SUN-478 The Role of Thimet Oligopeptidase (EP24.15) in Regulating Reproduction

For almost six decades, gonadotropin releasing hormone (GnRH) has been examined biochemically and physiologically in the hormonal control of reproduction. More recently, Kisspeptin (Kiss), neurokinin B (NKB) and dynorphin A (DynA) comprising KNDy neurons in the hypothalamus have been demonstrated to regulate gonadotropin releasing hormone (GnRH) release. To add to the complex circuitry of reproduction, and associated physiological processes, we sought to define the role of Phoenixin (Phx), a recently identified neuropeptide produced in the hypothalamus, that increases GnRH peptide, GnRH receptor and Kisspeptin mRNAs, increases GnRH secretion and mediates ovarian cyclicity. EP24.15 (EC 3.4.24.15, thimet oligopeptidase) is a neuropeptide processing metalloenzyme that is expressed throughout the hypothalamus and other brain regions. All neuropeptide hormones have their downstream signaling activity moderated enzymatically in the extracellular space though the specific enzymes remain unknown. EP24.15 is best known for regulating GnRH by degrading the hormone and thus, inactivating binding to the GnRH receptor. Likewise, we have proven that Kiss is likewise degraded by EP24.15 further pointing to a role in regulating reproduction. To validate both physiological and biochemical regulation of DynA and Phx, we determined if these neuropeptides and EP24.15 are co-expressed in the brain, (in addition to our previous studies of GnRH and Kiss) identifying a functional neuroendocrine circuit whereby EP24.15 could regulate reproduction. We have demonstrated Phx immunoreactivity in areas of the brain related to stress (amygdala and bed nucleus of the stria terminalis) and reproduction (hypothalamic arcuate nucleus). Biochemically, we used proteomics to identify new neuropeptide substrates, their binding to the active site and cleavage sites by high resolution 3D structural models, mass spectrometry, and enzyme kinetics. Two cleavage sites are present in Kiss and DynA whereas there is a single cleavage site in GnRH and Phx. Enzyme kinetic variables of DynA and Phx (Km and Vmax) are comparable to published values for GnRH and Kiss. The data demonstrates that EP24.15 cleaves DynA, and Phx in vivo as an additional layer to the elaborate mechanism of reproduction. Though NKB was co-expressed with EP24.1.5, it was not a substrate for the enzyme which allows for finer tuning of the reproductive axis. A better understanding of novel, regulated peptides that integrate stress and reproductive processes will provide insight into brain circuitry for moderating normal and aberrant reproductive function.