Expression of recombinant pro‐neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by ‘prohormone thiol protease’ (PTP)

Schiller, Martin; Kohn, Andrea B.; Mende‐Mueller, Liane; Miller, Kurt W.; Hook, Vivian

doi:10.1016/0014-5793(96)00083-x

Cited by 9 publications

(3 citation statements)

References 27 publications

(28 reference statements)

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“…This knowledge suggests that the PTP protease complex could possibly be involved in the proteolysis of multiple polypeptide substrates localized to secretory vesicles. In fact, PTP is capable of processing proenkephalin (10)(11)(12)(13), as well as proneuropeptide Y (47). Clearly, it will be of interest in future studies to define the role of the PTP protease complex in the production of biologically active neuropeptides that mediate intercellular communication in the nervous and endocrine systems.…”

Section: Acknowledgmentmentioning

confidence: 99%

Evidence for the Proenkephalin Processing Enzyme Prohormone Thiol Protease (PTP) as a Multicatalytic Cysteine Protease Complex: Activation by Glutathione Localized to Secretory Vesicles

et al. 1999

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The cysteine protease known as "prohormone thiol protease" (PTP) has been identified as a major proenkephalin processing enzyme in secretory vesicles of adrenal medulla (known as chromaffin granules). This study provides the first demonstration that PTP exists as a multicatalytic cysteine protease complex that can be activated by endogenous glutathione present in chromaffin granules. The high molecular mass nature of PTP, of approximately 185 kDa, was demonstrated by elution of a single peak of 35S-enkephalin precursor cleaving activity by Sephacryl S200 gel filtration chromatography and by a single band of 35S-enkephalin precursor cleaving activity detected on radiozymogram gels under native buffer conditions. Importantly, when 0.1% SDS was included in radiozymogram gels, PTP activity was resolved into three bands of proteolytic activity with apparent molecular masses of 88, 81, and 61 kDa. These activities were all cysteine proteases, since they were inhibited by the cysteine protease inhibitor E-64c but not by pepstatin A or EDTA that inhibit aspartyl protease and metalloprotease, respectively. Purification of native PTP by preparative gel electrophoresis indicated that PTP was composed of four polypeptides of 66, 60, 33, and 29 kDa detected on SDS-PAGE gels. These four protein subunits accounted for the three catalytic activities of PTP, as demonstrated on 35S-enkephalin precursor radiozymogram gels. Results also indicated that the electrophoretic mobilities of the four subunits differed under reducing compared to nonreducing conditions. The multicatalytic activities of the PTP complex all require reducing conditions for activity, which can be provided by endogenous reduced glutathione in chromaffin granules. These novel findings provide the first evidence for a role of a multicatalytic cysteine protease complex, PTP, in chromaffin granules that may be involved in the proteolytic processing of proenkephalin and perhaps other precursors into active neuropeptides.

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Section: Acknowledgmentmentioning

confidence: 99%

Evidence for the Proenkephalin Processing Enzyme Prohormone Thiol Protease (PTP) as a Multicatalytic Cysteine Protease Complex: Activation by Glutathione Localized to Secretory Vesicles

et al. 1999

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“…We generated a GST-fusion peptide Lv and a GST-fusion peptide II from E. coli (Fig. 6A) for the following electrophysiological experiments, since the GST fusion peptides can be easily expressed and purified from E. coli , and the recombinant peptides generated can mimic the activities of endogenous peptides [34], [35], [36], [37]. We took advantage of primary dissociated cell cultures of chicken photoreceptors for the following assays, because these cells have oil droplets present at the inner segments [22], which allow for easier identification under the microscope for patch-clamp recordings, and the cultures can be easily maintained in an incubator for several days.…”

Section: Resultsmentioning

confidence: 99%

Identification of Peptide Lv, a Novel Putative Neuropeptide That Regulates the Expression of L-Type Voltage-Gated Calcium Channels in Photoreceptors

Shi

Abbott

et al. 2012

PLoS ONE

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Neuropeptides are small protein-like signaling molecules with diverse roles in regulating neural functions such as sleep/wake cycles, pain modulation, synaptic plasticity, and learning and memory. Numerous drugs designed to target neuropeptides, their receptors, or relevant pathways have been developed in the past few decades. Hence, the discovery and characterization of new neuropeptides and their functions have received considerable attention from scientific research. Computational bioinformatics coupled with functional assays are powerful tools to address the difficulties in discovering new bioactive peptides. In this study, a new bioinformatic strategy was designed to screen full length human and mouse cDNA databases to search for novel peptides. One was discovered and named peptide Lv because of its ability to enhance L-type voltage-gated calcium channel (L-VGCC) currents in retinal photoreceptors. Using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), peptide Lv was detected in the culture media, which indicated that it was secreted from 661W cells transfected with the gene. In vitro treatments with either glutathione S-transferase (GST) fusion peptide Lv or synthesized peptide Lv enhanced L-VGCC channel activities in cone photoreceptors. At the molecular level, peptide Lv stimulated cAMP production, enhanced phosphorylation of extracellular signal-regulated kinase (ERK), and increased the protein expression of L-VGCCα1 subunits in cone photoreceptors. Therefore, the biological activities of peptide Lv may be very important in the modulation of L-VGCC dependent neural plasticity.

