Alignment of amino acid sequences of homologous proteins from different species show high evolution sequence conservation. Based on potential proteolytic cleavage sites, three C-terminal hypothetical peptides are predicted: Phoenixin-20 amide, Phoenixin15 (Phoenixin-14-Gly), and Phoenixin-14 amide.
Phoenixin (PNX) is a recently discovered neuropeptide shown to be involved in regulating the reproductive system, anxiety-related behaviors and pain though its receptor is still unknown. PNX-14, one of the endogenous active isoforms, is reported to regulate gonadotropin releasing hormone (GnRH) receptor expression and GnRH secretion. Because GnRH system is thought to be involved in the regulation of learning and memory processes, we hypothesized that PNX-14 might be mediate learning and memory. Here, we investigated the effects of PNX-14 in memory processes, using novel object recognition (NOR) and object location recognition (OLR) tasks. Our results revealed that intracerebroventricular (i.c.v.) injection of PNX-14 (25nmol) immediately after training not only facilitated memory formation, but also prolonged memory retention in both tasks. The memory-enhancing effects of PNX-14 were also seen when it was infused into the hippocampus. Moreover, these memory-improving effects of PNX-14 could be blocked by a GnRH receptor antagonist (Cetrorelix). The memory-improving effects of PNX-14 were not related to any effects on locomotor activity. Additionally, the results suggested that i.c.v. injection of PNX-14 mitigate the memory impairment induced by the amyloid-?1-42 (A?1-42) peptide and scopolamine. The present results indicate that PNX-14 facilitates memory formation and prolongs memory retention through activation of the GnRH receptor, and mitigates the memory-impairing effects of A?1-42 and scopolamine, suggesting that PNX-14 may be effective as a drug for enhancing memory and treating Alzheimer's disease.
The hypothalamus regulates a number of autonomic functions essential for homeostasis; therefore, investigations concerning hypothalamic neuropeptides and their functions and distribution are of great importance in contemporary neuroscience. Recently, novel regulatory factors expressed in the hypothalamus have been discovered, of which nesfatin-1 and phoenixin (PNX), show intriguing similarities in their brain distributions. There are currently few studies characterizing PNX expression, so it is imperative to accurately trace its localization, with particular attention to the hypothalamic nuclei and nesfatin-1 co-expression. Using fluorescence and classical immunohistochemical stainings on adult rat brain, we visualized the potential co-expression of nesfatin-1 and PNX immunoreactive cells. We have demonstrated a distinct PNX-immunoreactivity in 21-32% of cells in the arcuate nucleus, paraventricular nucleus, ventromedial and lateral hypothalamus. Nesfatin-1 expression reached 45-68% of all neurons in the same sites, while co-expression was strikingly seen in the vast majority (70-86%) of PNX-immunoreactive neurons in the rat hypothalamic nuclei. Our results demonstrate for the first time, a wide distribution of PNX in the hypothalamus which could implicate a potential functional relationship with nesfatin-1, possibly in the regulation of the hypothalamic-pituitary-gonadal axis or other autonomic functions, which require further study.
Phoenixin is an amidated neuropeptide, which is widely distributed in brain and periphery regions and is known for its key role in reproduction. Phoenixin-14 (PNX-14), one of the endogenous active isoforms, was reported to regulate pituitary gonadotrophin secretion by increasing the expression of the GnRH receptor mRNA. Studies showed that GnRH could regulate brain responses to anxiety. However, the role of PNX-14 in anxiety was largely unclear. Here, we investigated that the effects of PNX-14 in anxiety-related behavior in adult mice via the open field and elevated plus maze. PNX-14 was administered intracerebroventricularly (i.c.v.) in different doses (5, 10, 25 and 50nmol), and dose-dependently induced anxiolytic effects. Then this anxiolytic action was presented after PNX-14 injected into the anterior hypothalamic area (AHA), while PNX-14 infused into the amygdala did not exert anxiolytic effects. GnRH receptor antagonist (Cetrorelix) could significantly antagonize the anxiolytic effects of PNX-14, while Atosiban, a competitive vasopressin/oxytocin receptor antagonist could not. Moreover, PNX-14 could significantly lower the core temperature and Cetrorelix could block this effect of PNX-14. Additionally, the AHA infusion of PNX-14 (5nmol) increased the expression level of the GnRH mRNA in the hypothalamus and plasma concentrations of GnRH. Similarly, i.c.v. injection of PNX-20 also reduced the core temperature and exerted anxiolytic effects. Taken together, centrally injected PNX-14 generates anxiolytic effects in mice, via the activation of the AHA GnRH system.
