The authors have declared that no competing interests exist.
Conceived and designed the experiments: AJM AML AT AS. Performed the experiments: AJM AML AT. Analyzed the data: AJM AML AT. Contributed reagents/materials/analysis tools: AJM AML AT. Wrote the paper: AJM AT AS.
Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons. Proteolysis has long been suspected to regulate extracellular SP concentrations but data in support of this hypothesis is scarce. Here, we provide evidence that proteolysis controls SP levels in the spinal cord. Using peptidomics to detect and quantify endogenous SP fragments, we identify the primary SP cleavage site as the C-terminal side of the ninth residue of SP. If blocking this pathway increases SP levels, then proteolysis controls SP concentration. We performed a targeted chemical screen using spinal cord lysates as a proxy for the endogenous metabolic environment and identified GM6001 (galardin, ilomastat) as a potent inhibitor of the SP 1–9-producing activity present in the tissue. Administration of GM6001 to mice results in a greater-than-three-fold increase in the spinal cord levels of SP, which validates the hypothesis that proteolysis controls physiological SP levels.
A member of the tachykinin family of neuropeptides, substance P (SP) is an amidated undecapeptide (
A) An integrated approach that combines chemical screening and peptide profiling provides a new strategy to determine whether proteolysis plays a role in the regulation of endogenous SP levels. B) Initial experiments begin in tissue lysates and the data clearly shows that SP is processed by membrane proteases to generate a series of C-terminally truncated fragments, while the soluble proteome has little impact on SP processing.
Several mechanisms have been definitively shown to regulate SP. These include the differential expression of SP mRNA
One way to definitively determine that proteolysis regulates SP would be to block a proteolytic pathway
Recognizing that enzyme identification approaches are very time consuming given their reliance on extensive biochemical purification and confirmatory studies, we wondered whether there is an easier path to evaluating the hypothesis that proteolysis regulates SP. To this end, we devised a strategy that couples
Mouse SP was purchased from Anaspec, Inc. A protease inhibitor panel was obtained from Sigma Aldrich Inc.
Heavy-labeled SP1–7 (Pro containing five 13C and one 15N), SP1–9 (Phe containing eight 2H), and SP (Leu containing ten 2H) were synthesized manually using FMOC chemistry for solid-phase peptide synthesis. Crude peptides were purified by RP-HPLC (Shimadzu) using a C18 column (150×20 mm, 10 μm particle size, Higgins Analytical). The HPLC gradient varied depending on the peptide (Mobile Phase A: 99% H2O, 1% Acetonitrile, 0.1% TFA; Mobile Phase B: 90% Acetonitrile, 10% H2O, 0.07% TFA). HPLC fractions were analyzed for purity by MALDI-TOF (Waters) using α-cyano-4-hydroxycinnamic acid as the matrix. Pure fractions were combined and lyophilized. Concentrations of the purified peptides were determined by UV-vis using the extinction coefficient for phenylalanine.
Wild type (C57BL/6) mice used in this study were either purchased (Jackson Labs) or taken from a breeding colony.
Tissue peptide isolation and fractionation were previously described
SCX was performed using a PolySULFOETHYL ATM column (200×2.1mm, 5 µm, 300 Å; PolyLC INC.) connected to an Agilent Technologies 1200 series LC. All runs were operated at 0.3 mL/min. The SCX buffers (prepared with MS quality water) consisted of: A) 7 mM KH2PO4, pH 2.6, 25% ACN (vol/vol); B) 40 mM KCl, 7 mM KH2PO4, pH 2.6, 25% ACN (vol/vol); C) 100 mM KCl, 7 mM KH2PO4, pH 2.6, 25% ACN (vol/vol); D) 600 mM KCl, 7 mM KH2PO4, pH 2.6, 25% ACN (vol/vol). Prior to the SCX runs, all samples (N = 4) were dissolved in 900 µL buffer A (1 mL sample loop). A step-gradient was applied that included 60 min with Buffer A, 60 min with Buffer B, 60 min with Buffer C, and 60 min with Buffer D, with 1 min transitions between the different buffer conditions. Fractions were collected separately for each of the different buffer conditions (e.g., a buffer A fraction, a buffer B fraction, and so on). Fraction C was isolated because SP and the primary peptide products cleaved at the C-terminus are expected to be +3 charged at pH 2.6. This fraction was applied to a C18 Sep Pak cartridge, washed with water to desalt the samples, and then eluted with 1 mL of 70:30 H2O/ACN and concentrated using a speed vac. It is important to note that the Met on SP becomes nearly 100% oxidized following SCX. The peptide samples were dissolved in 0.1% aqueous formic acid (50 mg tissue/20 µL), normalized according to the original tissue weight, prior to LC-MS analysis.
