This project addresses the fundamental question of ?What `human' protease mechanisms are responsible for producing active peptide neurotransmitters, neuropeptides, that are a fundamental requirement for all brain and nervous system functions?? The unique hypothesis to address this question is that human-specific cathepsin V, combined with proteases identified in non-human animal models for neuropeptide production, participates in producing active neuropeptides. Fundamental knowledge of the human protease mechanisms for producing neuropeptides are relevant to neuropeptide regulation in neurological and mental health throughout aging, and which are impacted by environmental conditions including drugs of abuse. Studies in mice have demonstrated the important role of mouse cathepsin L in secretory vesicles for producing opioid and other neuropeptides. Interestingly, human-specific cathepsin V is the closest human homologue to mouse cathepsin L, suggesting a role for cathepsin V in neuropeptide production. The human-specific cathepsin V gene is not present in mouse or other species. We investigated human cathepsin V and found that it participates as a significant protease for enkephalin neuropeptide production. These findings lead to the goal of this `human focused' project to define the human protease mechanisms for neuropeptide production by cathepsin V, combined with cathepsin L and the PC1/3 & PC2 convertases that function in non-human species for neuropeptide production. Human induced pluripotent stem cell (hiPSC) neurons produce numerous neuropeptides and will be used as an innovative model for human protease mechanisms in neuropeptide biosynthesis. Parallel studies of neuropeptides and processing proteases in human brain regions rich in neuropeptides will support findings gained from the hiPSC model. Innovative neuropeptidomics mass spectrometry will identify neuropeptide profiles in an unbiased and high throughput manner.
The first aim will define the cellular role in hiPSC neurons of cathepsin V in the production of opioid neuropeptides with comparison to cathepsin L, PC1/3, and PC2 proteases by gene silencing and expression, combined with mass spectrometry peptide identifications.
The second aim will conduct in vitro biochemical studies of cathepsin V processing of opioid pro-neuropeptides compared to cathepsin L, PC1/3, and PC2, to define cleavage sites, peptide products, and kinetic efficiencies, with parallel analyses in human brain regions.
The third aim will investigate diverse neuropeptides by neuropeptidomics of hiPSC neurons and human brain tissues to evaluate the role of these human proteases in producing diverse neuropeptides. Results will define the basic human protease mechanisms for peptide neurotransmitter production that is fundamental for brain and nervous system functions in health and disease.
Neuropeptides are a fundamental requirement for chemical neurotransmission that regulates brain and nervous system functions in neurological and mental health through aging, and in responses to environmental factors including drugs. This project will asssess the hypothesis that human-specific protease pathways convert inactive protein precursors into active neuropeptides. Findings from this project will advance knowledge of the distinct `human protease mechanisms' utilized for producing peptide neurotransmitters in health and disease conditions.
| Hook, Vivian; Lietz, Christopher B; Podvin, Sonia et al. (2018) Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry. J Am Soc Mass Spectrom 29:807-816 |
| Lietz, Christopher B; Toneff, Thomas; Mosier, Charles et al. (2018) Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles. J Am Soc Mass Spectrom 29:935-947 |
| Hook, Vivian; Bandeira, Nuno (2015) Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems. J Am Soc Mass Spectrom 26:1970-80 |
| Podvin, Sonia; Bundey, Richard; Toneff, Thomas et al. (2015) Profiles of secreted neuropeptides and catecholamines illustrate similarities and differences in response to stimulation by distinct secretagogues. Mol Cell Neurosci 68:177-85 |
