University of California, San Diego | Skaggs School of Pharmacy and Pharmaceutical Sciences
RESEARCH
Proteases for Production of Active Peptides in Neurotransmission, Hormone Action,
and Neurodegenerative Diseases.
A. Research Summary.
 Figure 1. Protease Pathways for Biosynthesis of Peptide Neurotransmitters, and Toxic Peptides in Neurodegenerative Diseases: Applications to Drug Discovery. B. Description of Hook Lab Research. (1) Protease Pathways in Peptide Neurotransmitter and Hormone Production, Known as Neuropeptides, in Health and Disease. Health requires neuropeptides for coordinated cell-cell communication in the nervous and endocrine systems for regulation of physiological processes. For example, the neuropeptide enkephalin and ß-endorphin in the brain are required for endgenous pain regulation. These endogenous peptides modulate pain in infection and health conditions involving pain relief. The neurotransmitter known as NPY (neuropeptide Y) controls feeding behavior and obesity, a health condition linked to disease. In addition, peripheral neuropeptides function as hormones in the regulation of blood pressure, heart disease, arthritis, cancer, and other health conditions. The Hook laboratory is studying proteases for production of peptide hormones that regulate such physiological functions. Drug regulation of these proteases can impact therapeutic treatment of human diseases. Proteases for the Biosynthesis of Peptide Neurotransmitters and Hormones. Neuropeptides are produced by proteolytic processing of their respective protein precursors. We are investigating proteases and their endogenous protease inhibitors that are required for converting pro-neuropeptide precursors into biologically active peptides. These processing proteases are present in regulated secretory vesicles that synthesize, store, and release active neuropeptides that regulate specific CNS and neuroendocrine functions. The goal of our research is to understand the specificity and regulation of proteases that convert protein precursors into distinct peptides that function as neurotransmitters and hormones. Our research addresses the questions of (1) do processing proteases possess selectivity for different neuropeptide precursors, (2) are selective protease inhibitors involved in regulating these proteases, and (3) are there tissue-specific mechanisms for generating peptide neurotransmitters? Our results support the hypothesis that selective proteases and protease inhibitors are involved in the biosynthesis of peptide neurotransmitters, which can be regulated in a tissue-specific manner. Theses features for selective cellular utilization of protease pathways for neuropeptide production will be explored for pharmacological drug targets. Neuropeptide Production by the Novel Cathepsin L and Aminopeptidase B Protease Pathway, with Selective Inhibitor Regulation. Recent research has discovered a novel cysteine protease pathway for the conversion of protein precursors into active peptide neurotransmitters and hormones, termed ‘neuropeptides.’ The cysteine protease cathepsin L has been identified in neuropeptide-containing secretory vesicles by active-site directed affinity labeling, mass spectrometry, cell biological, and cathepsin L gene knockout studies to establish that cathepsin L functions as a proneuropeptide processing enzyme in secretory vesicles that store and secrete active peptide neurotransmitters and hormones. Furthermore, gene expression of cathepsin L in a neuronal cell line with proenkephalin (PE) results in conversion of PE to active enkephalin opioid peptide whose secretion is stimulated by nicotinic receptor activation of neuronal cells. The combined gene knockout and gene expression of cathepsin L demonstrate its function in neuropeptide biosynthesis. Following cathepsin L, aminopeptidase B represents a second exopeptidase step to remove basic residues from NH2-termini of peptide intermediates. The aminopeptidase B has been fully characterized via molecular cloning, gene expression, biochemistry, peptide substrates with kinetics, and subcellular localization with cathepsin L in neuropeptide-containing secretory vesicles. Regulation of neuropeptide production is important for the control of physiological functions mediated by neuropeptides, including enkephalin for pain relief, NPY in blood pressure control, and other neuropeptide functions. Therefore, novel serpin protease inhibitors, termed ‘endopins’, were discovered by the Hook laboratory by homology cloning, gene expression, protease and peptide biochemistry, and cell biological studies for secretory vesicle localization. Notably, the endopin 2 isoforms represent potent and selective inhibitors of cathepsin L in secretory vesicles. Gene expression of endopin 2 alters neuropeptide production with respect to conversion of POMC into active ACTH, beta-endorphin, and alpha-MSH. (2) Proteolytic Mechanisms in Neurodegenerative Diseases. Aberrant proteolytic mechansims are responsible for the development of many age-related neurodegenerative diseases, especially Alzheimer’s and Huntington’s diseases. Unique patterns of proteolytic processing of mutant gene products in these diseases results in neurotoxic peptide fragments that participate as major pathogenic mechanisms in the disease process. Investigations of the proteolytic mechansims responsible for Alzheimer’s and Huntington’s diseases will allow future investigation of protease inhibitors as therapeutic strategies for treatment of these diseases. Alzheimer’s Disease: Protease targets for production of neurotoxic Aß peptide that accumulates in Alzheimer’s disease brain. Production of the toxic ß-amyloid peptide (Aß) is a major factor in the development of Alzheimer’s disease. The ß-amyloid is generated by proteolytic cleavage of its protein precursor, the amyloid precursor protein (APP, to generate neurotoxic Aß. Secretases represent proteases that cleave at the NH2- and COOH-termini of Aß peptide within APP, by beta- and gamma-secretases, respectively. The secretases are believed to represent ideal drug targets for protease inhibitors to reduce production of Aß. It is, thus, important to identify the appropriate secretases for development of therapeutic drugs for Alzheimer’s disease. Proteolytic processing of APP into Aß occurs in the secretory pathway of neurons, consisting of distinct regulated and constitutive secretory pathways. The regulated secretory pathway provides stimulated secretion of neurotransmitters that allow neurons to