Positional Scanning Substrate Combinatorial Library (PS-SCL) Approach to Define Caspase Substrate Specificity

Poręba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Drąg, Marcin

doi:10.1007/978-1-4939-0357-3_2

Cited by 37 publications

(35 citation statements)

References 36 publications

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“…Similar to the recent work of Puorger et al., we opted for an initial screening strategy in which select positions within the canonical LPXTG motif were randomized during solid‐phase synthesis . Using a previously reported isokinetic coupling mixture, we generated a peptide pool in which position 5 of the substrate motif was randomized with one of 19 possible residues (Figure A). To avoid complications from undesired disulfide formation and thioether oxidation, cysteine was omitted from the isokinetic coupling mixture, and methionine was replaced with an isosteric norleucine (Nle) residue.…”

Section: Resultsmentioning

confidence: 99%

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

et al. 2017

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Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site-specific modification of the N-terminal serine residue in the naturally occurring antimicrobial peptide DCD-1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.

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Section: Resultsmentioning

confidence: 99%

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

et al. 2017

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“…To determine the optimum phytaspase hydrolysis motif, the PS-SCL approach was used (25). Tetrapeptides in P2-P4 libraries contained an aspartic acid residue in the P1 position linked to a fluorogenic ACC group.…”

Section: Resultsmentioning

confidence: 99%

“…In this study we defined the substrate specificity of rice (Oryza sativa) phytaspase using the positional scanning substrate combinatorial library (PS-SCL) approach (25). The preferred phytaspase cleavage sites identified turned out to be notably hydrophobic.…”

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confidence: 99%

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease

Galiullina

Kasperkiewicz

Chichkova

et al. 2015

Journal of Biological Chemistry

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Background:Phytaspase is a plant cell death-promoting protease capable of hydrolyzing a range of caspase substrates. Results: Phytaspase possesses an exclusive Asp specificity of hydrolysis and prefers a cleavage motif that is strikingly hydrophobic. Conclusion: Substrate specificities of plant and animal death proteases display both similarity and important distinction. Significance: Knowledge of the phytaspase specificity provides insight toward possible targets of the enzyme.

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“…Finally, the substrate specificity matrix was made based on the RFU/s value for each substrate, setting the highest value from each sublibrary to 100% and adjusting the other substrates accordingly. The precise procedure for the determination of protease substrate specificity profile using the PS-SCL approach can be found elsewhere (Poreba et al, 2014b). …”

Section: Methodsmentioning

confidence: 99%

Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes

Poręba

Solberg

Rut

et al. 2016

Cell Chemical Biology

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SUMMARY Legumain (AEP) is a lysosomal cysteine protease that is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP up-regulation is linked to a number of diseases including inflammation, arteriosclerosis and tumorigenesis. Thus legumain is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling.

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Positional Scanning Substrate Combinatorial Library (PS-SCL) Approach to Define Caspase Substrate Specificity

Cited by 37 publications

References 36 publications

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease

Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes

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