Pro-Gly-Pro Research Articles

Regulatory Peptide Pro-Gly-Pro Accelerates Neuroregeneration of Primary Neuroglial Culture after Mechanical Injury in Scratch Test

The scratch test is used as an experimental in vitro model of mechanical damage to primary neuronal cultures to study the mechanisms of cell death in damaged areas. The involvement of NMDA receptors in processes leading to delayed neuronal death, due to calcium dysregulation and synchronous mitochondrial depolarization, has been previously demonstrated. In this study, we explored the neuroregenerative potential of Pro-Gly-Pro (PGP)—an endogenous regulatory peptide with neuroprotective and anti-inflammatory properties and a mild chemoattractant effect. Mechanical injury to the primary neuroglial culture in the form of a scratch caused acute disruption of calcium homeostasis and mitochondrial functions. This was accompanied by neuronal death alongside changes in the profile of neuronal markers (BDNF, NSE and GFAP). In another series of experiments, under subtoxic doses of glutamate (Glu, 33 μM), delayed changes in [Ca2+]i and ΔΨm, i.e., several days after scratch application, were more pronounced in cells in damaged neuroglial cultures. The percentage of cells that restored the initial level of [Ca2+]i (p < 0.05) and the rate of recovery of ΔΨm (p < 0.01) were decreased compared with undamaged cells. Prophylactic application of PGP (100 μM, once) prevented the increase in [Ca2+]i and the sharp drop in mitochondrial potential [ΔΨm] at the time of scratching. Treatment with PGP (30 μM, three or six days) reduced the delayed Glu-induced disturbances in calcium homeostasis and cell death. In the post-glutamate period, the surviving neurons more effectively restored the initial levels of [Ca2+]i (p < 0.001) and Ψm (p < 0.0001). PGP also increased intracellular levels of BDNF and reduced extracellular NSE. In the context of the peptide’s therapeutic effect, the recovery of the damaged neuronal network occurred faster due to reduced astrogliosis and increased migration of neurons to the scratch area. Thus, the peptide PGP has a neuroprotective effect, increasing the survival of neuroglial cells after mechanical trauma in vitro by reducing cellular calcium overload and preventing mitochondrial dysfunction. Additionally, the tripeptide limits the post-traumatic consequences of mechanical damage: it reduces astrogliosis and promotes neuronal regeneration.

Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 μM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

Cell-liposome delivery system based on neuroinflammation to target the amygdala for ameliorating depressive-like behaviors

Depression is a serious psychiatric disorder with unsatisfactory outcomes due to difficulties in delivering therapeutic molecules from the periphery to the brain. Neuroinflammation plays a key role in neurobiology and the treatment of depression. Neutrophils can cross the blood–brain barrier (BBB) and infiltrate key brain regions related to the pathophysiology of depression during neuroinflammation. N-Acetyl Pro–Gly–Pro (PGP) peptides efficiently bind to CXCR2 receptors on the surface of neutrophils. The neuropeptide oxytocin demonstrated antidepressant properties in preclinical and clinical studies, but its inability to penetrate the BBB hampers its therapeutic applications. In this study, we established a novel drug delivery system based on neutrophil infiltration in key brain regions during neuroinflammation. PGP was used to modify oxytocin-loaded liposomes (PGP-OTL) as the target ligand. Systematic administration of PGP-OTL exhibited enhanced antidepressant properties resulting from elevated oxytocin concentrations, especially in the amygdala, a crucial depression-implicated brain region. Enhanced antidepressant effects of PGP-OTL, similar to the ones caused by central oxytocin infusion, were observed in behavioral measurement including forced swim and tail suspension tests. Our study demonstrated that PGP-OTL can “hitchhike” neutrophils and enhance delivery of therapeutics into the brain, thus providing the means for developing novel cell-liposome-based drug delivery strategies for depression therapy.