communicate with one another. Studies by the Hook laboratory discovered that the regulated secretory pathway provides the majority of secreted Aß, whereas basal, constitutive secretion provides a minor portion of extracellular Aß. It is known that different proteases reside within distinct secretory vesicles of the regulated secretory pathway compared to the constitutive secretory pathway. Therefore, our studies are investigating proteases for ß- and g-secretases in regulated secretory vesicles, obtained from model neuronal chromaffin cells obtained from the in vivo nervous system. Notably, recent results demonstrate that novel cysteine proteases contribute to the majority of ß-secretase activity in regulated secretory vesicles. These cysteine proteases are currently being investigated as possible drug targets for protease inhibitors of ß-secretase, to reduce production of the toxic Aß that accumulates in Alzheimer’s disease. These cysteine proteases, in combination with a methionine aminopeptidase, participate in two distinct proteolytic pathways for ß-secretase activity. These findings indicate that multiple proteases may be considered as targets for inhibition of ß-secretase to reduce production of Aß peptide in Alzheimer’s disease. Proteolysis of the mutant huntingtin protein in Huntington’s disease. Huntington’s disease (HD) is caused by mutation of the autosomal dominant IT15 gene product. The mutation is an expansion of CAG triplets within the coding region of the huntingtin (htt) protein gene product. The mutation causes an increase in the length of the polyglutamine domain of htt. The full-length huntingtin protein is cleaved to a toxic peptide fragment(s) that contains the polyglutamine mutation. This peptide fragment results in formation of pathological nuclear inclusions and behavioral abnormalities in HD. Our recent results have characterized and predicted ‘protease susceptible’ domains of normal and mutant huntingtin in human cortex and striatum. The current goal of this research will be to identify the protease(s) that generate the mutant peptide fragment, to gain knowledge of the proteolytic mechanisms that contribute to Huntington’s disease. (3) Genomic Features of Protease Functions: Pharmacogenetics, Protease SNPs in Disease. Pharmacogenetics. The Alzheimer’s disease (AD) project demonstrates clear pharmacogenetic differences in drug response in transgenic mice expression wild-type human APP (amyloid precursor protein), compared to mice expressing the Swedish mutant APP. Since the majority of AD patients express WT APP, the effective drugs identified are clearly relevant to the majority of the AD population. However, the identified drug inhibitors of cathepsin B have no effect in transgenic mice expressing the Swedish mutant form of human APP, a genetic form of AD present in one large family. The basis for the drug response differences in these different mouse genetic models of AD lies with the specificity of cathepsin B to cleave the WT beta-secretase site of APP, but not the Swedish mutant cleavage site of APP. These findings underscore the importance of selecting the animal model that represents the majority of the patient population for predicting drug efficacy in the majority of the AD patient population. Clearly, pharmacogenetic differences in drug responses can be defined in preclinical studies. Proteases and SNPs in Disease. Studies of cathepsin L for neuropeptide production in the sympathetic nervous system led to consideration of SNPs in the cathepsin L gene associated with genetic hypertension (collaboration with Dr. Danial O’Connor, Dept. of Medicine, UCSD). Genomic sequencing of DNAs from genetic hypertensive patients has reveals SNPs in the cathepsin L gene. These efforts are being facilitated by proteomic data of human neuropeptide secretory vesicls that allows analyses of SNPs identified in coding regions of candidate genes involved in in hypertension. Identified SNPs in protease genes related to hypertension will be analyzed by expression, for example, of cathepsin L gene SNP variants in mice, and in cellular systems, to evaluate the functional effects of variants of cathepsin L on production of neuropeptides known to regulate blood pressure, including NPY and catestatin. (4) Proteomics and Mass Spectrometry in Biomedical Research for Translation into Clinical Therapeutics. In the post-genomic era, it is now critical to understand the function of the gene products in studies of protein function and differential expression profiling of proteins and peptides. We are using several approaches in protein separation techniques, with quantitative labeling by fluorescent or isotopic reagents, for evaluation of proteomes and differential protein expression profiling. We are conducting a proteome study of secretory vesicles, to understand the protein components that provide for functional secretory vesicle function. This work is complemented with human protease gene bioinformatics and arrays to examine proteases within this organelle. In addition, neuropeptidomics to investigate profiles of active peptide neurotransmitters is being studied combined with peptide markers for applications to disease and drug therapeutic conditions. Proteomics will have impact in future clinical research to define new drug targets and therapeutics.
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Recent Publications:
Funkelstein L, Beinfeld M, Minokadeh A, Zadina J, Hook V. (2010)
Unique biological function of cathepsin L in secretory vesicles for biosynthesis of neuropeptides.
Neuropeptides 44, 457-66.
Hook V, Bark S, Gupta N, Lortie M, Lu WD, Bandeira N, Funkelstein L, Wegrzyn J, O'Connor DT, Pevzner P. (2010)
Neuropeptidomic components generated by proteomic functions in secretory vesicles for cell-cell communication.
AAPS J. 12, 635-45.
Gupta N, Bark SJ, Lu WD, Taupenot L, O'Connor DT, Pevzner P, Hook V. (2010)
Mass spectrometry-based neuropeptidomics of secretory vesicles from human adrenal medullary pheochromocytoma reveals novel peptide products of prohormone processing.
J Proteome Res. 9, 5065-75.
Highlighted Publications:
Hook, V., Funkelstein, L., Lu, D., Bark, S., Wegrzyn, J., and Hwang, S.R. (2008)
Proteases for processing proneuropeptides into peptide neurotransmitters and hormones.
Annu. Rev. Pharmacol. Tox. 48, 393-423.
Hwang, S-R., Garza, C., Mosier, C., Toneff, T., Wunderlich, E., Goldsmith, P., and Hook, V. (2007)
Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion.
J. Biol. Chem. 282, 9556-9563.