Fractionated spinal cord samples (N = 4) were analyzed using a nano flow LC (Nano LC-2D, Eksigent Technologies) system coupled to a linear ion trap mass spectrometer (LTQ, ThermoFinnigan) following a 10 uL injection. The analytical column (Self-pack picofrit column, 75 μm ID, New Objective) was packed 15 cm with 3 μm C18 (Magic C18 AQ 200A 3U, Michrom Bioresources Inc). The trap column was obtained pre-packed from New Objective Inc. (Integrafrit sample trap, C18 5 μm, 100 μm column ID). The samples were trapped at an isocratic flow rate of 2 μl/min for 10 minutes and eluted at a flow rate of 300 nl/min via a mobile phase gradient of 2–50% B in 180 min (Mobile Phase A: 0.1% formic acid in water, Mobile Phase B: 0.1% formic acid in acetonitrile). The peptides were detected in the positive mode and the mass range for data acquisition was set from m/z 400–1600. The data were collected in Top 6 MS2 mode (N = 4) with the dynamic exclusion set for 30s, the exclusion size list set to 200, and the normalized collision energy for CID set to 35%. The capillary spray voltage was set to 2.5 kV. All experiments were repeated multiple times to ensure reproducibility.
Peptide identification was performed via two methods using the SEQUEST algorithm. The first method applied a differential modification of methionine to its sulfoxide. The uniprotmus_frc.fasta mouse database, concatenated to a reversed decoy database, served to estimate a false discovery rate (FDR). Peptides were accepted within 1 Da of the expected mass, meeting a series of custom filters on ScoreFinal (Sf), −10 Log P, and charge state that attained an average peptide FDR of <2% across data sets. Manual inspection of spectra, FDR calculation, and protein inference were performed in Proteomics Browser Suite 2.23 (ThermoFisher Scientific).
In the second method that has been previously described
The heavy-label versions of SP1–7, SP1–9, and SP were spiked into spinal cord samples (N = 4) at the beginning of the peptide isolation process. After fine tuning the amount of spiked peptides, it was determined that a final concentration of 100 fmol/μL of SP1–7 and SP and 25 fmol/μL of SP1–9 would lead to adequate measurements of the peptides using Top 6 MS/MS. A comparison was made of the integrated area for specific, corresponding fragments of the +2 charge state of the endogenous and heavy labeled peptides detected in the positive mode. The mass range for data acquisition was set from m/z 400–700. The peptide levels were measured in pmol peptide/g of tissue. All experiments were repeated multiple times to ensure reproducibility.
Three mouse spinal cords were dounce homogenized in 1.1 mL phosphate buffered saline (PBS) on ice and then sonicated for 15 s at 4°C. Tissue debris was separated by centrifuging the sample at 5,000×g for 20 min at 4°C. The soluble fraction was collected after ultracentrifugation of the sample at 55,000×g for 1 h at 4°C. The membrane pellet that remained was washed 3x with 600 μL PBS. The pellet was then suspended in 100 μL PBS by sonication for 5 s at 4°C. The sample was ultracentrifugation at 55,000×g for 1 h at 4°C and the supernatant was separated as a wash membrane sample. The pellet was suspended in 100 μL of 1 mM sodium deoxycholate (Alfa Aesar) by sonication for 5 s at 4°C then stirred for 30 min at 4°C. Ultracentrifugation of the sample at 55,000×g for 1 h at 4°C separated the supernatant as a 1 mM deoxycholate-solubilized membrane fraction. The remaining pellet was washed 3x with 600 μL 1 mM deoxycholate solution. The pellet was subsequently suspended in 4, 12, and 24 mM deoxycholate following the same cycle. The final pellet was suspended in 100 μL PBS. The protein content in the soluble and membrane lysates was quantified by the Bradford assay. SP (100 μM) was incubated in 1 mg/mL soluble and membrane lysates diluted in 20 mM ammonium bicarbonate, pH 7.34 buffer for 1 h (determined to be the optimal incubation time) at 37°C. The reactions were quenched with neat formic acid and speed vac dried. The samples were dissolved in 0.1% formic acid (aq) and analyzed by MALDI-TOF MS for SP-degrading activity (i.e. formation of SP1–7 and SP1–9) using the method outlined in “MALDI-TOF MS and LC-MS/MS analysis of
The MEROPS database was utilized to devise a candidate list for the enzymes that could cleave SP, forming SP1–9 in mice
Western blotting was used to detect endothelin-converting enzyme 2 (ECE2) (Proteintech Group Inc.; rabbit polyclonal) and pitrilysin (Proteintech Group Inc.; rabbit polyclonal) in the mouse spinal cord membrane lysates prepared by deoxycholate solubilization.
