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Protease Activity Profiling via Programmable Phage Display of Comprehensive Proteome-Scale Peptide Libraries
Gabriel D Román-Meléndez1, Thiagarajan Venkataraman1, Daniel R Monaco1
1Institute for Cell Engineering, Immunology Division, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA 21205.
Cell Systems
|October 25, 2020
Summary
A new assay called SEPARATE enables proteomic-scale endopeptidase activity profiling. This method identified novel protease substrates, including a new target for caspase-1, advancing our understanding of protein cleavage in biological processes.
Area of Science:
- Biochemistry
- Proteomics
- Molecular Biology
Background:
- Endopeptidases are crucial enzymes involved in protein turnover, maturation, and signaling.
- Current assays lack the capacity for proteomic-scale characterization of endopeptidase activity.
- Understanding protease function is vital for numerous biological processes and disease states.
Purpose of the Study:
- To develop a novel assay for unbiased, proteomic-scale assessment of endopeptidase activity.
- To identify new substrates and characterize the activity of various human endopeptidases.
- To discover previously unknown roles of proteases in cellular functions.
Main Methods:
- Development of Sensing EndoPeptidase Activity via Release and recapture using flAnking Tag Epitopes (SEPARATE).
- Utilized monovalent phage display of the human proteome at 90-amino acid peptide resolution.
- Validated SEPARATE with human proteases including caspase-1, ADAM17, and thrombin.
Main Results:
- SEPARATE successfully detected known and identified novel substrates for tested proteases.
- Demonstrated compatibility with diverse human endopeptidases from different catalytic classes.
- Discovered HUWE1, an E3 ubiquitin ligase, as a novel non-canonical substrate of caspase-1.
- Identified HUWE1 as a key mediator in apoptotic cell death pathways.
Conclusions:
- SEPARATE provides an efficient and unbiased method for evaluating endopeptidase activity across the proteome.
- The assay facilitates the discovery of new protease-substrate interactions and functional roles.
- This work expands the understanding of caspase-1 activity and its involvement in apoptosis.
Keywords:
next generation DNA sequencingprogrammable phage displayprotease activity profilingproteomic analysis
