Revealing favorable and unfavorable residues in cooperative positions in protease cleavage sites

Qi, Enfeng; Wang, Dongyu; Li, Yang; Li, Guojun; Su, Zhengchang

doi:10.1016/j.bbrc.2019.09.056

Cited by 3 publications

(1 citation statement)

References 32 publications

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“…of P1 may be recognized by additional subsites (S2, S3 and S1’, S2’, respectively). Although this model implies that the interactions between the individual substrate residues and their corresponding subsites are relatively independent of one another, it is evident that constraints in size, geometry or charge may lead to interactions such that certain sequence combinations are favorable, others unfavorable (Ng, Pike, and Boyd 2009; Qi et al 2019) (Fig. 1A).…”

Section: Introductionmentioning

confidence: 99%

Massively parallel quantification of substrate turnover defines protease subsite cooperativity

Gudipati,

Gaidatzis,

Seebacher

et al. 2023

Preprint

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Substrate specificity determines protease functions in physiology and in clinical and biotechnological application. However, affordable and unbiased assays for large-scale quantification of substrate cleavage have been lacking. Here, we develop hiMAPS (high-throughputmapping of protease cleavage sites), a cheap, mass spectrometry-based assay, to derive cleavage motifs for humanDipeptidylPeptidase Four (DPP4), a key regulator of blood glucose levels. We recapitulate the known benefit of proline in the penultimate (P1) position from the substrate N-terminus, extend it to additional residues, and identify and quantify combinatorial interactions of P1 with its neighbors. These findings reveal extensive cooperativity among the enzyme’s active site subsites and allow us to derive a sequence motif that predicts substrate turnover. We show that this information provides new opportunities to engineer stabilized versions of a key substrate of DPP4, the small peptide hormone GLP-1, whose derivatives are used for clinical treatment of type 2 diabetes and obesity. Finally, structural and biochemical characterization of a DPP4 homologue, C. elegans DPF-3, reveal specific subsite differences and a distinct cleavage motif, providing insight into the mechanistic basis of the observed specificity. Collectively, our findings present a broadly applicable framework to high-throughput mapping of protease cleavage sites, extend our understanding of protease specificity, and provide a chemical space for substrate engineering of a clinically relevant family of exopeptidases.

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Section: Introductionmentioning

confidence: 99%

Massively parallel quantification of substrate turnover defines protease subsite cooperativity

Gudipati,

Gaidatzis,

Seebacher

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

Deep quantification of substrate turnover defines protease subsite cooperativity

Gudipati,

Gaidatzis,

Seebacher

et al. 2024

Mol Syst Biol

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Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4’s active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.

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Insights into protease sequence similarities by comparing substrate sequences and phylogenetic dynamics

Chen

Zhai

et al. 2021

Mathematical Biosciences and Engineering

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Revealing favorable and unfavorable residues in cooperative positions in protease cleavage sites

Cited by 3 publications

References 32 publications

Massively parallel quantification of substrate turnover defines protease subsite cooperativity

Massively parallel quantification of substrate turnover defines protease subsite cooperativity

Deep quantification of substrate turnover defines protease subsite cooperativity

Insights into protease sequence similarities by comparing substrate sequences and phylogenetic dynamics

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