Applications for chemical probes of proteolytic activity.
Recent genome sequencing projects have identified new peptidases in multiple organisms, many with unknown functions, suggesting the need for new tools to study these enzymes. This unit outlines selection and use of small-molecule and protein-based probes to covalently modify peptidases in complex cellular environments. These activity-based probes (ABPs) have been designed based on well characterized peptidase inhibitor scaffolds, but make use of new techniques to greatly enhance their utility for studying families of related peptidases. In particular, ABPs can be used to track activity of peptidases in crude cell extracts, intact cells, and in vivo, allowing rapid purification and identification of labeled targets. They can be used with libraries of small molecules to rapidly assess potency and selectivity of compounds in complex, physiologically relevant samples. Probe selection, probe tagging using reporters, labeling of recombinant targets, crude protein extracts, and peptidase targets in cell culture systems, affinity purification of targets, and inhibitor screening using affinity probes are outlined.
Bogyo
Matthew
M
Stanford University, Stanford, California.
Baruch
Amos
A
Celera Genomics, South San Francisco, California.
Jeffery
Douglas A
DA
Celera Genomics, South San Francisco, California.
Greenbaum
Doron
D
University of California, San Francisco, California.
Borodovsky
Anna
A
Harvard Medical School, Boston, Massachusetts.
Ovaa
Huib
H
Harvard Medical School, Boston, Massachusetts.
Kessler
Benedikt
B
Harvard Medical School, Boston, Massachusetts.
D005456:Fluorescent Dyes / Q000737:chemistry; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000737:chemistry; D015335:Molecular Probes; D010447:Peptide Hydrolases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D015335:Molecular Probes; D011506:Proteins; D010447:Peptide Hydrolases