Insight into Glyproline Peptides’ Activity through the Modulation of the Inflammatory and Neurosignaling Genetic Response Following Cerebral Ischemia–Reperfusion

Glyprolines are Gly-Pro (GP)- or Pro-Gly (PG)-containing biogenic peptides. These peptides can act as neutrophil chemoattractants, or atheroprotective, anticoagulant, and neuroprotective agents. The Pro-Gly-Pro (PGP) tripeptide is an active factor of resistance to the biodegradation of peptide drugs. The synthetic Semax peptide, which includes Met-Glu-His-Phe (MEHF) fragments of adrenocorticotropic hormone and the C-terminal tripeptide PGP, serves as a neuroprotective drug for the treatment of ischemic stroke. Previously, we revealed that Semax mostly prevented the disruption of the gene expression pattern 24 h after a transient middle cerebral artery occlusion (tMCAO) in a rat brain model. The genes of this pattern were grouped into an inflammatory cluster (IC) and a neurotransmitter cluster (NC). Here, using real-time RT-PCR, the effect of other PGP-containing peptides, PGP and Pro-Gly-Pro-Leu (PGPL), on the expression of a number of genes in the IC and NC was studied 24 h after tMCAO. Both the PGP and PGPL peptides showed Semax-unlike effects, predominantly without changing gene expression 24 h after tMCAO. Moreover, there were IC genes (iL1b, iL6, and Socs3) for PGP, as well as IC (iL6, Ccl3, Socs3, and Fos) and NC genes (Cplx2, Neurod6, and Ptk2b) for PGPL, that significantly changed in expression levels after peptide administration compared to Semax treatment under tMCAO conditions. Furthermore, gene enrichment analysis was carried out, and a regulatory gene network was constructed. Thus, the spectra of the common and unique effects of the PGP, PGPL, and Semax peptides under ischemia-reperfusion were distinguished.

Substrate-dependent modulation of the leukotriene A4 hydrolase aminopeptidase activity and effect in a murine model of acute lung inflammation.

The aminopeptidase activity (AP) of the leukotriene A4 hydrolase (LTA4H) enzyme has emerged as a therapeutic target to modulate host immunity. Initial reports focused on the benefits of augmenting the LTA4H AP activity and clearing its putative pro-inflammatory substrate Pro-Gly-Pro (PGP). However, recent reports have introduced substantial complexity disconnecting the LTA4H modulator 4-methoxydiphenylmethane (4MDM) from PGP as follows: (1) 4MDM inhibits PGP hydrolysis and subsequently inhibition of LTA4H AP activity, and (2) 4MDM activates the same enzyme target in the presence of alternative substrates. Differential modulation of LTA4H by 4MDM was probed in a murine model of acute lung inflammation, which showed that 4MDM modulates the host neutrophilic response independent of clearing PGP. X-ray crystallography showed that 4MDM and PGP bind at the zinc binding pocket and no allosteric binding was observed. We then determined that 4MDM modulation is not dependent on the allosteric binding of the ligand, but on the N-terminal side chain of the peptide. In conclusion, our study revealed that a peptidase therapeutic target can interact with its substrate and ligand in complex biochemical mechanisms. This raises an important consideration when ligands are designed to explain some of the unpredictable outcomes observed in therapeutic discovery targeting LTA4H.

ACTH6-9-PGP improves memory consolidation processes in rats

Introduction: The His-Phe-Arg-Trp sequence corresponding to the 6-9th amino acid residue of the adrenocorticotropic hormone molecule (ACTH6-9) is the critical pharmacophore of all endogenous melanocortin receptor agonists. In order to effects prolongation it may be stabilized by the addition of the amino acid sequence Pro-Gly-Pro (PGP) to the C-terminus. The aim of this work was to study the effect of ACTH6-9-PGP (HFRWPGP) on the processes of memory consolidation in the model of passive avoidance conditioning in comparison with ACTH4-7-PGP effects.
 Materials and methods: The study was carried out on the model of passive avoidance conditioning. The effects of ACTH6-9-PGP were studied after its intraperitoneal injection to male Wistar rats at doses of 0.5, 5, 50, 150, and 450 μg/kg 15 minutes before the experiment, whereas the effects of ACTH4-7-PGP– under the similar conditions at doses of 50, 150, and 450 μg/kg.
 Results and discussion: It was found that ACTH6-9-PGP had a pronounced stimulating effect on the memory consolidation process in the dose range from 0.5 μg/kg to 150 μg/kg, significantly increasing the latent period of an animal entering the dark chamber. Administration of ACTH4-7-PGP led to an improvement in the consolidation processes of the acquired conditioned reflex at the doses of 50 μg/kg and 450 μg/kg.
 Conclusion: The range of effective doses of ACTH6-9-PGP is lower than that of ACTH4-7-PGP, which indicates the greater activity of HFRWPGP sequence in relation to memory consolidation processes and allows considering this peptide as a promising molecule for creating nootropic pharmacological drugs.

