Characterization of endoproteolytic processing of dynorphins by proprotein convertases using mouse spinal cord S9 fractions and mass spectrometry
Introduction
In mammals, the presence of tissue-damaging stimuli is sensed by primary afferent nociceptors. The sensation of pain produced by a noxious stimulus is not always consistent and depends on numerous factors influencing the neurophysiology of pain transmission (Julius and Basbaum, 2001, Gold and Gebhart, 2010). The nervous system has developed very complex mechanisms that control how noxious sensory input is perceived by the organism (Basbaum et al., 2009). It has been shown that extensive modulation of sensory information happens in the dorsal horn of the spinal cord, more specifically during the first synapse (Honore et al., 2000, Levine et al., 1993). There are various molecular events contributing to the transmission of the sensory information during the first synapse and several major neuropeptides were identified including dynorphin, enkephalin and tachykinin peptides (Kuner, 2010, Mika et al., 2011, Felippotti et al., 2012, Pailleux et al., 2013, Ferland et al., 2011).
Neuropeptides are either neurotransmitters or neuromodulators at various levels in the central nervous system and play a critical role in pain transmission (Levine et al., 1993, Seybold, 2009). Many members of the tachykinin family (e.g. Substance P) are mostly pro-nociceptive neuropeptides and have been known to play a fundamental role in central sensitization leading to hyperalgesia and allodynia (Lecci et al., 2000, Teodoro et al., 2013). Opioid peptides (i.e., endomorphins, enkephalins and dynorphins) have potent analgesic effects in the central nervous system (CNS) and play a fundamental role in endogenous pain inhibition (Machelska, 2007, Wahlert et al., 2013, Bali et al., 2014). They are interacting with μ, κ and δ-opioid receptors expressed widely in the brain and in the spinal cord (Carr and Lovering, 2000, Stanojevic et al., 2008, Mulder et al., 1989).
Dynorphin A (Dyn A), one of the major proteolytic fragments of prodynorphin (Civelli et al., 1985, Dores et al., 1985), is an endogenous ligand of the κ and μ-opioid receptors (Beaulieu, 2005, Mika et al., 2011). The agonist activity of Dyn A significantly reduces neuronal activity (Chavkin et al., 1982, Mizoguchi et al., 2006). However, the concentration of Dyn A in the spinal cord can be regulated rapidly by endoproteolysis (Cho and Basbaum, 1989). Neuropeptides are derived from larger protein precursors (i.e., proneuropeptides), and their primary structures include the sequence of the active form of at least one neuropeptide within its full-length (Funkelstein et al., 2010). It has been revealed that many neuropeptides are synthesized by the actions of proprotein convertases (PCs) and endopeptidases within dense core vesicle (Harrison and Geppetti, 2001). Particularly, recent studies outlined the significant contribution of PC1 and PC2 into the proteolytic processing of proneuropeptides (Zheng et al., 1994, Cui et al., 1998, Jin et al., 2005, Miller et al., 2003). Proprotein convertases, particularly PC1 and PC2, are widely expressed in the CNS and specifically cleave at C-terminal of either a pair of basic amino acids (KR, RR, RK and KK), or a single basic residue (R or K). Early prodynorphin (PDYN) endoproteolytic studies suggest that PCs play a significant role in PDYN processing (Berman et al., 2000, Day et al., 1998). These preliminary results generated using indirect methods suggest that the action of PC2 is needed for the formation of Big dynorphin (BDyn), Dyn A and dynorphin B (Dyn B) (Day et al., 1998). The proteolysis control of endogenous BDyn and Dyn A levels has a profound impact on pain perception and the role of PCs remain unclear. As shown in Fig. 1, both peptides contain paired or single basic residues and the action of PCs could be involved in C-terminal processing of BDyn and Dyn A leading to several important N-terminal metabolites. Interestingly, BDyn, Dyn A and all N-terminal metabolites encode one copy of Leu-enkephalin (Leu-Enk), an important opioid peptide. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild-type (WT), PC1−/+ and PC2−/+ animals.
Section snippets
Chemicals and reagents
Big dynorphin (BDyn), dynorphin A (Dyn A), dynorphin 1–13 (Dyn 1–13), dynorphin 1–11 (Dyn 1–11), dynorphin 1–10 (Dyn 1–10), dynorphin 1–9 (Dyn 1–9), dynorphin 1–7 (Dyn 1–7), dynorphin 1–6 (Dyn 1–6) and leu-enkephalin (Leu-Enk) were purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). Dynorphin 1–19 (Dyn 1–19) and deuterium-labeled analog peptides were synthesized de novo (CanPeptide, Inc., Pointe-Claire, QC, Canada). Other chemicals, including acetonitrile, hexane, formic acid,
Mass spectrometry and isotopic dilution method
Full-scan and product ion mass spectra for all peptides and internal standards were obtained in positive ion mode. The full-scan electrospray mass spectrum of targeted peptides displayed the formation of characteristic pseudo molecular ions [M + nH]n +, and the fragment ions observed in MS/MS spectra were annotated based on the Roepstorff and Fohlman nomenclature (Roepstorff and Fohlman, 1984). Details on MS parameters and MRM transitions are reported in Table 1. Full-scan and product ion mass
Conclusion
In neuronal cells, almost all neuropeptides are produced through endoproteolysis specifically at C-terminal pairs of basic residues during their transport along the secretory pathway. Interestingly, it is recognized that proneuropeptides are cleaved using a cell-specific mechanism by members of the family of calcium-dependent subtilisin-like endoproteases (Steiner, 1998, Seidah and Chretien, 1999, Hook et al., 2008). PC1 and PC2 are predominantly expressed in neural cells and our results
Acknowledgements
This project was funded by the National Sciences and Engineering Research Council of Canada (F. Beaudry discovery grant no. 386637-2010). The analyses were performed using analytical instruments acquired with an NSERC Research Tools and Instruments grant (F. Beaudry NSERC-RTI grant no. 439748-2013). Thermo Fisher Scientific provided generous access to a Q-Exactive Orbitrap Mass Spectrometer. A. Ruiz received a scholarship from Agència de Gestió d'Ajuts Universitaris i de Recerca
References (55)
- et al.