The membrane fraction of spinal cord lysates (1 mg/mL) were pre-incubated at 37°C for 30 minutes separately with each of the following inhibitors (N = 4): 10 μM E-64 (cysteine protease), 1 mM iodoacetamide (cysteine protease), 1 mM o-phenanthroline (metalloprotease), 10 μM pepstatin A (aspartyl protease), 1 mM phenylmethylsulfonyl fluoride (PMSF, serine protease), 1 mM diisopropylfluorophosphate (serine protease), and vehicle (PBS with DMSO for 5% DMSO final concentration in reaction). After the pre-incubation with each inhibitor, SP was added to 100 μM final concentration. The reactions proceeded at 37°C for 1 h. Dried samples were dissolved in 0.1% formic acid (aq) and analyzed by LC-MS/MS for SP-degrading activity (i.e. formation of SP1–7 and SP1–9) using the method outlined in “MALDI-TOF MS and LC-MS/MS analysis of
The membrane fraction of spinal cord lysates (1 mg/mL) were pre-incubated at 37°C for 30 minutes separately with each of the following inhibitors (N = 4): SM-19712 (ECE1), phosphoramidon (neprilysin; ECE2), MMP9 inhibitor, TIMP2 (MMP inhibitor), GM6001 (MMP and neprilysin broad inhibitor), chymostatin (pitrilysin), captopril (ACE), enalaprilat (ACE), actinonin (Meprin 1A), and vehicle (PBS with DMSO for 5% DMSO final concentration in reaction). All inhibitors were present at 100 μM except for TIMP2, which was present at 4 μM. After the pre-incubation with each inhibitor, either SP or SP1–9 was added to 100 μM final concentration. The reactions proceeded at 37°C for 1 h. Dried samples were dissolved in 0.1% formic acid (aq) and analyzed by LC-MS/MS for SP or SP1–9-degrading activity. The same comparative reactions were performed with the deoxycholate-solubilized membrane fractions but only with the GM6001 inhibitor.
The levels of SP in WT and
MALDI-TOF MS was performed with α-cyano-4-hydroxycinnamic acid as the matrix using 2 μL of a 50 μM reconstituted degradation reaction solution (based on initial SP quantities). Data were acquired on a Waters MALDI micro MX instrument operated in reflectron positive mode.
For LC-MS analysis, an Agilent 6220 LC-ESI-TOF instrument was used in the positive mode. A Bio-Bond C18 (5 μm, 150×2.1 mm) column was used together with a precolumn (C18, 3.5 μm, 2×20 mm). Following injection of 25 μL of 5 μM solutions, the samples were trapped at an isocratic flow rate of 0.1 ml/min for five minutes and eluted at a flow rate of 0.25 mL/min via a mobile phase gradient of 2–100% B in 40 min (Mobile Phase A: 0.1% formic acid in water, Mobile Phase B: 0.1% formic acid in acetonitrile). MS analysis was performed with an electrospray ionization (ESI) source. The capillary voltage was set at 4.0 kV and the fragmentor voltage to 100 V. The drying gas temperature was 350°C, the drying gas flow rate was 10 L/min, and the nebulizer pressure was 45 psi. Data was collected in the centroid mode using a mass range of 100–500 Da. The peptides were analyzed by mass extraction of the +3 charge state.
For GM6001 injection experiments, 3–4 month old female WT mice (N = 4) were fasted overnight. GM6001 was dissolved at a high concentration in DMSO. Intraperitoneal (i.p.) injections were performed with a 10 μL/g injection of either vehicle (5% DMSO, 95% saline) or 10 mg/mL GM6001 in 5% DMSO, 95% saline for a final dose of 100 mg/kg GM6001. Mice were allowed to return to their cages for three hours and then spinal cords were isolated as described in the ‘Animal studies’ section. IDMS was used to measure differences in the levels of SP in the inhibitor treated and untreated samples.
All data will be made available upon request.
To determine the candidate proteolytic pathway or pathways for SP degradation in the spinal cord, we separated tissue lysates from mouse spinal cords into membrane and soluble fractions, incubated them with full-length SP for varying lengths of time (15, 60, 240 min) and then analyzed the quenched reactions by MALDI mass spectrometry to identify any SP fragments that had been produced. No discernable fragments appeared in the soluble fraction, while ions corresponding to SP1–10, SP1–9, SP1–8, and SP1–7 were all produced in the membrane fraction (
Lysate experiments are imperfect because the compartmentalization of a tissue is disrupted, which can lead to the production of SP fragments that are not physiologically relevant. Therefore, we complement these lysate experiments with LC-MS/MS experiments to determine which of these SP fragments, if any, are present in vivo
Peptides | SPC (pmol/g) |
SP | 105.9±8.5 |
SP1–10 | n.d. |
SP1–9 | 2.1±0.5 |
SP1–8 | n.d. |
SP1–7 | 1.6±0.5 |
To identify the enzyme class that mediates SP processing we relied on a screen using class-specific peptidase inhibitors coupled to quantitative MS analysis. The inhibitors used in this screen include compounds such as the metallopeptidase inhibitor O-phenanthroline
A) Different class-selective inhibitors were tested for their ability to slow SP degradation in spinal cord membrane lysates. The most effective compound at inhibiting SP degradation in this assay is O-phenanthroline, a metalloprotease inhibitor. B) O-phenanthroline was also the most potent inhibitor of SP1–9 production in these experiments. C) Multiple class-selective inhibitors regulate SP1-7 production including O-phenanthroline, pepstatin A and PMSF. (Statistical significance calculated by a Student's t-test; p-value <0.05, *; p-value <0.01, **; p-value <0.001, ***, N = 4).
