It has been established that proteins (and thus, their peptide products) are ribosomally translated using only L-amino acids, and so it was assumed that D-amino acids did not naturally exist in animal peptides. However, physiological D-amino containing-peptides (DAACP) do exist, first identified in frogs, resulting from a post-translationa modification: peptide isomerization. However, because they have no associated mass shift, DAACPs are historically very difficult to identify, causing a gap in mass spectrometry-driven peptide characterization studies. The objective of the proposed work aims to bridge this gap by developing tools to detect DAACPs by taking advantage of their unique properties, including their resistance to degradation by peptidases. In addition, formation of a DAACP alters the shape of a peptide, which can change its ability to bind to a receptor. This is especially important for neuropeptides, cell-cell signaling molecules that are bioactive by binding to their cognate receptor. Thus, in specific aim 1, the tools to identify DAACPs will be developed using neuropeptides from a simpler model animal, where one DAACP has already been identified. This system of DAACP discovery involves chiral analysis: breaking down an endogenous neuropeptide into its component amino acids and assaying those amino acids for their chirality using analytical chemistry methods. The long-term objective of this work is to identify functional DAACPs in the mammalian nervous system. As part of the National Institute on Drug Abuse (NIDA) program areas, it is interested in the function of neurotransmitters (like neuropeptides) in endogenous systems. In spite of intensive study, more than 100 human G-protein coupled receptors have no known binding partner. DAACPs may explain, in part, the discrepancy between the number of neuropeptides known through genomic and peptidomic studies and the large number of orphan receptors. For the proposed work, in specific aim 2, neuropeptides will be studied for peptide isomerization in the context of the suprachiasmatic nucleus (SCN), a hypothalamic group of neurons with a known role in circadian rhythms. The SCN is targeted because there are extensive studies on time of day release of neuropeptides and a number of peptidase-resistant peptides have already been identified in the SCN. This work is significant because NIDA has identified sleep disorders as having a link to drug abuse, so the implication of an overlooked PTM can result in many missed neuropeptide functions in our sleep/wake cycles. The discovery of DAACPs as a physiological phenomenon in mammalian neuropeptides ultimately helps reach the goal of understanding drug abuse by identifying the functional properties of neural circuits underlying drug abuse.
Neuropeptides are important signaling molecules in the nervous system that influence mood and behavior, and many drugs of abuse act on neuropeptide signaling systems. A neuropeptide's final form is critical for its action and our work seeks to characterize an unusual change in neuropeptides, known as peptide isomerization. Peptide isomerization may be involved in neuropeptides related to sleep disorders, which affect roughly 50 million Americans and are linked to higher rates of drug abuse.
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