Kisspeptin and Kisspeptide Synthesis

Kisspeptin and Kisspeptides

A Kiss is needed for humans and mammals to grow up.

Kisspeptin was originally discovered as a metastasis-suppressor gene in 1996. The name KISS was given to the gene because of its role as a suppressor sequence (SS). The letters “KI” were added as a prefix because it was discovered at Hershey, Pennsylvania, which is the home of the “Hershey Chocolate Kiss.” The 54-amino acid product was called metastin and the family of neuropeptides coded by the Kiss1 gene were given the name kisspeptins.

Kisspeptin 1 (human)

Kisspeptin is a protein that is encoded by the KISS1 gene in humans. The recent literature suggests that the terms KISS1 and Kiss1 should be used for human and non-human kisspeptin genes, respectively. Furthermore, it is also recommended to use the term KISS1R for the human kisspeptin receptor gene. 

In 1996 Lee et al. identified the cDNA coding for the metastasis-suppressor KiSS-1 preproprotein gene as a human melanoma metastasis suppressor gene. This gene suppresses metastases of melanomas and breast carcinomas, without affecting tumorigenicity from malignant melanoma cells that had been suppressed for metastatic potential by the introduction of human chromosome 6. The research group used subtractive hybridization and northern blot analysis to compare mRNAs from a panel of human melanoma cells. The results revealed that KiSS-1 mRNA expression occurred only in nonmetastatic melanoma cells. No mRNA expression was detected in normal heart, brain, liver, lung, and skeletal muscle. A weak expression was found in the kidney and pancreas, but the highest expression was observed in the placenta.

The KiSS-1 cDNA encodes a predominantly hydrophilic, 164 amino acid protein with a polyproline-rich domain, indicative of an SH3 ligand that may bind to the homology 3 domain of the oncoprotein Src and a putative protein kinase C-alpha phosphorylation site.

Furthermore, the researchers showed that transfection of a full-length KiSS-1 cDNA into C8161 melanoma cells suppressed metastasis in an expression-dependent manner. It was suggested that the encoded protein may inhibit chemotaxis and invasion, and thereby attenuate metastasis in malignant melanomas. Further studies suggest a putative role in the regulation of events downstream of cell-matrix adhesion, perhaps involving cytoskeletal reorganization. A protein product of this gene, kisspeptin, stimulates gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulates the pubertal activation of GnRH neurons. A polymorphism in the terminal exon of this mRNA results in two protein isoforms. An adenosine present at the polymorphic site represents the third position in a stop codon. When the adenosine is absent, a downstream stop codon is utilized and the encoded protein extends for an additional seven amino acid residues.

GnRH, also known as luteinizing-hormone-releasing hormone (LHRH) and luliberin, is a peptide which was identified in 1977 by Nobel Laureates Roger Guillemin and Andrew V. Schally. GnRH has the sequence: pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂.

In 2001, kisspeptin was identified as a high-affinity RFamide (Arg-Phe-NH2) peptide ligand for a then orphan G protein-coupled membrane receptor, GPR54. This receptor is now called “Kiss1r” for its role as a kisspeptin receptor. The receptor was first found in the rat, followed by the identification of the human homolog of GPR54, KISS1R (also referred to as AXOR12 or hOT7T175). It was suggested that KiSS-1 may be a useful marker for distinguishing metastatic melanomas from nonmetastatic melanomas.

In the year 2003, it was discovered that mutations in KISS1R were associated with idiopathic hypothalamic hypogonadism and impaired pubertal maturation in patients. Hypergonadotropic hypogonadism, in mammals and humans is caused by a lower activity of the gonads – the testes in males and the ovaries in females - which results in the synthesis of lower amounts of sex hormones and impaired gamete production and/or regulation. The impaired response of the gonads to the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) results in a lack of sex steroid production and elevated gonadotropin levels. Low level of androgens (e.g. testosterone) are called hypoandrogenism and low levels of estrogens (e.g., estradiol) are called hypoestrogenism. Other hormones that the gonads produce may also be decreased by hormones. These hormones include progesterone, DHEA, anti-Müllerian hormone, activin, and inhibin. Their synthesis may also be impaired. This condition may impair spermatogenesis in males and ovulation in females which can result in partial or complete infertility.