O-phenanthroline inhibited the degradation of SP to the largest extent with SP values twice as high as the vehicle control, indicating that a metallopeptidase contributes the most to SP processing in these tissue lysates. Modest increases in SP levels were also seen for pepstatin and PMSF, but these differences were not statistically significant. O-phenanthroline was also the strongest inhibitor of SP1–9 production, and the overall pattern of SP1–9 production correlates with the inhibitor specificity for SP degradation. Specifically, O-phenanthroline was the best inhibitor but pepstatin and PMSF had a small effect on SP1–9 production. The correlation between SP1–9 production and SP degradation indicates that the conversion of SP to SP1–9 may be the key step in the conversion of SP in spinal cord lysates. By contrast, the inhibitor sensitivity of SP1–7 production is markedly different from that of SP degradation.
Having characterized the conversion of SP to SP1–9 as the key step in SP degradation, we turned our attention toward the identification of a chemical inhibitor of this step. SP is often used as a model substrate for peptidases and proteases and therefore there are a number of metallopeptidases reported to cleave SP and produce SP1–9
Using the MEROPS database, we identified eight mammalian peptidases reported to cleave SP to produce SP1–9 (
A) Utilizing the MEROPS and Allen Brain Map databases a number of candidate metallopeptidases in the nervous system that are capable of cleaving SP to produce SP1–9 are identified. Inhibitors against these peptidases were then used in lysates to evaluate their affect on SP degradation and SP1–9 production. B) The matrix metalloprotease (MMP) inhibitor GM6001 was the most effective compound at preventing SP degradation. C) GM6001 is also the best inhibitor of SP1–9 production. (Statistical significance calculated by a Student's t-test; p-value <0.05, *; p-value <0.01, **; p-value <0.001, ***, N = 4).
Each of these inhibitors was added to spinal cord membrane lysate along with SP. After incubation, the relative levels of SP and SP1–9 were assessed in the presence of each inhibitor. This assay revealed three compounds that could inhibit SP degradation and formation of SP1–9: phosphoramidon, actinonin and the MMP inhibitor GM6001
Based on the inhibitor sensitivities of SP degradation (
A) Phosphoramidon slows SP1–9 production in tissue lysates, which suggests that NEP might have a role in SP processing. Experiments in NEP+/+ and NEP–/– spinal cord lysates reveals no significant difference in SP degradation. B) Likewise, no difference in endogenous SP levels is observed in spinal cords from NEP+/+ and NEP–/– mice. C) Acute treatment of mice with GM6001 results in a 3-fold elevation of SP in the spinal cord to reveal a GM6001-sensitive pathway for SP regulation. (Statistical significance calculated by a Student's t-test; p-value <0.001, ***, N = 4).
To determine whether ECE-2 is responsible for the SP-degrading activity we detect in tissue lysates, we solubilized the proteome with different concentrations of deoxycholate and then tested these fractions for SP-degrading activity. In addition, we performed activity assays on each of these fractions to ascertain whether ECE-2 levels correlate with SP-degrading activity. These data clearly show that ECE-2 and SP-degrading activity are not connected and this data disfavors any role for ECE-2 in SP-degradation in tissue lysates (
Typical approaches for testing enzymes or pathways for a role in peptide regulation
The hypothesis that SP is regulated by proteolysis originated with the earliest discoveries of potent SP-degrading activity in nervous tissue, which occurred over three decades ago
Recently, we developed an advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide profiling strategy to elucidate the proteolysis of bioactive peptides
A series of lysate experiments with class selective inhibitors showed that SP is regulated by a metallopeptidase and that the levels of SP and SP1–9 are inversely correlated (
At this point, a typical approach would call for the identification of the enzyme responsible for this step followed by perturbation of the protein
Fortuitously, GM6001 is bioavailable and had previously been shown to permeate the central nervous system (CNS)
We expect the strategy described herein to be applicable to all bioactive peptides
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