Phoenixin-14 amide, herein referred to as phoenixin, is a newly identified peptide from the rat brain. Using a previously characterized rabbit polyclonal antiserum against phoenixin, enzyme-immunoassay detected a high level (>4.5 ng/g tissue) of phoenixin-immunoreactivity (irPNX) in the rat spinal cords. Immunohistochemical studies revealed irPNX in networks of cell processes in the superficial dorsal horn, spinal trigeminal tract and nucleus of the solitary tract; and in a population of dorsal root, trigeminal and nodose ganglion cells. The pattern of distribution of irPNX in the superficial layers of the dorsal horn was similar to that of substance P immunoreactivity (irSP). Double-labeling the dorsal root ganglion sections showed that irPNX and irSP express in different populations of ganglion cells. In awake mice, intrathecal injection of phoenixin (1 or 5 ?g) did not significantly affect the tail-flick latency as compared to that in animals injected with artificial cerebrospinal fluid (aCSF). Intrathecal administration of phoenixin (0.5, 1.25 or 2.5 ?g) significantly reduced the number of writhes elicited by intraperitoneal injection of acetic acid (0.6%, 0.3 ml/30 g) as compared to that in mice injected with aCSF. While not affecting the tail-flick latency, phoenixin antiserum (1:100) injected intrathecally 10 min prior to the intraperitoneal injection of acetic acid significantly increased the number of writhes as compared to mice pre-treated with normal rabbit serum. Intrathecal injection of non-amidated phoenixin (2.5 ?g) did not significantly alter the number of writhes evoked by acetic acid. Our result shows that phoenixin is expressed in sensory neurons of the dorsal root, nodose and trigeminal ganglia, the amidated peptide is bioactive, and exogenously administered phoenixin may preferentially suppress visceral as opposed to thermal pain.
Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones luteinising hormone and follicle-stimulating hormone is regulated primarily by hypothalamically-derived gonadotrophin-releasing hormone (GnRH), although other releasing factors (RF) have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotrophin secretion by modulating the expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using small interfering RNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretary vesicles to pituitary membranes.
SUMMARY: Bioinformatic analysis of evolutionarily conserved sequences in the hypothetical proteins human LOC389203 and rat LOC501923 predict the possibility of peptides which we name Phoenixin-20 amide, Phoenixin-15 (Phoenixin-14-Gly), and Phoenixin-14 amide. Based on the conserved sequence, these peptides were synthesized and rabbit polyclonal antisera against them were generated. After purification of heart and hypothalamus homogenate extracts from both bovine and rat tissues, a HPLC fraction showed an above ng/ml concentration of immunoreactive Phoenixin (ir-Phoenixin) in the immunoassays and indicated the Phoenixins were present in tissues. From HPLC purification and analysis, the natural peptides corresponding to the predicted Phoenixin sequence were revealed. The sequences were also confirmed by mass-spectrometry analysis and corresponding profiles of synthetic peptides. In summary, high levels of Phoenixins for the hypothalamus and heart homogenates can be quantified by immunoassay. Localization of ir-Phoenixin cells also has been identified with Immunohistochemical (IHC) staining on various tissues, including brain. In vivo and In vitro functional assays of Phoenixin have been under investigation. The Phoenixin peptide hormones have been discovered in brain/heart tissues and found to have diverse cellular function and physiological activities.
INTRODUCTION: There is considerable interest in the discovery of new endogenous ligands such as peptides that modulate the homeostasis of multicellular organisms. Based on bioinformatic analysis of signal peptide and proteolytic processing sites, we predicted the existence of several peptides in the uncharacterized protein LOC389203 and LOC501923. As a first step towards realizing the function of these peptides, we first isolated the peptides and generated antibodies against the putative peptides. The identified peptides, named Phoenixin-20 amide, Phoenixin-15 (Phoenixin-14-Gly), and Phoenixin-14 amide are found in the heart, and certain regions in the brain.