Глипролины — регуляторные пептиды с интегративным действием

Glyprolines (short proline and glycine containing peptides) are a new type of regulatory peptides. They are of interest to both fundamental physiology and medicine (possible use for medical purposes). The main advantages of glyprolines are their relative resistance to biodegradation and the integrative effect on human body. Initially, the amino acid sequence Pro-Gly-Pro (PGP) was used to increase the stability of an in vivo unstable peptide such as adrenocorticotropic hormone. The study of the intrinsic properties of PGP, as well as its possible metabolites — GP and PG — showed that all the three peptides have a wide range of physiological effects. Recently, biological activity has also been demonstrated for N-acetylated PGP, cyclic prolyl-hydroxyproline, cycloprolylglycine, di-, tri- and tetrapeptides containing proline and glycine with additional inclusion of arginine or leucine in various positions, as well as for the N-terminal fragment of the insulin-like growth factor-1 — Gly-Pro-Glu. The obtained data allowed classifying short proline and glycine containing peptides into a separate class of regulatory peptides — glyprolines. Endogenous PGP and cycloprolylglycine have been found. Collagen and insulin-like growth factor-1, respectively, are considered the most likely sources of these peptides in human body. Glyprolines have a neuroprotective effect, protect the gastric mucosa from damage, regulate inflammation and regeneration processes, have a positive effect on hemostasis, and exhibit hypolipidemic and normoglycemic effects. The article discusses the physiological effects of glyprolines, their possible endogenous and exogenous sources, mechanisms of action, and prospects for use in medicine.

Влияние аргининсодержащих пептидов на антикоагулянтную и фибринолитическую активность плазмы крови крыс в условиях <i>in vitro</i>

Исследовано действие олигопептидов глипролинового ряда — His-Phe-Arg-Trp-Pro-Gly-Pro (АКТГ(6-9)PGP), Thr-Lys-Pro-Arg-Pro-Gly-Pro (селанк) и Pro-Gly-Pro (PGP) – в различных концентрациях от 1 до 10 – 5 мг/мл в условиях in vitro на плазму крови крыс посредством тестов АЧТВ (активированное частичное тромбопластиновое время) и определения пластин нестабилизированного фибрина. Удлинение времени свертывания плазмы (по тесту АЧТВ) от 158 до 210 % в зависимости от концентрации пептидов при p < 0,01 указывает на гипокоагуляцию под действием АКТГ(6-9)PGP и селанка. Выявлены суммарные фибринолитические эффекты пептидов в концентрациях от 1 до 10–5 мг/мл методом измерения зоны лизиса на пластинах нестабилизированного фибрина в норме и под влиянием пептидов. Наибольшей фибриндеполимеризационной активностью по данным суммарной фибринолитической активности обладал пептид АКТГ(6-9)PGP от 117 до 148 % в концентрациях от 10–3 до 1 мг/мл при p < 0,05.

Changes in the nociceptive response to thermal stimulation in rats following administration of N-terminal analogs of the adrenocorticotropic hormone

Melanocortins (MCs) are an increasingly studied class of regulatory peptides exerting a wide range of biological effects. All naturally occurring MCs share a His-Phe-Arg-Trp fragment (HFRW) corresponding to the sequence of amino acid residues 6–9 of the adrenocorticotropic hormone (ACTH6-9), which is also a central active component of ACTH. Attaching the Pro-Gly-Pro (PGP) sequence to the C-end of the peptide extends the duration of the peptide’s effect. The aim of this study was to investigate the effects of ACTH6-9-PGP (HFRWPGP) on the spinal and supraspinal mechanisms involved in the nociceptive response in rats and to compare them to those of its structural analog ACTH4-7-PGP (MEHFPGP). ACTH6-9-PGP effects were studied following the intraperitoneal administration of the peptide at doses 0.5, 1.5, 5, 15, 50, 150, or 450 μg/kg 15 minutes before the hot plate and tail flick tests. ACTH4-7-PGP effects were studied under the same conditions at the following doses: 50, 150 and 450 μg/kg. We found that ACTH6-9-PGP administered intraperitoneally at 5 or 150 μg/kg induced a pronounced reduction in pain sensitivity 15 and 45 minutes after the injection (p = 0.04); this effect was implemented via supraspinal mechanisms. In the tail flick test, 150 μg/kg ACTH6-9-PGP increased pain sensitivity, with the participation of segmental spinal mechanisms (p = 0.04). ACTH4-7-PGP did not have any effect on the studied mechanisms of pain sensitivity. Thus, unlike ACTH4-7-PGP, ACTH6-9-PGP can both increase pain sensitivity and exert an analgesic effect.

