The Enzymology and Intracellular Organization of Peptide Precursor Processing: The Secretory Vesicle Hypothesis

Abstract

The ‘secretory vesicle hypothesis of precursor processing’ states that the initial endopeptidase cleavages which excise the nascent, biologically active peptides from their protein precursors occur primarily in secretory vesicles (or granules). Hence, all the processing steps subsequent to these cleavages must also occur within these organelles. Two types of evidence are presented in support of this view: (1) cell biological studies which implicate the secretory vesicle as the site of precursor conversion to peptides, and (2) enzymological studies which locate and characterize putative processing enzymes in secretory vesicles. The processing enzymes reviewed include the ‘prohormone-converting enzymes’ which cleave at pairs of basic amino acids, other endopeptidases, carboxypeptidase-B-like enzymes and aminopeptidase, and N-acetylation and α-amidation enzymes. The properties of these enzymes in relation to the nature of the processing micro-environment in the secretory vesicles is discussed.

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

“…In this study affinity labeling with the activity-based probe DCG-04, combined with peptide sequencing by MS, provided molecular identification of secretory vesicle cathepsin L as the cysteine protease responsible for PE-processing activity. The identified cathepsin L fulfills the criteria expected of a proneuropeptide or prohormone-processing protease (1,2,25). Cathepsin L, similar to native PTP activity, produced active ME by cleaving enkephalin-containing peptide substrates at paired basic and monobasic processing sites.…”

“…The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of T he biosynthesis of enkephalin opioid peptides as well as numerous peptide neurotransmitters and hormones requires proteolytic processing of respective proprotein precursors within regulated secretory vesicles (1)(2)(3)(4). The mature, processed enkephalin peptide is stored within these vesicles and undergoes stimulated secretion to mediate neurotransmission and cell-cell communication in the regulation of analgesia, behavior, and immune-cell functions.…”

A 29 kDa Intracellular Chloride Channel p64H1 Is Associated with Large Dense-Core Vesicles in Rat Hippocampal Neurons

“…After leaving the Golgi apparatus, the electron dense-core continues to mature by progressive aggregation of the vesicle contents, a process that also is promoted by low pH (Tooze and Stinchcombe, 1992). More importantly, to produce f unctionally active peptide, the neuropeptide precursor has to be cleaved by endo/exoproteases that generally have acidic pH optima (for review, see Gainer et al, 1985). The f unction of LDC Vs is consistent with the notion that the internal pH of LDC Vs is acidic, pH 5-6 (L oh et al, 1984).…”

“…Several enzymes (e.g., acetyltransferase and ␣-amidating enzymes), which f urther modif y neuropeptides after proteolytic cleavage, show maximal activity at neutral pH and are inhibited at acidic pH (for review, see Gainer et al, 1985). Aggregated proteins also are thought to be solubilized at more neutral pH before release (Tooze and Stinchcombe, 1992;Dartsch et al, 1998).…”

Evidence for a PGF2α auto-amplification system in the endometrium in mares

“…In mares, OT is secreted from hypothalamic nerve cells via the posterior pituitary (Gainer et al 1985) and the endometrium (Stout et al 2000, Watson et al 2000. The temporal relationship between PGF and OT secretion is an important aspect of the luteolytic mechanism in ruminants (McCracken et al 1999) and perhaps in mares (Shand et al 2000, Utt et al 2007).…”