Fluorogenic Peptide Substrates for Serine and Threonine Phosphatases

2010 ◽  
Vol 12 (9) ◽  
pp. 1936-1939 ◽  
Author(s):  
Fengtian Xue ◽  
Christopher T. Seto
Keyword(s):  
Peptide Substrates ◽  
Fluorogenic Peptide

scholarly journals Development of Chemical Tools to Monitor Human Kallikrein 13 (KLK13) Activity

2019 ◽  
Vol 20 (7) ◽  
pp. 1557 ◽  
Author(s):  
Natalia Gruba ◽  
Ewa Bielecka ◽  
Magdalena Wysocka ◽  
Anna Wojtysiak ◽  
Magdalena Brzezińska-Bodal ◽  
...  
Keyword(s):  
Serine Protease ◽  
Biological Samples ◽  
Substrate Binding ◽  
Breast Tumors ◽  
Active Enzyme ◽  
Design Synthesis ◽  
Peptide Substrates ◽  
Binding Subsites ◽  
Fluorogenic Peptide ◽  
Selective Activity

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.

scholarly journals Changes in Activity and Kinetic Properties of the Proteasome in Different Rat Organs during Development and Maturation

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
A. Petersen ◽  
A. Honarvar ◽  
M. Zetterberg
Keyword(s):  
Rat Brain ◽  
Structural Changes ◽  
Specific Activity ◽  
Kinetic Properties ◽  
Loss Of Function ◽  
Proteolytic System ◽  
Peptide Substrates ◽  
Menten Constant ◽  
Fluorogenic Peptide ◽  
Soluble Fractions

The proteasome is considered the most important proteolytic system for removal of damaged proteins with aging. Using fluorogenic peptide substrates, the chymotrypsin-like, the trypsin-like, and the peptidylglutamyl peptidase activities of the proteasome were measured in the soluble fractions of liver, brain, and lens rat homogenates. Specific activity was significantly decreased in liver and brain homogenates with maturation of the animal, that is, from newborn (7 days old) to fertile rats (2–4 months old). Rat lens homogenate exhibited an increase in activity with maturation and also with aging. Chymotrypsin-like activity was stimulated by calcium and this proteolytic activity was significantly decreased with maturation of the rat brain. The Michaelis-Menten constant (Km) increased with age in rat liver and lens, indicating a loss of affinity for its substrates by the proteasome in the animal with maturation and aging. The present data suggest that the loss of function of the proteasome with maturation may be due to structural changes of the proteasome or a decreased content of regulatory components.

A Proteasome Specific Binding Assay for Quantitation of Constitutive and Immunoproteasome Active Sites.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2472-2472
Author(s):  
Mark K. Bennett ◽  
Monette A. Aujay ◽  
Tonia J. Buchholz ◽  
Susan D. Demo ◽  
Guy J. Laidig ◽  
...  
Keyword(s):  
Active Sites ◽  
Antigen Processing ◽  
Specific Binding ◽  
Peripheral Blood Mononuclear ◽  
Peptide Substrates ◽  
Enzyme Preparations ◽  
Proteolytic Activities ◽  
Fluorogenic Peptide

Abstract The ubiquitin-proteasome pathway constitutes a major intracellular system for protein degradation. Substrates for this pathway include misfolded or unassembled proteins as well as short-lived regulatory proteins that play key roles in signaling and proliferative pathways. The majority of cell types express the standard, or “constitutive”, form of the proteasome, while cells of the immune system also express the immunoproteasome, a form of the proteasome that contributes to class I major histocompatibility complex restricted antigen processing. Non-immune cells can also express immunoproteasome in response to interferon gamma exposure. The immunoproteasome retains the same structural subunits as the constitutive proteasome but has three different catalytic subunits. The catalytic activities of both forms of the proteasome have been traditionally characterized with purified enzyme preparations and fluorogenic peptide substrates. Such fluorogenic peptide substrates suffer from two characteristics that limit their utility in measuring proteasome activities in complex cell or tissue lysates: 1) they cannot distinguish proteasome activities from other proteolytic activities within the lysate; and 2) they can not distinguish between constitutive and immunoproteasome activities. We have developed an ELISA-based proteasome-specific binding (PSB) assay that can detect and quantify the chymotryptic-like proteasome active sites of the beta-5 constitutive proteasome subunit and the LMP7 immunoproteasome subunit. The assay utilizes a biotin-modified peptide epoxyketone probe that covalently and irreversibly interacts with the active site threonine present in catalytic proteasome subunits. Once bound to the probe, the labeled subunits are recovered on streptavidin-conjugated beads and detected with subunit-specific antibodies. The PSB assay is both quantitative and sensitive. We have demonstrated that the assay is capable of measuring constitutive proteasome and immunoproteasome binding activity in human whole blood and peripheral blood mononuclear cell preparations, respectively. In experiments with the epoxyketone-based proteasome inhibitor PR-171, the dose response for inhibition of the PSB assay is equivalent to that measured with a conventional fluorogenic peptide proteasome substrate. In addition, the PSB assay can effectively measure the level of PR-171 mediated inhibition of both the constitutive and immunoproteasome in the RPMI-8226 multiple myeloma cell line that co-expresses both proteasome types. Thus, the PSB assay overcomes the limitations of conventional fluorogenic substrate-based proteasome activity assays when applied to cell or tissue lysates that contain multiple proteolytic activities or mixtures of constitutive and immunoproteasomes. Potential applications of the PSB assay include the measurement of the pharmacodynamic response to proteasome inhibitors and the evaluation of constitutive vs. immunoproteasome selectivity of inhibitors both in vitro and in vivo.