Proline-containing peptides-New insight and implications: A Review.

The family of regulatory proline-containing peptides (PCPs), also known as glyprolines, exhibit significant biological activity. The group of glyprolines includes Gly-Pro (GP), Pro-Gly-Pro (PGP), cyclic Gly-Pro (cGP), as well as PGP derivatives, for example, N-acetylated PGP (N-a-PGP) and N-methylated PGP (N-m-PGP). PCPs are engaged in various biological processes including the proinflammatory neutrophil chemoattraction in lung diseases, inflammatory bowel diseases or ischemic stroke. Glyprolines have been also postulated to play an important role as atheroprotective and anticoagulant agents, exhibit neuroprotective effects in Parkinson's disease, as well as regulate insulin-like growth factor (IGF) homeostasis. It was also noticed that PCPs inhibit proliferation and migration of keratinocytes in wound healing, protection of the gastric mucosa and stimulation of its regeneration. The regulatory glyprolines are derived from endogenous and exogenous sources. Most PCPs are derived from collagen or diet protein degradation. Recently, great interest is concentrated on short proline-rich oligopeptides derived from IGF-1 degradation. The mechanism of PCPs biological activity is not fully explained. It involves receptor-mediated mechanisms, for example, N-a-PGP acts as CXCR1/2 receptor ligand, whereas cGP regulates IGF-1 bioavailability by modifying the IGF-1 binding to the IGF-1 binding protein-3. PGP has been observed to interact with collagen-specific receptors. The data suggest a promising role of PGP as a target of various diseases therapy. This review is focused on the effect of PCPs on metabolic processes in different tissues and the molecular mechanism of their action as an approach to pharmacotherapy of PCPs-dependent diseases.

Benzyloxycarbonyl-proline-prolinal (ZPP): Dual complementary roles for neutrophil inhibition

Neutrophil influx and activation contributes to organ damage in several major lung diseases. This inflammatory influx is initiated and propagated by both classical chemokines such as interleukin-8 and by downstream mediators such as the collagen fragment cum neutrophil chemokine Pro-Gly-Pro (PGP), which share use of the ELR + CXC receptor family. Benzyloxycarbonyl-proline-prolinal (ZPP) is known to suppress the PGP pathway via inhibition of prolyl endopeptidase (PE), the terminal enzyme in the generation of PGP from collagen. However, the structural homology of ZPP and PGP suggests that ZPP might also directly affect classical glutamate-leucine-arginine positive (ELR+) CXC chemokine signaling. In this investigation, we confirm that ZPP inhibits PE in vitro, demonstrate that ZPP inhibits both ELR + CXC and PGP-mediated chemotaxis in human and murine neutrophils, abrogates neutrophil influx induced by murine intratracheal challenge with LPS, and attenuates human neutrophil chemotaxis to sputum samples of human subjects with cystic fibrosis. Cumulatively, these data demonstrate that ZPP has dual, complementary inhibitory effects upon neutrophil chemokine/matrikine signaling which make it an attractive compound for clinical study of neutrophil inhibition in conditions (such as cystic fibrosis and chronic obstructive pulmonary disease) which evidence concurrent harmful increases of both chemokine and matrikine signaling.

Surface modification of PGP for a neutrophil–nanoparticle co-vehicle to enhance the anti-depressant effect of baicalein

Exploiting cells as vehicles combined with nanoparticles combined with therapy has attracted increasing attention in the world recently. Red blood cells, leukocytes and stem cells have been used for tumor immunotherapy, tissue regeneration and inflammatory disorders, and it is known that neutrophils can accumulate in brain lesions in many brain diseases including depression. N-Acetyl Pro–Gly–Pro (PGP) peptide shows high specific binding affinity to neutrophils through the CXCR2 receptor. In this study, PGP was used to modify baicalein-loaded solid lipid nanoparticles (PGP-SLNs) to facilitate binding to neutrophils in vivo. Brain-targeted delivery to the basolateral amygdala (BLA) was demonstrated by enhanced concentration of baicalein in the BLA. An enhanced anti-depressant effect was observed in vitro and in vivo. The mechanism involved inhibition of apoptosis and a decrease in lactate dehydrogenase release. Behavioral evaluation carried out with rats demonstrated that anti-depression outcomes were achieved. The results indicate that PGP-SLNs decrease immobility time, increase swimming time and climbing time and attenuate locomotion in olfactory-bulbectomized (OB) rats. In conclusion, PGP modification is a strategy for targeting the brain with a cell–nanoparticle delivery system for depression therapy.