• The peptides, Phoenixin-20 amide, Phoenixin-15, and Phoenixin-14 amide have been identified in brain and heart tissues.
• The amounts of Phoenixin-20 amide and Phoenixin-14 amide are about 150 pg/mg protein both in the rat heart and bovine hypothalamus.
• Phoenixin-14 amide appears to be able to compete with Phoenixin-20 amide for binding to pituitary cells.
• The stimulation of cAMP production for pituitary cells implies those peptides might have an important extracellular function and physiological activities.
Rong-Ming Lyu, Xiang-Qun Chen, Qing Tian, Oliver Jahraus, Nae J. Dun and Jaw-Kang Chang: Isolation, identification, and distribution of Phoenixin, Presented at the 22nd American Peptide Symposium, 2011. (Manuscript has been submitted for publication in "proceedings of the 22nd American Peptide Symposium".)
The level of immunoreactive Phoenixin in porcine (P) or bovine (B) tissues were measured by using a specific RIA kit that recognizes both Phoenixin-20 amide and Phoenixin-14 amide.
HPLC profile and immunoreactivity of isolated peptides- In the heart homogenate extracts, the major peaks of phoenixin immunoreactive fractions were from the elutes at 26 and 27 min of the 1st HPLC column. The major immunoreactive fraction at 27 min of 1st HPLC contains peptide ions at MW 1583, 1641 and 2187 which were corresponding to the theoretical molecular weight of the peptides, Phoenixin-14 amide, Phoenixin-15 and Phoenixin-20 amide. In addition, the synthetic peptides, Phoenixin-14 amide, Phoenixin 15 and Phoenixin-20 amide eluted from the HPLC at 27 min and showed the same peak positions around the MW of 1583, 1641 and 2184 in mass spectrometry.
The 2nd HPLC profile, immunoreactivity and Mass spectrometry to identify the isolated peptides- The fractions with highest immunoreactivity in 2nd HPLC were from the peak between 26 min and 27 min. The fraction collected at 26 min, the isolated peptide identified by Mass spectrometer were at MW 1582.3 and 1641 which represent Phoenixin-14 amide and Phoenixin-15.
|079-03||Phoenixin-20 amide (Human, Rat, Mouse, Porcine, Bovine, Canine)||200 µg||$153|
|079-01||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine)||200 µg||$122|
|H-079-01||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - Antibody||100 µl||$357|
|B-079-01||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - Biotin Labeled||100 µg||$357|
|B-G-079-01||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - Biotin Labeled Purified IgG||50 µg||$357|
|EK-079-01||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - EIA Kit||96 wells||$458|
|FEK-079-01||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - Fluorescent EIA Kit||96 wells||$500|
|FEK-079-01CE||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - Fluorescent EIA Kit, CE Mark Certified||96 wells||$520|
|MB-079-01||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - MagBead (Magnetic Bead Linked Antibody)||1 ml||$638|
|G-079-01||Phoenixin-14 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - Purified IgG Antibody||200 µg||$459|
|079-02||Phoenixin-15 (Human, Rat, Mouse, Porcine, Bovine, Canine)||200 µg||$133|
|B-079-02||Phoenixin-15 (Human, Rat, Mouse, Porcine, Bovine, Canine) - Biotin Labeled||100 µg||$357|
|B-079-03||Phoenixin-20 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - Biotin Labeled||100 µg||$357|
|EK-079-03||Phoenixin-20 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - EIA Kit||96 wells||$458|
|RK-079-03||Phoenixin-20 amide (Human, Rat, Mouse, Porcine, Bovine, Canine) - RIA Kit||125 tubes||$588|
|079-30||Phoenixin-45 amide / C4orf52 (E5RH91) precursor amide (Human)||100 µg||$357|
|079-13||Phoenixin-5 amide / C4orf52(Q8N5G0-2) (60-64) amide (Human, Rat, Mouse, Porcine, Bovine, Canine)||200 µg||$61|
|079-26||Phoenixin-8 amide / C4orf52(Q8N5G0-2) (57-64) amide (Human, Rat, Mouse, Porcine, Bovine, Canine)||200 µg||$122|