Comparison of Kiss-1 isoforms

The alignment of metastasis-suppressor KiSS-1 preproteins is shown below.


The amino acid sequences for the KISS-1 protein isoforms CRA a and CRA b from humans are compared to the sequence of a synthetic construct, the malignant melanoma metastasis suppressor protein and the KISS-1 sequence for horse. 

The initial product of the Kiss 1 gene is a protein that can vary in sequence size from 129 to 153 amino acids. The first isolated form, the malignant melanoma suppressor protein from human cell lines was reported to have a length of 145 amino acids. The cleavage of this protein results in kisspeptin-54, a 54-amino acid protein. The sequence of kisspeptin-54 is surrounded by pairs of basic residues. It is thought that enzymes such as furin or prohormone convertase cleaves the protein to release shorter peptides called kisspeptin-10, kisspeptin-13, and kisspeptin-14, as depicted in the following illustrations.

All four kisspeptin peptides exhibit the same affinity and efficacy for the kisspeptide receptor, Kiss1r. This may be an indication that the C-terminal end of the peptide is responsible for the binding and activation of the receptor. It has been reported that the production of specific anti sera has been a challenge because the RFamide peptide family often share several identical C-terminal amino acid residues. This may cause antibody staining results of brain tissue to produce misleading results. 

Model of neural depolarization by kisspeptin

A model of the possible mechanism of neural depolarization by kisspeptin binding to its receptor, Kiss1r, is illustrated here. The binding of kisspeptin to Kiss1r activates the G protein, Gaq, and phospholipase C (PLC) to cleave phosphatidylinositol 4,5-biphosphate (PIP2) into IP3 and diacylglycerol (DAG). DAG activates a signal cascade by activating PKC, wheras IP3 mobilizes calcium ions. Membrane depolarization is caused by activating nonselective transient receptor potential-canonical (TRPC) cation channels and inhibition of potassium channels. (Oakley et al., 2009)

Pharmacophore mimetics

The use of peptide structure-activity relationship derived pharmacophores combined with nuclear magnetic resonance, receptor binding, and functional assays allowed for the identification of metastin (KiSS-1) mimetics (Orsini et al. 2007). The authors demonstrated that kisspeptin-13 has a relatively stable helix conformation that includes residues 7 to 13.The three key residues for functionality, Phe9, Arg12, and Phe13, lie on one face of the helix and define its pharmacophore site. Guitierrez-Pascual et al. in 2009 identified alanine at positions 6 and 10, critical for kisspeptin-10 interaction at Kiss1r by using an amino acid substitution screen.

The findings of this research could lead to new functional kisspeptin analogs. Many more approaches have been reported to design kisspeptin mimetics for the development of therapeutic drugs. The distribution and physiological role of kisspeptin-Kiss1r signaling has been studied in many species. It has been observed that the location, developmental timing and patterns of expression differ among species for this pathway. However, this pathway plays an important role in reproduction in many vertebrates. Studies of families in humans that have idiopathic hypogonadotropic hypogonadism point to mutations in KISS1R.

This suggests that the receptor is essential for normal GnRH secretion and the development of normal puberty. Transcripts for the receptor’s ligand (KISS1 mRNA) have been identified in human placenta, testis, pancreas, liver and small intestine. Within the hypothalamus, KISS1 neurons are present predominantly in the infundibular nucleus and in sparse and indiscrete foci in the medial preoptic area. The finding that KISS1 and KISS1R are expressed throughout the body suggest that kisspeptin plays a role in cell proliferation, metastasis, GnRH/gonadotropin secretion, as well as in other physiological processes in various organ systems.

Kisspeptins and the Hypothalamus

The location of the hypothalamus within a human brain is shown to the left. The graphic to the right illustrates how kisspeptin stimulates the neuroendocrine reproductive axis. Sex steroids differentially regulate the expression of Kiss1 mRNA in different nuclei within the forebrain. GnRH release is stimulated by kisspeptin that itself is released by neurons in the anteroventral periventricular nucleus(AVPV). GnRH on the other hand induces the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The gonads respond to gonadotropins by secreting sex steroids. The steroids feed back to regulate the activity of kisspeptin neurons by inhibiting Kiss1 expression in the arcuate nucleus (ARC) and inducing its expression in the AVPV. The figure to the right shows how kisspeptin neurons may act as central processors for relaying signals from periphery to GnRH neurons. Metabolic and environmental factors regulate reproductive function. This ensures that reproduction proceeds only when metabolic and environmental conditions are favorable. The expression of Kiss1 may be induced by leptin. Other factors that may have regulatory function are also depicted.