Feasibility and physiological relevance of designing highly potent aminopeptidase-sparing leukotriene A4 hydrolase inhibitors

Leukotriene A4 Hydrolase (LTA4H) is a bifunctional zinc metalloenzyme that comprises both epoxide hydrolase and aminopeptidase activity, exerted by two overlapping catalytic sites. The epoxide hydrolase function of the enzyme catalyzes the biosynthesis of the pro-inflammatory lipid mediator leukotriene (LT) B4. Recent literature suggests that the aminopeptidase function of LTA4H is responsible for degradation of the tripeptide Pro-Gly-Pro (PGP) for which neutrophil chemotactic activity has been postulated. It has been speculated that the design of epoxide hydrolase selective LTA4H inhibitors that spare the aminopeptidase pocket may therefore lead to more efficacious anti-inflammatory drugs. In this study, we conducted a high throughput screen (HTS) for LTA4H inhibitors and attempted to rationally design compounds that would spare the PGP degrading function. While we were able to identify compounds with preference for the epoxide hydrolase function, absolute selectivity was not achievable for highly potent compounds. In order to assess the relevance of designing such aminopeptidase-sparing LTA4H inhibitors, we studied the role of PGP in inducing inflammation in different settings in wild type and LTA4H deficient (LTA4H KO) animals but could not confirm its chemotactic potential. Attempting to design highly potent epoxide hydrolase selective LTA4H inhibitors, therefore seems to be neither feasible nor relevant.

The Matrikine Acetylated Proline-Glycine-Proline Couples Vascular Inflammation and Acute Cardiac Rejection

The extracellular matrix (ECM) is a dynamic, bioactive structure critical to organ development, structure and function. Excessive remodeling of the ECM is a hallmark of a variety of inflammatory conditions including vascular disease. Endothelin-1 (ET1) synthesis is understood to promote cardiovascular diseases including acute cardiac transplant rejection; however, the contribution of ECM-derived chemokines (matrikines) to vascular inflammation remains poorly understood. Herein we report that the matrikine acetylated Pro-Gly-Pro (PGP) stimulates vascular inflammation through activation of endothelial CXC Chemokine Receptor 2 (CXCR2) and production of endothelin-1 both in vitro and in vivo. As a proof of hypothesis, we demonstrate that coronary PGP levels associate with both circulating endothelin-1 and acute rejection in cardiac transplant patients (sensitivity of 100% and specificity of 86%). These findings establish PGP as a novel mediator in cardiovascular disease, and implicate bioactive matrix fragments as underappreciated agents potentially active in numerous conditions propagated by progressive vascular inflammation.

Self-assembly of a Peptide [2]Catenane through Ω-Loop Folding

In this work, a peptide [2]catenane was created by exploiting the strong tendency of the Pro-Gly-Pro (PGP) sequence to fold into an Ω-loop. Ditopic heptapeptide ligand 1, which includes the PGP sequence, formed peptide [2]catenane 2 on complexation with Ag+. Moreover, addition/removal of Na+ triggered a reversible structural change between 2 and twisted macrocycle 3.

The development of novel LTA4H modulators to selectively target LTB4 generation

The pro-inflammatory mediator leukotriene B4 (LTB4) is implicated in the pathologies of an array of diseases and thus represents an attractive therapeutic target. The enzyme leukotriene A4 hydrolase (LTA4H) catalyses the distal step in LTB4 synthesis and hence inhibitors of this enzyme have been actively pursued. Despite potent LTA4H inhibitors entering clinical trials all have failed to show efficacy. We recently identified a secondary anti-inflammatory role for LTA4H in degrading the neutrophil chemoattractant Pro-Gly-Pro (PGP) and rationalized that the failure of conventional LTA4H inhibitors may be that they inadvertently prevented PGP degradation. We demonstrate that these inhibitors do indeed fail to discriminate between the dual activities of LTA4H, and enable PGP accumulation in mice. Accordingly, we have developed novel compounds that potently inhibit LTB4 generation whilst leaving PGP degradation unperturbed. These novel compounds could represent a safer and superior class of LTA4H inhibitors for translation into the clinic.

