Activity-based protein profiling guided identification of urine proteinase 3 activity in subclinical rejection after renal transplantation.

BACKGROUND: The pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection. METHODS: Serine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine. RESULTS: In-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group. CONCLUSIONS: There appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process.

Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.

New selective peptidyl di(chlorophenyl) phosphonate esters for visualizing and blocking neutrophil proteinase 3 in human diseases.

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl(P)(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.

The circadian clock protein Cryptochrome 1 is a direct target and feedback regulator of the Hippo pathway.

Circadian clock controls daily behavior and physiology. The activity of various signaling pathways affects clock gene expression. Here, we show that the core circadian clock gene CRY1 is a direct target of the Hippo pathway effector YAP. YAP binds to TEADs and occupies the proximal promoter regions of CRY1, positively regulating its transcription. Interestingly, we further identified that CRY1 acts in a feedback loop to fine-tune Hippo pathway activation by modulating the expression of YAP and MOB1. Indeed, loss of CRY1 results in enhanced YAP activation. Consistently, we found that YAP levels and activity control clock gene expression and oscillation in synchronized cells. Furthermore, in breast cancer cells, CRY1 downregulation causes YAP/TAZ hyperactivation and enhanced DNA damage. Together, our findings provide a direct mechanistic link between the Hippo pathway and the circadian clock, where CRY1 and Hippo components form an orchestrated signaling network that influences cell growth and circadian rhythm.

Regulation of Cell Polarity by Posttranslational Protein Palmitoylation.

Cell polarity is a common feature of many living cells, especially epithelial cells, and plays important roles in development, tissue homeostasis, and diseases. Therefore, the signaling pathways involved in establishing and maintaining cell polarity are tightly controlled. Protein S-palmitoylation has been recently recognized as an important posttranslational modification involved in cell polarity, via dynamic covalent attachment of fatty acyl groups to the cysteine residues of cell polarity proteins. Here, we describe the methods to study the function and regulation of S-palmitoylation of cell polarity proteins.

Exploiting the S4-S5 Specificity of Human Neutrophil Proteinase 3 to Improve the Potency of Peptidyl Di(chlorophenyl)-phosphonate Ester Inhibitors: A Kinetic and Molecular Modeling Analysis.

The neutrophilic serine protease proteinase 3 (PR3) is involved in inflammation and immune response and thus appears as a therapeutic target for a variety of infectious and inflammatory diseases. Here we combined kinetic and molecular docking studies to increase the potency of peptidyl-diphenyl phosphonate PR3 inhibitors. Occupancy of the S1 subsite of PR3 by a nVal residue and of the S4-S5 subsites by a biotinylated Val residue as obtained in biotin-VYDnVP(O-C6H4-4-Cl)2 enhanced the second-order inhibition constant kobs/[I] toward PR3 by more than 10 times ( kobs/[I] = 73000 ± 5000 M-1 s-1) as compared to the best phosphonate PR3 inhibitor previously reported. This inhibitor shows no significant inhibitory activity toward human neutrophil elastase and resists proteolytic degradation in sputa from cystic fibrosis patients. It also inhibits macaque PR3 but not the PR3 from rodents and can thus be used for in vivo assays in a primate model of inflammation.

Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases.

Cathepsin C (CatC) is a tetrameric cysteine dipeptidyl aminopeptidase that plays a key role in activation of pro-inflammatory serine protease zymogens by removal of a N-terminal pro-dipeptide sequence. Loss of function mutations in the CatC gene is associated with lack of immune cell serine protease activities and cause Papillon-Lefèvre syndrome (PLS). Also, only very low levels of elastase-like protease zymogens are detected by proteome analysis of neutrophils from PLS patients. Thus, CatC inhibitors represent new alternatives for the treatment of neutrophil protease-driven inflammatory or autoimmune diseases. We aimed to experimentally inactivate and lower neutrophil elastase-like proteases by pharmacological blocking of CatC-dependent maturation in cell-based assays and in vivo. Isolated, immature bone marrow cells from healthy donors pulse-chased in the presence of a new cell permeable cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites. These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases, which opens new perspectives for therapeutic applications in humans.