The anteroventral periventricular nucleus (AVPV), a small brain region that is abundant in nuclear hormone receptors in a sexually dimorphic manner, is strongly implicated, in rat models, as being neonatally imprinted and is subsequently controlling sex-typical physiology and behaviors. The AVPV plus the periventricular nucleus make up the rostral periventricular region of the third ventricle (RP3V) in rats and mice. This area is full of kisspeptin expressing neurons. The arcuate nucleus is an assembly of neurons in the mediobasal hypothalamus, adjacent to the third ventricle and the median eminence.

Adult humans and mice with nonfunctional KISS1R (or GPR54) both exhibit low plasma levels of gonadotropins and sex steroids, have underdeveloped gonads and are infertile. Despite being prepubertal, these individuals are otherwise normal and apparently healthy.

It is now recognized that nutritional status and availability of energy stores can have an effect on reproduction. Fasting and food restriction have an inhibitory effect on fertility caused by a decreased circulation of gonadotropins anovulation, which is a menstrual cycle during which the ovaries do not release an oocyte. Therefore, ovulation does not take place. Castellano et al., showed that expression of Kiss1 mRNA is decreased in fasting rats compared with rats fed ad libitum. The resulting suppression of pubertal onset could be corrected by the administration of kisspeptin.

All the reported experimental evidence so far indicates that Kiss1/Kiss1r signaling in the ARC mediates the negative feedback regulation of GnRH/LH secretion in the male. However, the signaling in the female appears to be more complex.

Recently, it has become clear that the peptide family of kisspeptins play a critical role in the regulation of the hypothalamic-pituary-gonadal axis, making them key players in the regulation of fertility and reproduction.

Jayasena and colleagues in 2010 used single kisspeptin-54 (KP-54) injections to show that the peptide stimulates the release of reproductive hormones in women with hypothalamic amenorrhea (HA), a commonly occurring condition characterized by absence of menstruation. The same group compared the effects of kisspeptin-10 administration on gonadotropin release in healthy men and women in 2012. They administered IV bolus injections of kisspeptin-10 to men and women. The researchers collected blood samples at regular time periods for 4 hours after injection to measure plasma luteinizing hormone (LH) levels. The results showed that that Kisspeptin-10 stimulates gonadotropin release in men as well as women during the preovulatory phase of menstrual cycle, but failed to stimulate gonadotropin release in women during the follicular phase. This sexual dimorphism of the reaction of healthy men and women to kisspeptin-10 administration has important clinical implications for the potential use of kisspeptin-10 for the treatment of disorders of reproduction.

Furthermore, Milton et al. suggested in 2012 that kisspeptin peptides are neuroprotective against Alzheimer disease (AD). The group reported that kisspeptin peptides inhibit the neurotoxicity of Aβ, IAPP, and PrP peptides via a receptor independent action involving direct binding to the amyloid peptides. This indicates that kisspeptin peptides may be useful for the treatment of Alzheimer and prion type diseases.

Key words
 

Activin; Alzheimer; prion; amino acid substitution screen; amyloid peptides; androgens (e.g. testosterone); anti-Müllerian hormone; DHEA; estrogens (e.g., estradiol); follicle-stimulating hormone (FSH); gamete; G protein-coupled membrane receptor, gonads; gonadotropin; GPR5; GPR54;high-affinity RFamide (Arg-Phe-NH2) peptide ligand; hypergonadotropic; hypoestrogenism; gonadotropins; luteinizing hormone (LH); hypoandrogenism; hypogonadism; idiopathic hypothalamic hypogonadism; impaired pubertal maturation; infertility; inhibin; KISS; Kiss1 gene; Kisspeptin; kisspeptins; Kiss1r; kisspeptin receptor; marker; metastasis-suppressor gene; metastatic melanomas; metastin; neuropeptides; testes; ovaries; ovulation; progesterone; receptor; sex hormones; spermatogenesis.

References for Kisspeptin and Metastasis