Semax, an analog of ACTH(4-7), regulates expression of immune response genes during ischemic brain injury in rats.

Brain stroke continues to claim the lives of million people every year. To build the effective strategies for stroke treatment it is necessary to understand the neuroprotective mechanisms that are able to prevent the ischemic injury. Consisting of the ACTH(4-7) fragment and the tripeptide Pro-Gly-Pro (PGP), the synthetic peptide Semax effectively protects brain against ischemic stroke. However, the molecular mechanisms underlying its neuroprotection and participation of PGP in them are still needed to be clarified. To reveal biological processes and signaling pathways, which are affected by Semax and PGP, we performed the transcriptome analysis of cerebral cortex of rats with focal cerebral ischemia treated by these peptides. The genome-wide biochip data analysis detected the differentially expressed genes (DEGs) and bioinformatic web-tool Ingenuity iReport found DEGs associations with several biological processes and signaling pathways. The immune response is the process most markedly affected by the peptide: Semax enhances antigen presentation signaling pathway, intensifies the effect of ischemia on the interferon signaling pathways and affects the processes for synthesizing immunoglobulins. Semax significantly increased expression of the gene encoding the immunoglobulin heavy chain, highly affects on cytokine, stress response and ribosomal protein-encoding genes after occlusion. PGP treatment of rats with ischemia attenuates the immune activity and suppresses neurotransmission in the CNS. We suppose that neuroprotective mechanism of Semax is realized via the neuroimmune crosstalk, and the new properties of PGP were found under ischemia. Our results provided the basis for further proteomic investigations in the field of searching Semax neuroprotection mechanism.

Effect of Proline-Containing Oligopeptides PGP and RGP on Proliferative and Protein-Synthesizing Activity of Cultured Pulmonary Fibroblasts under Conditions of Oxidative Stress.

We studied the effect of glyprolines Pro-Gly-Pro (PGP) and Arg-Gly-Pro (RGP) on the primary culture of pulmonary fibroblasts from newborn albino rats under normal conditions and during oxidative stress. Under physiological conditions, the peptides had no effect on parameters of cell division. Hydrogen peroxide induced intensive oxidative stress accompanied by suppression of protein-synthesizing function. When hydrogen peroxide was added to the culture containing the test peptides, correction of the oxidative status was observed accompanied by activation of DNA-synthesizing activity and inhibition of lucigenin-dependent chemiluminescence.

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

Dual anti-inflammatory and selective inhibition mechanism of leukotriene A4 hydrolase/aminopeptidase: insights from comparative molecular dynamics and binding free energy analyses

Human leukotriene A4 hydrolase/aminopeptidase (LTA4H) is a zinc metalloenzyme with a dual catalytic activity; conversion of LTA4 into LTB4 and degradation of chemotactic tripeptide Pro-Gly-Pro (PGP). Existing inhibitors, such as SC-57461A, block both catalytic activities of the enzyme, leading to drug failures. Recently, a novel compound, ARM1, was reported to selectively inhibit the hydrolase activity of LTA4H while sparing its aminopeptidase activity. However, the molecular understanding of such preferential inhibitory mechanism remains obscure. The discovery of ARM1 prompted us to further explore its binding theme and provide more insight into the structural and dual mechanistic features of LTA4H protein. To accomplish this, we embarked on wide range of computational tools, including comparative molecular dynamics (MDs) simulations and postdynamic analyses for LTA4H and in complex with ARM1, PGP, ARM1-PGP, and SC-57461A. MD analysis reveals that the binding of ARM1 exhibits a more stable active site and overall stable protein conformation when compared to the nonselective inhibitor SC-57461A. In addition, MM/GBSA-binding free energy calculation also reveals that ARM1 exhibit a lower binding affinity, when compared to the nonselective inhibitor SC-57461A – which is in a great agreement with experimental data. Per residue energy decomposition analysis showed that Phe314, Val367, Tyr378, Trp311, Pro382, and Leu369 are key residues critical for the selective inhibition of the epoxide hydrolase activity of LTA4H by ARM1. Findings from this report will not only provide more understanding into the structural, dynamic, and mechanistic features of LTA4H but would also assist toward the rational design of novel and selective hydrolase inhibitors of LTA4H as anti-inflammatory drugs.