Processing of prohormones

The proteases involved in the processing of prohormones and many other cellular precursor molecules are called converting enzymes or convertases, e.g., KEX[1,2] proteins of yeast (34), human furin, and mPC[l,2] proteins (or mouse prohormone convertases) (35). These proprotein convertases commonly recognize and cut within a basic amino acid sequence motif -Lys/Arg-Arg. 

FIGURE 18-7 Processing of the proopiomelanocortin (POMC) precursor proceeds in an ordered, stepwise fashion. Cleavage of the POMC precursor occurs at seven sites, with some of the reactions being tissue-specific. The circled numbers indicate the temporal order of cleavage in tissues where these proteolytic events occur. ACTH, adrenocorticotropic hormone CLIP, corticotropin-like intermediate lobe peptide JP, joining peptide LPH, lipotropin MSH, melanocyte-stimulating hormone PC, prohormone convertase. 

Many secreted proteins, as well as smaller peptide hormones, are acted upon in the endoplasmic reticulum by tryptases and other serine proteases. They often cut between pairs of basic residues such as KK, KR, or RR.214-216 A substilisin-like protease cleaves adjacent to methionine.217 Other classes of proteases (e.g., zinc-dependent carboxypeptidases) also participate in this processing. Serine carboxypeptidases are involved in processing human prohormones.218 Among the serine carboxypeptidases of known structure is one from wheat219 and carboxypeptidase Y, a vacuolar enzyme from yeast.220 Like the pancreatic metallocarboxypeptidases discussed in Section 4, these enzymes remove one amino acid at a time, a property that has made carboxypeptidases valuable reagents for determination of amino acid sequences. Carboxypeptidases may also be used for modification of proteins by removal of one or a few amino acids from the ends. 

Plants do not only possess the tools for the perception of peptide signals, they also have enzymes potentially involved in the processing of peptide prohormones. The existence of such enzymes provides additional indirect evidence for a general role of peptide hormones in plant signal transduction processes. 

Cockayne, S.E., Rassl, D.M., and Thomas, S.E., 2000, Squamous cell carcinoma arising in Hailey-Hailey disease of the vulva. Br. J. Dermatol. 142, 540-542 Creemers, J.W., Jackson, R.S., and Hutton, J.C., 1998, Molecular and cellular regulation of prohormone processing. Semin. Cell. Dev. Biol. 9, 3—10... 

Based on a current model, the processing of proinsulin is initiated by prohormone convertase 1 /3 (PCI /3) cleavage at the B-chain to C-peptide junction followed by PC2 cleavage at the C-peptide to A-chain junction. PCI/3 and PC2 cleave prohormones C-termi-nally to basic amino acid residues, that are further trimmed off by Cpe to eventually release mature insulin into the circulation. 

Brakch, N., Rist, B., Beck-Sickinger, A. G., et al. (1997) Role of prohormone convertases in pro-neuropeptide Y processing coexpression and in vitro kinetic investigations. Biochemistry 36, 16,309-16,320. 

Figure 1 Peptide neurotransmitters in the brain. Neuropeptides in the brain function as peptide neurotransmitters to mediate chemical communications among neurons. Neuropeptides are synthesized within secretory vesicles that are transported from the neuronal cell body via the axon to nerve terminals. The proneuropeptide (or prohormone) is packaged with the newly formed secretory vesicle in the cell body, and proteolytic processing of the precursor protein occurs during axonal transport and maturation of the secretory vesicle. Mature processed neuropeptides are contained within secretory vesicles at the synapse where activity-dependent, regulated secretion of neuropeptides occurs to mediate neurotransmission via neuropeptide activation of peptidergic receptors.

Proteases are essential for the conversion of inactive proprotein precursors into the active neuropeptides. Two main protease pathways have been elucidated for processing proneuropeptides and hormones the recently discovered cysteine protease cathepsin L with aminopeptidase B and the well-established subtilisin-like serine proteases that consist of prohormone con-vertases 1 and 2 followed by carboxypeptidase E/H. Endogenous regulators modulate these two protease pathways as endogenous peptide inhibitors, activators, and in vivo secretory vesicle proteins. Neuropeptides in CSE (cerebrospinal fluid) in neurological diseases can monitor brain nervous activity because neuropeptides represent active neurotransmission (93, 94). 

Schiller MR, Mende-Mueller L, Moran K, Meng M, Miller KW, Hook VY. Prohormone thiol protease (PTP) processing of recombinant proenkephalin. Biochemistry 1995 34 7988-7995. 

Azaryan AV, Hook VYH. (1994) Unique cleavage specificity of prohormone thiol protease related to proenkephalin processing. FEBS Lett. 1994 341 197-202. 

Rehfeld JP, Lindberg I, Priis-Hansen L. Increased synthesis but decreased processing of neuronal proCCK in prohormone convertase... 

Allen RG, Peng B, Pellegrino MJ, Miller ED, Grandy DK, Lundblad JR, Washburn CL, Pintar JE. Altered processing of pro-orphanin PQ/nociceptin and pro-opiomelanocortin-derived peptides in the brains of mice expressing defective prohormone 62. convertase 2. J. Neurosci. 2001 21 5864-5870. 

Severe block in processing of proinsulin to insulin accompanied by elevation of des-64,65 proinsulin intermediates in islets of mice lacking prohormone convertase 1/3. Proc. Natl Acad. Sci. U.S.A. 66. 2002 99 10299-10304. 

Figure 25-12 Processing of proinsuiin.The enzymes prohormone convertase ) and 2 (PCI and PC2) act on proinsulin to form the appropriate split proinsulins. Carboxypeptidase-H (CPH) removes the two exposed basic amino acid residues (circles).

Like all neuropeptides, NT is synthesized as part of a larger precursor that also contains neuromedin N (NN), a 6 amino acid neurotensin-like peptide (Table 1). Pro-NT/NN is processed in the regulated secretory pathway of neuroendocrine cells by prohormone convertases PCI, PC2 and PC5-A that belong to a larger family of proprotein convertases. Due to differential cleavage specificity and tissue distribution of the convertases, pro-NT/NN processing gives rise to approximately a 1 1 and a 5 1 ratio of NT over NN content in the brain and gut, respectively. The peptides are stored in secretory vesicles and released from neuroendocrine cells in a Ca2+-dependent manner. NT and NN actions are terminated by desensitization of the... 

P cells of the pancreatic islets in combination with atoms of zinc, but when required to regulate blood glucose concentration, the prohormone is cleaved and functional insulin is released into the circulation along with the C-peptide. This example of post-translational processing is mediated by peptidases which are contained in the vesicles along with the proinsulin. The fusion of the secretory vesicles with the cell membrane and activation of the peptidase prior to exocytosis of the insulin are prompted by an influx of calcium ions into the P-cell in response to the appropriate stimulus. Similarly, catecholamines are synthesized and held within the cell by attachment to proteins called chromogranins. 

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