We are interested in elucidating the proteolytic mechanisms required to convert protein precursors into active peptide neurotransmitters. These studies are aimed at defining the key proteases and protease inhibitors involved in processing protein precursors into active neuropeptides including enkephalin and ß-endorphin opioid peptides. Integrated approaches are utilized in biochemistry, molecular biology, cell biology, and neuroanatomy.
Molecular Cloning of Novel Processing Enzymes: (1) The Cysteine Protease 'Prohormone Thiol Protease' (PTP), and (2) A 70 KDA Aspartyl Protease. Structural comparison of neuropeptide precursor processing enzymes will indicate if unique proteases are involved in the production of peptide neurotransmitters and peptide hormones. Multiple molecular cloning approaches include RT-PCR, complementary oligonucleotides as probes, and expression cloning with specific antibodies. Expression of cloned processing proteases and precursors will identify PTP and 70 kDa aspartyl proteases.
Coordinate Regulation of Proenkephalin and Processing Enzyme Genes by cAMP and Other Second Messengers. Precursor and processing enzyme genes must be coordinately regulated to provide the neuron with the required precursors and proteases needed to generate active peptide neurotransmitters. Thus, these genes must possess common regulatory mechanisms to allow cells to co-express precursors and processing enzymes. Coordinate regulation by cAMP of proenkephalin and PTP in chromaffin cells is being investigated to understand the phosphorylating mechanisms, at the transcriptional or post-translational levels, involved in the regulation of peptide neurotransmitter biosynthesis.
Antisense Expression of Processing Enzymes to Block Peptide Neurotransmitter Biosynthesis. If cellular levels of the primary processing protease(s) for proenkephalin or proopiomelanocortin (POMC) are reduced by antisense expression of the enzyme genes, then precursor processing and production of active neuropeptides should be lowered. Comparison of antisense enzyme expression in different model cell lines and primary cell cultures will be performed to test the hypothesis that different proteases are required for processing different neuropeptide precursors.
Molecular Cloning and Expression of Isoforms of the Protease Inhibitor Alpha1-Antichymotrypsin (ACT) as an Endogenous Inhibitor of Neuropeptide Precursor Processing. The PTP and PC (PC = prohormone converteases) proteases involved in processing neuropeptide precursors are inhibited by isoforms of ACT that possess differential potencies against processing enzymes. Molecular cloning and expression will characterize the neuroendocrine-specific isoforms of ACT. This topic is relevant to Alzheimer's Disease (AD) in which ACT is overexpressed in senile plaques. Knowledge of potential target proteases of ACT will enhance knowledge of ACT's role in AD.
Overall, elucidation of the proteases and protease inhibitors required in the formation of peptide neurotransmitters will further our understanding of normal brain function and implicate molecular mechanisms in neurologic disease.
Hwang, S.R., Kohn, A.B., and Hook, V.Y.H. (1994). Molecular cloning reveals isoforms of bovine a1-antichymotrypsin. Proc. Natl. Acad. Sci. USA 91: 9579-9583.
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Azaryan, A.V., Krieger, T.J., and Hook,V.Y.H. (1995). Characteristics of the candidate prohormone processing proteases, PC2 and PC1/3, from bovine adrenal medulla chromaffin granules. J. Biol. Chem. 270: 8201-8208.
Tezapsidis, N., Noctor, S., Kannan, R., Krieger, T.J., Mende-Mueller, L., and Hook, V.Y.H. (1995). Stimulation of 'prohormone thiol protease' (PTP) and (Met)enkephalin by forksolin: blockade of elevated (Met)enkephalin by a cysteine protease inhibitor of PTP. J. Biol. Chem. 270: 13285-13290.
Schiller, M.R., Mende-Mueller, L., Miller, K.W., and Hook, V.Y.H. (1995). 'Prohormone thiol protease' (PTP) processing of recombinant proenkephalin. Biochemistry 34: 7988-7995.
Hook, V.Y.H., Schiller, M.R., and Azaryan, A.V. (1996). The processing proteases 'prohormone thiol protease' (PTP), PC1/3 and PC2, and 70 kDa aspartic proteinase show preferences among proenkephalin, pro-neuropeptide Y, and proopiomelanocortin substrates. Arch. Biochem. Biophys. 328: 107-114.
Hook, V.Y.H., Azaryan, A.V., Schiller, M.R., and Tezapsidis, N. (1996). Prohormone processing enzymes in chromaffin granules. Ann. N.Y. Acad. Sci. 780: 121-133.