Inhibition Of The Deubiquitinating Enzymes UCHL5 and USP14 Is Lethal To Waldenströms Macroglobulinemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1823-1823 ◽  
Author(s):  
Kasyapa S. Chitta ◽  
Aneel Paulus ◽  
Sharoon Akhtar ◽  
Maja Kuranz ◽  
Vivek Roy ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Annexin V ◽  
26S Proteasome ◽  
Deubiquitinating Enzymes ◽  
Catalytic Core ◽  
Peptide Substrates ◽  
Proteasomal Activity ◽  
Dub Inhibitor ◽  
Fluorogenic Peptide

Abstract Background Waldenströms macroglobulinemia (WM) is a lymphoplasmacytic subtype of Non-Hodgkins lymphoma in which specific molecular changes lead to unrestricted cell proliferation and overproduction of immunoglobulin-M. These processes require exquisite coordination between growth promoting transcription factors (NFkB) and cell homeostatic systems. The bioavailability of various transcription factors and cell cycle proteins is primarily regulated by the ubiquitin proteasome system (UPS). Moreover, the proteolytic machinery ensures proper elimination of unwanted or misfolded proteins, which when disrupted leads to protein accumulation and apoptotic cell death. The 26S proteasome is a barrel shaped structure with a 20S catalytic core that is flanked by 19S caps on either side. 26S proteasomes are critical for WM cell survival, which is clinically evident, as up to 80% of WM patients treated with the proteasome inhibitor (PI) bortezomib have derived significant benefit. Traditional PI such as bortezomib and carfilzomib have been designed to target the B5 catalytic core within the 26S proteasome. However, proteasomes are capable of altering conformation of their proteolytic subunits in response to inflammation and stress, which can lower the effectiveness of these PI. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14 results in cell death in a variety of tumor types but more so in cancer cells that rapidly divide or have a high protein turnover rate. Aim To define the anti-WM activity of the novel DUB inhibitor, b-AP15 in preclinical models of WM. Methods The WM cell lines BCWM.1, RPCI-WM1 and MWCL-1 were used for experiments in this analysis. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Apoptosis was determined by annexin-V/PI staining and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiling was done via immunoblot analysis. Results As reported by D’Arcy et al in their characterization of the b-AP15 molecule, inhibition of UCHL5 and USP14 by b-AP15 does not disrupt proteasomal activity conferred by the b-catalytic subunits. We confirmed these findings by treating WM cells with single agent bortezomib (10nM), carfilzomib (10nM), b-AP15 (100nM) or all three agents in combination to assess chymotrypsin-like, caspase-like and trypsin-like proteasomal activity using fluorogenic peptide substrates. Further, when used in combination with the other PI, PI-mediated chymotrypsin-like activity remained inhibited in the presence of b-AP15. Next, we sought to determine if b-AP15 treatment could induce WM cell death. Annexin-v staining showed that b-AP15 (500uM) induced apoptosis in all WM cell lines (28 – 60% cell death) with variable responses noted among the 3 models. Treatment with b-AP15 increased MOMP while also inducing the cleavage of caspases-9 and 3 and PARP-1 suggesting that the anti-WM activity of b-AP15 is mediated through the intrinsic apoptotic pathway. As components of the NFkB signaling cascade lie downstream to the UPS, we examined the effects of b-AP15 on this pathway. When treated with the DUB-inhibitor, nuclear translocation of NFkB was reduced in a time dependent manner. Interestingly, treatment with b-AP15 increased the phosphorylation of the mitogen activated protein kinase-p38 (p38) in all the WM cell lines tested. Treatment of WM cells with b-AP15 in presence of the p38 inhibitor, SB203580, resulted in a synergistic induction of cell death. Conclusion b-AP15 is a novel DUB inhibitor that disrupts two regulatory enzymes (UCHL5 and USP14) present in the 19S proteasomal cap. Results presented here demonstrate that b-AP15 induces apoptosis in in vitro models of WM and with in vivo analysis underway, is a potential therapeutic in WM. We would like to acknowledge the Waterfall Waldenström Macroglobulinemia Research Fund, the Leukemia and Lymphoma Society and the International Waldenström Macroglobulinemia Foundation for their continued support. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A novel high-yield synthesis of aminoacyl p-nitroanilines and aminoacyl 7-amino-4-methylcoumarins: Important synthons for the synthesis of chromogenic/fluorogenic protease substrates

