular stresses. MDPs (Humanin, MOTS-c and SHLP-2) are present in testes. Humanin, the first MDP, is predominantly expressed in Leydig cells, and moderately in germ cells and seminal plasma. The administration of synthetic humanin peptide agonist HNG protects male germ cells against apoptosis induced by intratesticular hormonal deprivation, testicular hyperthermia, and chemotherapeutic agents in rodent testes. Humanin interacting with IGFBP-3 and/or Bax (pro-apoptotic proteins) prevents the activation of germ cell apoptosis. Humanin participates in the network of IL-12/IL-27 family of cytokines to exert the immune-modulation of the testicular environment. Humanin and other MDPs may be important in the amelioration of testicular stress and prevention of cell injury with possible implications for male infertility, fertility preservation and contraceptive development.     

Solution structure of Ser14Gly-humanin, a potent rescue factor against neuronal cell death in Alzheimer's disease

The NMR solution study of Ser14Gly-humanin (S14G-HN), a 1000-fold more potent derivative of humanin (HN), is reported. HN is 24-residue peptide that selectively suppresses neuronal cell death caused by Alzheimer's disease (AD)-specific insults and offers hope for the development of a cure against AD. In aqueous solution the NMR data show that S14G-HN is a flexible peptide with turn-like structures in its conformational ensemble distributed over an extensive part of its sequence from Pro3 to Glu15. In the more lipophilic environment of 30% TFE, an alpha-helical structure spanning residues Phe6 to Thr13 is identified. Comparison of these findings to the NMR structure of the parent HN and to existing structure-function relationship literature data outlines the important for activity structural features for this class of neuroprotective peptides, and brings forth flexibility as an important characteristic that may facilitate interactions with functional counterparts of the neuroprotection pathway.     

Biological implications of SNPs in signal peptide domains of human proteins

Proteins destined for secretion or membrane compartments possess signal peptides for insertion into the membrane. The signal peptide is therefore critical for localization and function of cell surface receptors and ligands that mediate cell-cell communication. About 4% of all human proteins listed in UniProt database have signal peptide domains in their N terminals. A comprehensive literature survey was performed to retrieve functional and disease associated genetic variants in the signal peptide domains of human proteins. In 21 human proteins we have identified 26 disease associated mutations within their signal peptide domains, 14 mutations of which have been experimentally shown to impair the signal peptide function and thus influence protein transportation. We took advantage of SignalP 3.0 predictions to characterize the signal peptide prediction score differences between the mutant and the wild-type alleles of each mutation, as well as 189 previously uncharacterized single nucleotide polymorphisms (SNPs) found to be located in the signal peptide domains of 165 human proteins. Comparisons of signal peptide prediction outcomes of mutations and SNPs, have implicated SNPs potentially impacting the signal peptide function, and thus the cellular localization of the human proteins. The majority of the top candidate proteins represented membrane and secreted proteins that are associated with molecular transport, cell signaling and cell to cell interaction processes of the cell. This is the first study that systematically characterizes genetic variation occurring in the signal peptides of all human proteins. This study represents a useful strategy for prioritization of SNPs occurring within the signal peptide domains of human proteins. Functional evaluation of candidates identified herein may reveal effects on major cellular processes including immune cell function, cell recognition and adhesion, and signal transduction.     

The neuropeptide PACAP attenuates beta-amyloid (1-42)-induced toxicity in PC12 cells

Pituitary adenylate cyclase activating polypeptide (PACAP) modulates neurotransmission in the central and peripheral nervous systems. In vitro and in vivo studies have shown the protective effects of PACAP against neuronal damage induced by ischemia and agonists of NMDA-type glutamate receptors. Here, we demonstrated that PACAP also protected against neuronal toxicity induced by beta-amyloid (Abeta) peptide, aggregation of which is a causative factor for Alzheimer's disease. PACAP (10(-9)M) rescued 80% of decreased cell viability and 50% of elevated caspase-3 activity that resulted from exposure of PC12 cells to Abeta. PACAP was at least 10(4)-fold more effective than other neuropeptides including vasoactive intestinal peptide (VIP) and humanin, which correlated with the level of cAMP accumulation. Thus, our results suggested that PACAP attenuates Abeta-induced cell death in PC12 cells through an increase in cAMP and that caspase-3 deactivation by PACAP is involved in the signaling pathway for this neuroprotection.     

An IgA-binding peptide derived from a streptococcal surface protein.

Surface proteins that bind to the Fc part of human IgA are expressed by many strains of Streptococcus pyogenes, a major human pathogen. Studies of these proteins have been complicated by their size and by their ability to bind human plasma proteins other than IgA. Here, we describe a synthetic 50-residue peptide, derived from streptococcal protein Sir22, that binds human IgA but not any of the other plasma proteins known to bind to Sir22. The peptide binds serum IgA and secretory IgA and binds IgA of both subclasses. Evidence is presented that the peptide folds correctly both in solution and when it is immobilized and that it readily renatures after denaturation. Together, these data indicate that the peptide corresponds to a protein domain that binds IgA with high specificity. This is the first report of an IgA-binding domain that retains its properties in isolated form.     

Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis

Humanin (HN) is a 24 amino acids peptide with potent neuro-survival properties that protects against damage associated with Alzheimer's disease. In the present report, we have demonstrated by immunohistochemical analysis and Western blotting the pattern of expression of rat humanin (HNr) in the testis of 10- to 60-day-old rats. The Leydig cells of 10- and 40- day-old rats expressed this peptide at high levels; and in the testis of 60-day-old rats the expression of HNr expanded to include Leydig, endothelial, peritubular and germ cells. As monitored by Western blotting, HNr was released into the medium of cultures of Leydig cells isolated from 10-, 40-, and 60-days-old rats. HNr stimulated the incorporation of [(3)H]TdR into DNA of Leydig cells from 10-days-old rats, in a manner that indicated promotion of cell survival rather than an increase in the rate of cell multiplication. This peptide also enhanced steroidogenesis by cultured Leydig cells from 10- to 40-day-old rats both alone and synergistically with IGF-I. The expression of HNr in cultured Leydig cells increased in response to GH and IGF-I. In summary, we demonstrated here that HNr was expressed at all stages of maturation in the rat testis. This peptide promoted the survival of Leydig cells in culture and interacted with IGF-I to stimulate DNA synthesis and steroidogenesis. We propose that HNr is a novel testicular anti-apoptotic factor.