Three Wavelength Substrate System of Neutrophil Serine Proteinases

Wysocka, Magdalena; Lesner, Adam; Gruba, Natalia; Korkmaz, Brice; Gauthier, Francis; Kitamatsu, Mizuki; Łęgowska, Anna; Rolka, Krzysztof

doi:10.1021/ac301684w

Cited by 24 publications

(27 citation statements)

References 23 publications

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“…The calculated initial hydrolysis rates were used as a measure of the substrate activity of the investigated peptides. All details of kinetic studies and the method of calculating kinetic parameters have been described elsewhere [44].…”

Section: Determination Of Kinetic Parametersmentioning

confidence: 99%

Bladder cancer detection using a peptide substrate of the 20S proteasome

et al. 2016

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The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2‐aminobenzoic acid (ABZ)‐Val‐Val‐Ser‐Tyr‐Ala‐Met‐Gly‐Tyr(3‐NO2)‐NH2, is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 105 m−1·s−1) and a high kcat (8 s−1). Using this peptide, we identified chymotrypsin‐like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods.

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Section: Determination Of Kinetic Parametersmentioning

confidence: 99%

Bladder cancer detection using a peptide substrate of the 20S proteasome

et al. 2016

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“…Other chemicals, including heparin–agarose type I, were purchased from Sigma‐Aldrich and were of the highest quality and purity. The substrate peptides NWVSAAKFESTDGSTDYGIYQV (22‐peptide), whose sequence was based on a peptide described in , AbzVLDPILF

Y^{{normalNO}_{2}}

‐O2Oc‐O2Oc‐IFQI (PepPEG2), AbzKASPVSL

Y^{{normalNO}_{2}}

G (reporter) and NWVSAAKFE‐O2Oc‐O2Oc‐IYQV (PepPEG3) were synthesized in the Department of Chemistry, University of Gdansk as described in and . The Abz (aminobenzoic acid) served as a fluorescent probe,

Y^{{normalNO}_{2}}

(3‐nitrotyrosine) served as a fluorescence quencher.…”

Section: Methodsmentioning

confidence: 99%

The LD loop as an important structural element required for transmission of the allosteric signal in the HtrA (DegP) protease from Escherichia coli

et al. 2016

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High-temperature requirement A (HtrA; DegP) from Escherichia coli, an important element of the extracytoplasmic protein quality-control system, is a member of the evolutionarily conserved family of serine proteases. The characteristic feature of this protein is its allosteric mode of activation. The regulatory loops, L3, L2, L1 and LD, play a crucial role in the transmission of the allosteric signal. Yet, the role of LD has not been fully elucidated. Therefore, we undertook a study to explain the role of the individual LD residues in inducing and maintaining the proteolytic activity of HtrA. We investigated the influence of amino acid substitutions located within the LD loop on the kinetics of a model substrate cleavage as well as on the dynamics of the oligomeric structure of HtrA. We found that the mutations that were expected to disturb the loop's structure and/or interactions with the remaining regulatory loops severely diminished the proteolytic activity of HtrA. The opposite effect, that is, increased activity, was observed for G174S substitution, which was predicted to strengthen the interactions mediated by LD. HtrAG174S protein had an equilibrium shifted toward the active enzyme and formed preferentially high-order oligomeric forms.

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“…Structural differences between PR3 and HNE at S3, S2, S19, and S29 have been exploited to develop highly sensitive and specific synthetic FRET peptide substrates for human PR3 that allow its detection in complex biologic samples (Hajjar et al, 2006;Korkmaz et al, 2007;Popow-Stellmaszyk et al, 2013;Sinden and Stockley, 2013;Hinkofer et al, 2015). In addition, a cell-permeable selective PR3 substrate, O 2 Oc-K(HMC)-YYAbu-Orn(CM3), was recently developed (Wysocka et al, 2012).…”

Section: B Substrate Binding Sitesmentioning

confidence: 99%

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

Korkmaz¹,

Lesner²,

Guarino³

et al. 2016

Pharmacol Rev

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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 alpha-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 alpha-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

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Three Wavelength Substrate System of Neutrophil Serine Proteinases

Cited by 24 publications

References 23 publications

Bladder cancer detection using a peptide substrate of the 20S proteasome

Bladder cancer detection using a peptide substrate of the 20S proteasome

The LD loop as an important structural element required for transmission of the allosteric signal in the HtrA (DegP) protease from Escherichia coli

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

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