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The Regulation of Mammalian Reproduction by the Neuropeptide Processing Enzyme EP24.15
Başlık:
The Regulation of Mammalian Reproduction by the Neuropeptide Processing Enzyme EP24.15
Yazar:
Woitowich, Nicole C., author.
ISBN:
9780438014459
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (270 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Marc J. Glucksman; Janice H. Urban Committee members: Kenneth E. Neet; Monica Oblinger; James L. Roberts.
Özet:
Reproductive function is maintained by the coordinated efforts of the hypothalamic-pituitary-gonadal (HPG) axis along with peripheral metabolic and environmental cues. The neuropeptide, gonadotropin releasing hormone (GnRH), is the central elicitor of the HPG axis. Pulsatile GnRH secretion from the hypothalamus stimulates the production and subsequent secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary. In turn, LH and FSH stimulate the gonads to facilitate ovulation, sperm production, and production of the sex steroids. Recently, the 52-54 amino acid neuropeptide kisspeptin (Kiss1, metastin) has emerged as an upstream regulator of GnRH secretion. Mutations in the Kiss1 gene or receptor can lead to delayed or accelerated puberty, hypogonadotropic hypogonadism, and infertility.
Neuropeptides, unlike other small molecule neurotransmitters, do not utilize a reuptake mechanism. Instead, they rely on neuropeptide processing enzymes to cleave and ultimately terminate their signal. The enzyme EP24.15 (thimet oligopeptidase, TOP, or EC 3.4.24.15) is a neuropeptide processing enzyme responsible for the cleavage of GnRH. Over the last decade, numerous studies have examined the actions of Kiss1 within the HPG axis, however, little is known about how Kiss1 is metabolized. The goal of this dissertation is to explore the regulation of Kiss1 and other neuropeptides implicated in the regulation of reproduction by EP24.15 using an interdisciplinary approach utilizing biochemical, biophysical, cellular, and physiological-based tools to examine the regulation of reproduction.
Initial studies utilizing in silico molecular modeling implied that the decapeptide form of Kiss1, termed Kiss10, a product of the larger Kiss1 peptide, was a putative substrate of EP24.15. Co-incubation of EP24.15 and Kiss1 revealed that both Kiss1, a peptide of 52 amino acids in length, and Kiss10 are substrates of EP24.15. Furthermore, EP24.15 metabolizes both the rodent and human isoforms of Kiss10, which differ in the C-terminal sequence, at rates that are physiologically relevant. Immunohistochemical studies indicated that EP24.15 and Kiss1 are co-expressed within the hypothalamus, which suggest that EP24.15 could cleave Kiss1 in vivo as well as in vitro.
A population of Kiss1 neurons in the arcuate nucleus co-expressing the neuropeptides neurokinin B (NKB) and dynorphin A (DynA), also known as KNDy neurons, have been implicated in the regulation of pulsatile gonadotropin secretion. We sought to determine if in addition to Kiss1, the other KNDy peptides, NKB and DynA, could also serve as substrates for EP24.15, implying a functional neuroendocrine circuit regulated by EP24.15 to influence the expression of KNDy mediated gonadotropin secretion. Biochemical analyses revealed that DynA(1-8) and DynA(1-17) are both substrates of EP24.15 and while EP24.15 cleaves DynA(1-8) at rates that are physiologically relevant, the larger isoform, DynA(1-17), is likely a poor substrate. Interestingly, EP24.15 does not cleave NKB. However, immunohistochemical studies indicated that EP24.15 is co-expressed with both DynA and NKB in the arcuate nucleus, which might suggest that EP24.15 is expressed within KNDy neurons. In addition, physiological studies were conducted in order to determine if inhibition of EP24.15 in vivo could alter gonadotropin secretion or if co-administration of EP24.15 and Kiss1 could potentiate Kiss1-induced LH release in intact male rats. However, inhibition of EP24.15 did not increase plasma LH levels, nor did it increase the amplitude of a Kiss1-induced LH response.
The final studies examined the intramolecular interactions between EP24.15 and substrates Kiss10 and DynA(1-8) on the atomic level. Utilizing X-ray crystallography, two co-crystal structures of EP24.15 with Kiss10 and DynA(1-8) were solved at 2.1 and 1.9 A resolution, respectively. Structural analyses revealed that upon substrate binding, EP24.15 undergoes a dynamic conformational change which encompasses the substrate. Furthermore, these structures identified key enzyme residues involved in substrate binding and provide insight into mechanisms describing the broad substrate specificity of EP24.15.
In summary, this work explored the regulation of Kiss1 and Kiss1-associated peptides by EP24.15 from the level of the atom to the animal. These studies provide insight into the complex regulation of neuropeptides by neuropeptide processing enzymes within the HPG axis and implicate EP24.15 in the regulation of reproduction. Specific therapeutic agents can then be targeted to modulate neuropeptide signaling and utilized to treat reproductive disorders.
Notlar:
School code: 1489
Mevcut:*
Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(677859.1) | 677859-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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