Molecular and neuroanatomical properties of the endogenous opioid system: implications for treatment of opiate addiction
Semin. Neurosci.
(1997) - et al.
Interplay between RAS and opioids: opening the Pandora of complexities
Neuropeptides
(2014) - et al.
Cellular and molecular mechanisms of pain
Cell
(2009) - et al.
Mu and delta opioid receptor regulation of pro-opiomelanocortin peptide secretion from the rat neurointermediate pituitary in vitro
Neuropeptides
(2000) - et al.
Dynorphin peptides differentially regulate the human κ opioid receptor
Life Sci.
(2007) - et al.
Prodynorphin processing by proprotein convertase 2 cleavage at single basic residues and enhanced processing in the presence of carboxypeptidase activity
J. Biol. Chem.
(1998) - et al.
Analysis of opioid and non-opioid end products of pro-dynorphin in the substantia nigra of the rat
Neuropeptides
(1985) - et al.
Endogenous opioid peptide-mediated neurotransmission in central and pericentral nuclei of the inferior colliculus recruits μ 1-opioid receptor to modulate post-ictal antinociception
Neuropeptides
(2012) - et al.
Determination of specific neuropeptides modulation time course in a rat model of osteoarthritis pain by liquid chromatography ion trap mass spectrometry
Neuropeptides
(2011) - et al.
Unique biological function of cathepsin L in secretory vesicles for biosynthesis of neuropeptides
Neuropeptides
(2010)
Substance p
Int. J. Biochem. Cell Biol.
Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons
Neuroscience
Proprotein covertases are responsible for proteolysis and inactivation of endothelial lipase
J. Biol. Chem.
Peripheral actions of tachykinins
Neuropeptides
Targeting of opioid-producing leukocytes for pain control
Neuropeptides
The role of nociceptin and dynorphin in chronic pain: implications of neuro–glial interaction
Neuropeptides
Selective roles for the PC2 processing enzyme in the regulation of peptide neurotransmitter levels in brain and peripheral neuroendocrine tissues of PC2 deficient mice
Neuropeptides
Cathepsin L participates in dynorphin production in brain cortex, illustrated by protease gene knockout and expression
Mol. Cell. Neurosci.
Selectivity of various opioid peptides towards delta-, kappa; and mu-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the brain
Neuropeptides
Targeted liquid chromatography quadrupole ion trap mass spectrometry analysis of tachykinin related peptides reveals significant expression differences in a rat model of neuropathic pain
Neuropeptides
Liquid chromatography-electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in Tac1 −/− mouse spinal cords reveal significant lower concentration of opioid peptides
Neuropeptides
Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides
Brain. Res.
Methionine-enkephalin modulation of hydrogen peroxide (H2O2) release by rat peritoneal macrophages involves different types of opioid receptors
Neuropeptides
Peripheral substance P and neurokinin-1 receptors have a role in inflammatory and neuropathic orofacial pain models
Neuropeptides
Spinal astrocytes produce and secrete dynorphin neuropeptides
Neuropeptides
Dynorphin reduces voltage-dependent calcium conductance of mouse dorsal root ganglion neurons
Neuropeptides
Dynorphin-(1–8) inhibits the release of substance P-like immunoreactivity in the spinal cord of rats following a noxious mechanical stimulus
Eur. J. Pharmacol.
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2020, NeuropharmacologyCitation Excerpt :Proteinases involved in cleaving dynorphins were also described in the membrane fraction of neuroblastoma cell, and in the primary neuronal culture of cerebral cortex (Silberring and Nyberg, 1989; Silberring et al., 1992b). It is suggested, that prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2), which process prodynorphin, leading to the generation of high molecular weight sequences, are also involved in the processing of shorter peptides, including dynorphins (Berman et al., 1999a; Orduna and Beaudry, 2016). Moreover, the potential role of PACE4 in dynorphin B conversion has been proposed by Watanabe et al. (2015) and Kest et al. (1992).
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2017, NeuropeptidesCitation Excerpt :Moreover, the N-terminal sequence of Dyn A encodes specifically Leu-Enk. As we demonstrated previously, the proteolytic processing of Pdyn and Dyn A leads to the formation of Leu-Enk and its processing depends on the proteolytic activity of both, PC1 and PC2 [Orduna Ruiz and Beaudry, 2016]. As shown in Fig. 6, the levels observed for Dyn A, Leu-Enk and Met-Enk in PC1−/+ and PC2−/+ mouse spinal cords are coherent with the assertion.