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Elucidating the Reproductome: System‐wide Regulation of Reproductive Neuropeptides
Author(s) -
Glucksman Marc,
Philibert Keith,
Woitowich Nicole,
Urban Janice,
DeJoseph Gina
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.936.15
Subject(s) - kisspeptin , neurokinin b , hypothalamus , medicine , endocrinology , median eminence , neuropeptide , dynorphin , biology , gonadotropin releasing hormone , receptor , chemistry , microbiology and biotechnology , hormone , luteinizing hormone , substance p , opioid peptide , opioid
Kisspeptin (Kiss), neurokinin B (NKB) and dynorphin A (DynA), collectively known as the KNDy neurons in the hypothalamus, drive the activity of the reproductive axis through the regulation of gonadotropin releasing hormone (GnRH) release. These neurons project to the rostral hypothalamus and median eminence to relay peripheral signals such as estrogen levels and energy homeostasis to the GnRH neuron. As neuropeptides, the actions of Kiss and DynA are required to be modified enzymatically in the extracellular space to modulate downstream signaling, though the regulator of this process is unknown. Furthermore, Phoenixin, a recently identified neuropeptide produced in the hypothalamus, increases GnRH, GnRH receptor and Kisspeptin mRNAs, increases GnRH secretion and mediates ovarian cyclicity. EP24.15 (EC 3.4.24.15, thimet oligopeptidase) is a metallo‐endopeptidase, expressed throughout the hypothalamus and other brain regions, that cleaves GnRH and inactivates binding to its cognate receptor. To authenticate a biochemical and physiological regulatory mechanism for the functioning of Kiss and DynA, we utilized a high throughput workflow schema by first identifying potential active site binding and cleavage sites both in silico and by mass spectrometry; measured enzymatic parameters; performed structural studies. Finally, we determined if EP24.15 and both Kiss and DynA are co‐expressed in the brain, leading to the elucidation of a functional neuroendocrine circuit whereby EP24.15 could regulate Kiss‐ or DynA ‐induced reproductive hormone secretion. EP24.15 and Kiss were co‐incubated and subjected to MALDI‐TOF mass spectrometry to confirm that EP24.15 cleaves full length Kiss as well as the fully functional Kiss10 decapeptide between S5–F6 in vitro (Vmax = 2.37±0.09 pmol/min/ng, Km = 19.68±2.53 μM). Dynorphin A (1–8) is cleaved between F4–L5 and L5–R6 (Vmax = 30.9±0.48 pmol/min/ng, Km = 14.62±0.89 μM). Phoenixin is cleaved between K8–V9 in vitro (Vmax = 2.90±0.20 pmol/min/ng, Km = 25.81±5.40 μM). These novel substrates are cleaved at rates comparable to other known neuropeptides. We have also demonstrated that both Kiss and DynA immunoreactivities are co‐expressed and localized with EP24.15 in the hypothalamus. These data demonstrate that EP24.15 cleaves Kiss, DynA, and Phoenixin in vivo to unveil an additional layer on the exquisite control mechanism of the mammalian reproductome. Further understanding the role of EP24.15 in neuropeptide metabolism will provide insight into the circuitry within the hypothalamus and provide targets for modulating various neuroendocrine axes, including reproductive function. Support or Funding Information Supported by NIH OD010662 (MJG).