Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library

Byzia, Anna; Szeffler, Agata; Kalinowski, Leszek; Drąg, Marcin

doi:10.1016/j.biochi.2015.09.035

Cited by 17 publications

(16 citation statements)

References 32 publications

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“…Moreover, the use of unnatural amino acids in proteases screening does not have to be limited to combinatorial peptide mixtures. HyCoSuL was published in 2014, however before that our group presented HyCoSuL-related strategies where individual substrate libraries with unnatural amino acids can be successfully applied in protease substrate screens (H2N-P1-ACC for aminopeptidases 39,40 or H2N-P2-P1-ACC for dipeptidyl dipeptidases 41 ). Recently we have also reported on the use of tripeptide libraries with unnatural amino acids tailored for ClpP proteases 42,43 .…”

Section: Application / Case Study 2 (Human Apoptotic Caspases)mentioning

confidence: 99%

Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Poręba¹,

Salvesen²,

Drąg³

2017

Nat Protoc

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108

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Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4-P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1' position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4-P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4-8 weeks (HyCoSuL synthesis: 3-5 weeks; HyCoSuL screening per enzyme: 4-8 d; and activity-based probe synthesis: 1-2 weeks).

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Section: Application / Case Study 2 (Human Apoptotic Caspases)mentioning

confidence: 99%

Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Poręba¹,

Salvesen²,

Drąg³

2017

Nat Protoc

Self Cite

108

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“…L-Leu-pNA and L-Glu-pNA were purchased from Bachem. Microsomes containing porcine kidney cortex glutamyl aminopeptidase (pAPA) and human placental APN (hAPN) were prepared as described [22], based on a previous report [23]. LAP (hLAP) was prepared from human placenta as described [22].…”

Section: Materials and Animalsmentioning

confidence: 99%

Biochemical evidences for M1-, M17- and M18-like aminopeptidases in marine invertebrates from Cuban coastline

Alonso

Méndez

Valdés‐Tresanco

et al. 2020

Zeitschrift Für Naturforschung C

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AbstractMetallo-aminopeptidases (mAPs) control many physiological processes. They are classified in different families according to structural similarities. Neutral mAPs catalyze the cleavage of neutral amino acids from the N-terminus of proteins or peptide substrates; they need one or two metallic cofactors in their active site. Information about marine invertebrate’s neutral mAPs properties is scarce; available data are mainly derived from genomics and cDNA studies. The goal of this work was to characterize the biochemical properties of the neutral APs activities in eight Cuban marine invertebrate species from the Phyla Mollusca, Porifera, Echinodermata, and Cnidaria. Determination of substrate specificity, optimal pH and effects of inhibitors (1,10-phenanthroline, amastatin, and bestatin) and cobalt on activity led to the identification of distinct neutral AP-like activities, whose biochemical behaviors were similar to those of the M1 and M17 families of mAPs. Additionally, M18-like glutamyl AP activities were detected. Thus, marine invertebrates express biochemical activities likely belonging to various families of metallo-aminopeptidases.

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“…A recent study of different aminopeptidases examined an array of natural and unnatural amino acid substrates and determined the specificity towards APN. The unnatural amino acids, styryl-Ala, hCha, and Nle ( Figure 5 ), were found to have the highest selectivity for APN [ 44 ]. The results also suggested that an assay based on multiple substrates can produce a library fingerprint that identifies the responsible aminopeptidase more accurately than a single-substrate assay.…”

Section: Measuring Apn Activity Using Reactive Substratesmentioning

confidence: 99%

Molecular Imaging of Aminopeptidase N in Cancer and Angiogenesis

Schreiber

Smith

2018

Contrast Media & Molecular Imaging

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This review focuses on recent advances in the molecular imaging of aminopeptidase N (APN, also known as CD13), a zinc metalloenzyme that cleaves N-terminal neutral amino acids. It is overexpressed in multiple cancer types and also on the surface of vasculature undergoing angiogenesis, making it a promising target for molecular imaging and targeted therapy. Molecular imaging probes for APN are divided into two large subgroups: reactive and nonreactive. The structures of the reactive probes (substrates) contain a reporter group that is cleaved and released by the APN enzyme. The nonreactive probes are not cleaved by the enzyme and contain an antibody, peptide, or nonpeptide for targeting the enzyme exterior or active site. Multivalent homotopic probes utilize multiple copies of the same targeting unit, whereas multivalent heterotopic molecular probes are equipped with different targeting units for different receptors. Several recent preclinical cancer imaging studies have shown that multivalent APN probes exhibit enhanced tumor specificity and accumulation compared to monovalent analogues. The few studies that have evaluated APN-specific probes for imaging angiogenesis have focused on cardiac regeneration. These promising results suggest that APN imaging can be expanded to detect and monitor other diseases that are associated with angiogenesis.

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Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library

Cited by 17 publications

References 32 publications

Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Biochemical evidences for M1-, M17- and M18-like aminopeptidases in marine invertebrates from Cuban coastline

Molecular Imaging of Aminopeptidase N in Cancer and Angiogenesis

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