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“…For in vitro screening of potential agonists and antagonists, a combination of recombinant expression and synthetic biology approaches can be employed for high‐throughput and large‐scale synthesis of neuropeptide analog libraries. Due to the low abundance of neuropeptides in vivo and their high proteolytic degradation vulnerability, multiple neuropeptides have been expressed at high levels in E. coli , e.g., an NPY precursor [ 284 ] and NTS. [ 285 ] Additionally, large‐scale peptide production usually relies on solid phase synthesis, by which diversities of ligands are restricted or time/cost‐consuming to achieve.…”

Section: Current Research Strategies and Future Directionsmentioning

confidence: 99%

Neuropeptides in Cancer: Friend and Foe?

Berisha

Borniger

2022

Advanced Biology

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highly dynamic network of various cell types (e.g., cancer cells, endothelial cells, immune cells, fibroblasts) that exert differential effects on neuronal activity within the local environment. Thus, complex signaling between cancer and stromal cells in the TME results in altered firing rates of local nerves. Reciprocally, increased neuronal activity and subsequent release of classical neurotransmitters and/or neuropeptides within the TME results in enhanced cancer progression and subsequent metastasis in various preclinical models and clinical studies. [5][6][7][8][9] Nerves interact with multiple stromal cells in the TME where they indirectly promote tumor growth, progression, and subsequent metastasis. [10] Several studies have demonstrated that sympathetic nerve innervation and subsequent release of the neurotransmitter norepinephrine (NE) is increased in breast tumors. [6,8,9] However, recent work has demonstrated that there is an inverse relationship between tumor weight and norepinephrine content (i.e., larger the size of the tumor, the lower the levels of NE), despite increased innervation of sympathetic nerves within the TME. [11] These findings may be attributed to a relatively unexplored phenomenon: neuropeptide release within the TME. Neuropeptides are molecular messengers that regulate a variety of functions in the central and peripheral nervous systems via binding G-protein-coupled receptors (GPCRs) on target cells. One of the many functions of neuropeptides is serving as growth factors for normal cells via the activation of the heterotrimeric G protein Gq and subsequent synthesis of second messengers and engagement of tyrosine phosphorylation cascades. Neuropeptides act as neuromodulators, in that they can alter the response of neurons to neurotransmitters and other circulating signals, such as leptin, ghrelin, glucose, and insulin-all of which are affected during cancer progression. [12][13][14][15][16][17] For example, both neuropeptide Y (NPY) and hypocretin/orexin neurons appear to mediate some of the orexigenic effects of centrally administered ghrelin as administration of NPY receptor antagonists attenuated ghrelininduced feeding. Similarly, ghrelin-induced feeding was suppressed in orexin knockout mice, indicating that ghrelin may stimulate feeding through both the orexin and NPY systems. [18] However, neuropeptide signaling is exploited within cancer cells and many studies demonstrate the contribution of neuropeptides in tumor cell proliferation and migration, [19] with many major neuropeptides also potentially contributing to cancer processes (see Table 1). Additionally, neuropeptides exert direct Neuropeptides are small regulatory molecules found throughout the body, most notably in the nervous, cardiovascular, and gastrointestinal systems. They serve as neurotransmitters or hormones in the regulation of diverse physiological processes. Cancer cells escape normal growth control mechanisms by altering their expression of growth factors, receptors, or intracellular signals, and neuropeptid...

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Expression of recombinant pro‐neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by ‘prohormone thiol protease’ (PTP)

Cited by 9 publications

References 27 publications

Evidence for the Proenkephalin Processing Enzyme Prohormone Thiol Protease (PTP) as a Multicatalytic Cysteine Protease Complex: Activation by Glutathione Localized to Secretory Vesicles

Evidence for the Proenkephalin Processing Enzyme Prohormone Thiol Protease (PTP) as a Multicatalytic Cysteine Protease Complex: Activation by Glutathione Localized to Secretory Vesicles

Identification of Peptide Lv, a Novel Putative Neuropeptide That Regulates the Expression of L-Type Voltage-Gated Calcium Channels in Photoreceptors

Neuropeptides in Cancer: Friend and Foe?

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