2011 ◽  
Vol 7 ◽  
pp. 1030-1035 ◽  
Author(s):  
Xinghua Wu ◽  
Yu Chen ◽  
Herve Aloysius ◽  
Longqin Hu
Keyword(s):  
Amino Acids ◽  
High Yield ◽  
Easy Access ◽  
General Applicability ◽  
Peptide Substrates ◽  
Peptide Chemistry ◽  
Amino Acid Peptide ◽  
Hydroxysuccinimide Esters ◽  
Fluorogenic Peptide

Aminoacyl p-nitroaniline (aminoacyl-pNA) and aminoacyl 7-amino-4-methylcoumarin (aminoacyl-AMC) are important synthons for the synthesis of chromogenic/fluorogenic protease substrates. A new efficient method was developed to synthesize aminoacyl-pNA and aminoacyl-AMC derivatives in excellent yields starting from either amino acids or their corresponding commercially available N-hydroxysuccinimide esters. The method involved the in situ formation of selenocarboxylate intermediate of protected amino acids and the subsequent non-nucleophilic amidation with an azide. Common protecting groups used in amino acid/peptide chemistry were all well-tolerated. The method was also successfully applied to the synthesis of a dipeptide conjugate, indicating that the methodology is applicable to the synthesis of chromogenic substrates containing short peptides. The method has general applicability to the synthesis of chromogenic and fluorogenic peptide substrates and represents a convenient and high-yield synthesis of N α-protected-aminoacyl-pNAs/AMCs, providing easy access to these important synthons for the construction of chromogenic/fluorogenic protease substrates through fragment condensation or stepwise elongation.

Fluorogenic Peptide Substrates For Proteases In Blood Coagulation, Kallikrein-kinin And Fibrinolysis Systems: Substrate For Plasmin And Factor XIa

1979 ◽  
Author(s):  
S Iwanaga ◽  
H Kato ◽  
I Maruyama ◽  
N Adachi ◽  
Y Ohno ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Factor Xa ◽  
Molar Ratio ◽  
Assay Method ◽  
Plasma Kallikrein ◽  
Urinary Kallikrein ◽  
Fluorogenic Substrates ◽  
Peptide Substrates ◽  
Factor Xia ◽  
Fluorogenic Peptide

Fluorogenic peptide substrates, peptidyl-4-methylcoumarin amides (MCA), are now commercially available and proved to be useful for the assay of α-thrombin, Factor Xa, urokinase, Limulus clotting enzyme, plasma and urinary kallikreins. We have newly synthesized the fluorogenic substrates for plasmin and Factor XIa and tested for their possible use as specific substrates for these enzymes. Among six peptidyl-Lys-MCA, Boc-Glu-Lys-Lys-MCA and Boc-Val-Leu-Lys-MCA were good substrates for bovine and human plasmins, which were activated by urokinase. Streptokinase-activated plasmin complex (molar ratio with 1 to 1) hydrolyzed the former but not the latter. Using Boc-Phe-Ser-Arg-MCA and Boc-Leu-Thr-Arg-MCA, a specific assay method for Factor XIa activated with Factor XHa in the presence of HMW kininogen and kaolin, was developed. The kinetic parameters of various proteinases towards the specific substrates were as follows: α-Thrombin (Boc-Val-Pro-Arg-MCA, Km=2.1x10-5M), Factor Xa (Boc-Ile-Glu-Gly-Arg-MCA, 1.6x10-4), Plasma kallikrein (Z-Phe-Arg-MCA, 2.4xl0-4M), Pancreatic kallikrein (Pro-Phe-Arg-MCA, 1.6x10-4M), Urinary kallikrein (Pro-Phe-Arg-MCA, 2.2x10-4M), Urokinase (Glutaryl-Gly-Arg-MCA, 4.4xl0-5M), Limulus clotting enzyme (Boc-Leu-Gly-Arg-MCA, 2.7X10-5M), Plasmin (Boc-Glu-Lys-Lys-MCA, 6.7xl0-4M and Boc-Val-Leu-Lys-MCA, 2.5x10-4M).

Sign in / Sign up

Export Citation Format

Share Document