Positional Scanning Peptide Libraries for Kinase Substrate Specificity Determinations: Straightforward and Reproducible Synthesis Using Pentafluorophenyl Esters

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1′–P2′ residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

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Substrate specificity of schistosome versus human legumain determined by P1–P3 peptide libraries

Molecular and Biochemical Parasitology ◽

10.1016/s0166-6851(02)00026-9 ◽

2002 ◽

Vol 121 (1) ◽

pp. 99-105 ◽

Cited By ~ 32

Author(s):

Mary A. Mathieu ◽

Matthew Bogyo ◽

Conor R. Caffrey ◽

Youngchool Choe ◽

Jewok Lee ◽

...

Keyword(s):

Substrate Specificity ◽

Peptide Libraries

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Investigation of Thylakoid Protein Kinase Substrate Specificity Using Synthetic Peptides

Current Research in Photosynthesis ◽

10.1007/978-94-009-0511-5_394 ◽

1990 ◽

pp. 1727-1730

Author(s):

Ian R. White ◽

Paul A. Millner ◽

John B. C. Findlay

Keyword(s):

Protein Kinase ◽

Substrate Specificity ◽

Synthetic Peptides ◽

Kinase Substrate ◽

Thylakoid Protein ◽

Thylakoid Protein Kinase

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Bimodal evolution of Src and Abl kinase substrate specificity revealed using mammalian cell extract as substrate pool

10.1101/2020.08.12.248104 ◽

2020 ◽

Author(s):

Patrick Finneran ◽

Margaret Soucheray ◽

Christopher Wilson ◽

Renee Otten ◽

Vanessa Buosi ◽

...

Keyword(s):

Substrate Specificity ◽

Mammalian Cell ◽

Tyrosine Kinases ◽

Cell Extract ◽

Peptide Sequence ◽

Last Common Ancestor ◽

Sequence Specificity ◽

Kinase Substrate ◽

Evolutionary Trajectories ◽

Substrate Proteins

AbstractThe specificity of phosphorylation by protein kinases is essential to the integrity of biological signal transduction. While peptide sequence specificity for individual kinases has been examined previously, here we explore the evolutionary progression that has led to the modern substrate specificity of two non-receptor tyrosine kinases, Abl and Src. To efficiently determine the substrate specificity of modern and reconstructed ancestral kinases, we developed a method using mammalian cell lysate as the substrate pool, thereby representing the naturally occurring substrate proteins. We find that the oldest tyrosine kinase ancestor was a promiscuous enzyme that evolved through a more specific last common ancestor into a specific human Abl. In contrast, the parallel pathway to human Src involved a loss of substrate specificity, leading to general promiscuity. These results add a new facet to our understanding of the evolution of signaling pathways, with both subfunctionalization and neofunctionalization along the evolutionary trajectories.

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Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: A framework for anti–COVID-19 drug design

Science Advances ◽

10.1126/sciadv.abd4596 ◽

2020 ◽

Vol 6 (42) ◽

pp. eabd4596 ◽

Cited By ~ 8

Author(s):

Wioletta Rut ◽

Zongyang Lv ◽

Mikolaj Zmudzinski ◽

Stephanie Patchett ◽

Digant Nayak ◽

...

Keyword(s):

Substrate Specificity ◽

Crystal Structures ◽

Drug Repurposing ◽

Activity Profiling ◽

Irreversible Inhibitors ◽

Inhibitory Mechanisms ◽

Promising Target ◽

Therapeutic Value ◽

Positional Scanning ◽

High Degree

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.

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