Proteolysis Controls Endogenous Substance P Levels

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

The Biochemistry and Physiology of Peptidases

Peptidases regulate important physiological processes by controlling levels of bioactive peptides and occasionally through noncatalytic processes. This thesis presents a study of prolyl endopeptidase-like (PREPL), which is a peptidase involved in several human deletion syndromes, including hypotonia-cystinuria syndrome (HCS). Phenotypes tentatively attributed to PREPL deletion include hypotonia and decreased growth hormone (GH) levels. However, little is known about the mechanisms by which PREPL deletion causes these phenotypes. To better understand PREPL catalytic activity, we used an activity-based protein profiling fluorescence polarization screen to identify the first specific PREPL inhibitors. We proceeded to demonstrate the activity of these inhibitors in cells and discovered several classes of cell-active PREPL inhibitors. Further, one of these inhibitors, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile, was able to enter mouse brains. To characterize PREPL substrate specificity, we performed several substrate profiling experiments, but no substrates could be identified, in line with reports from other groups who used related approaches to attempt to identify PREPL substrates. To characterize any noncatalytic functions of PREPL, we used an affinity purification-mass spectrometry approach (AP-MS) to search for any protein-protein interactions of PREPL. We identified brain-expressed X-linked 2 (BEX2) as a novel interactor of PREPL, and confirmed this interaction by immunoblot. Several other proteins identified in the AP-MS experiment, including several members of the STRIPAK complex are being further investigated for possible PREPL interaction. To determine whether HCS phenotypes are in fact due to PREPL deletion and to delineate the molecular pathways involved, we generated a conditional PREPL knockout mouse. These mice were visibly smaller than wildtypes and growth curve analysis verified that from week three of life, there was a significant difference in weight between wildtype and knockout mice. Initial surface righting task experiments also indicate that PREPL knockout pups may have a hypotonia phenotype. In summary, we have developed several new tools for studying PREPL catalytic and noncatalytic function, demonstrated that PREPL deletion causes a GH-related growth deficiency and possible hypotonia and thus moved several steps closer to understanding the molecular mechanisms underlying PREPL deletion phenotypes.Chemistry and Chemical Biolog

Phosphoproteomics-based characterization of prostaglandin E2 signaling in T cells

Prostaglandin E2 (PGE2) is a key lipid mediator in health and disease and serves as a crucial link between the immune response and cancer. With the advent of cancer therapies targeting PGE2 signaling pathways at different levels, there has been increased interest in mapping and understanding the complex and interconnected signaling pathways arising from the four distinct PGE2 receptors. Here, we review phosphoproteomics studies that have investigated different aspects of PGE2 signaling in T cells. These studies have elucidated PGE2’s regulatory effect on T cell receptor signaling and T cell function, the key role of protein kinase A in many PGE2 signaling pathways, the temporal regulation of PGE2 signaling, differences in PGE2 signaling between different T cell subtypes, and finally, the crosstalk between PGE2 signaling pathways elicited by the four distinct PGE2 receptors present in T cells

Proinflammatory and immunoregulatory roles of eicosanoids in T cells

Eicosanoids are inflammatory mediators primarily generated by hydrolysis of membrane phospholipids by phospholipase A2 to ω-3 and ω-6 C20 fatty acids that next are converted to leukotrienes (LTs), prostaglandins (PGs), prostacyclins (PCs), and thromboxanes (TXAs). The rate-limiting and tightly regulated lipoxygenases control synthesis of LTs while the equally well-controlled cyclooxygenases 1 and 2 generate prostanoids, including PGs, PCs, and TXAs. While many of the classical signs of inflammation such as redness, swelling, pain, and heat are caused by eicosanoid species with vasoactive, pyretic, and pain-inducing effects locally, some eicosanoids also regulate T cell functions. Here, we will review eicosanoid production in T cell subsets and the inflammatory and immunoregulatory functions of LTs, PGs, PCs, and TXAs in T cells

Deletion of prepl causes growth impairment and hypotonia in mice

Genetic studies of rare diseases can identify genes of unknown function that strongly impact human physiology. Prolyl endopeptidase-like (PREPL) is an uncharacterized member of the prolyl peptidase family that was discovered because of its deletion in humans with hypotonia-cystinuria syndrome (HCS). HCS is characterized by a number of physiological changes including diminished growth and neonatal hypotonia or low muscle tone. HCS patients have deletions in other genes as well, making it difficult to tease apart the specific role of PREPL. Here, we develop a PREPL null (PREPL(-/-)) mouse model to address the physiological role of this enzyme. Deletion of exon 11 from the Prepl gene, which encodes key catalytic amino acids, leads to a loss of PREPL protein as well as lower Prepl mRNA levels. PREPL(-/-) mice have a pronounced growth phenotype, being significantly shorter and lighter than their wild type (PREPL(+/+)) counterparts. A righting assay revealed that PREPL(-/-) pups took significantly longer than PREPL(+/+) pups to right themselves when placed on their backs. This deficit indicates that PREPL(-/-) mice suffer from neonatal hypotonia. According to these results, PREPL regulates growth and neonatal hypotonia in mice, which supports the idea that PREPL causes diminished growth and neonatal hypotonia in humans with HCS. These animals provide a valuable asset in deciphering the underlying biochemical, cellular and physiological pathways that link PREPL to HCS, and this may eventually lead to new insights in the treatment of this disease.status: publishe

Metallopeptidase inhibitors potently block SP degradation in spinal cord lysates.

<p>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 SP_1–9 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).</p

Absolute quantities of SP and SP fragments in the spinal cord as measured by isotope dilution mass spectrometry (IDMS).

<p>Absolute quantities of SP and SP fragments in the spinal cord as measured by isotope dilution mass spectrometry (IDMS).</p

SP is regulated by proteolysis but not by NEP.

<p>A) Phosphoramidon slows SP_1–9 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).</p

GM6001 is significantly more effective than other metallopeptidase inhibitors at preventing SP degradation and conversion of SP to SP_1–9.

<p>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 SP_1–9 are identified. Inhibitors against these peptidases were then used in lysates to evaluate their affect on SP degradation and SP_1–9 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 SP_1–9 production. (Statistical significance calculated by a Student's t-test; p-value <0.05, *; p-value <0.01, **; p-value <0.001, ***, N = 4).</p

C-terminal processing is the primary mode of SP degradation.

<p>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.</p