MON-057 - Endocrinomics in and Beyond the Brain: Proteomics and Structural Approaches for Neuropeptide Discovery and Recategorizing Substrate Specificity

Neuropeptides modulate intercellular communication for proper metabolic activity, cell differentiation and growth. Alterations in expression or processing of neuropeptides produces deleterious consequences. Neuropeptide processing enzymes form a nexus in a synapse for regulating signaling through control of peptide neurotransmitters in the extracellular milieu. Our Discovery Pipeline to examine novel substrates compared two closely related neuropeptide processing enzymes: EC3.4.24.15 (EP24.15; thimet oligopeptidase) and EC3.4.24.16 (EP24.16; neurolysin). These metalloenzymes are currently and erroneously distinguished solely by an alteration in the bond hydrolyzed in one neuropeptide, neurotensin; hence, the designation Neurolysin. These enzymes are homologous to angiotensin converting enzyme 2 (ACE2), the SARS CoV2 cellular receptor, and share angiotensin as a substrate.
Through our discovery workflow, novel substrates were unveiled for processing enzymes as the repertoire of neuropeptide regulation has expanded beyond the initial half dozen reported in the literature. Utilizing multiple technological approaches: biochemical, structural, and systems-based proteomics including in silico molecular modeling, mass spectrometry, enzyme kinetics, and X-Ray crystallography, we have discovered novel substrates for EP24.16 that differ substantially from the closely related EP 24.15 both in detailed cleavage sites and in the enzyme active site as a true substrate. Several examples include the EP 24.15 cleaved reproductive and reproductive-related and cardiac peptide Phoenixin and the neuroprotective, hybrid neuropeptide substrate Colivelin. Additionally, the gonadotropin inhibitory neuropeptide RFRP-3 exhibits different cleavage sites and is a novel peptide. Interestingly, Xenin 8, a gut peptide, with a C-terminal sequence very similar to Neurotensin, is cleaved at the same site with both EP24.15 and EP24.16. Arg-Vasopressin (AVP) involved in regulating water absorption and blood pressure is a substrate for only one of the two processing enzymes.
With the discovery and functions of novel neuropeptides utilizing advanced technological methodologies, one must be cautious with utilizing older, misleading enzyme designations and limited numbers of substrates for the processing of the expanding repertoire of medically relevant neuropeptides. An emergent theme with regards to the expanding repertoires of neuropeptides in the brain (and beyond) is several processing enzymes can act on the same neuropeptide, and a given neuropeptide can be a substrate for several neuropeptide processing enzymes. Traditionally viewed to be mere degradation products, the multiplicity of physiological functions are extended to the smaller peptides produced by these processing enzymes that have roles beyond those of the parent substrate.

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