Proteolysis plays an important role in the regulation of diverse biological processes. Unfortunately, current methods for monitoring proteolytic events in complex samples suffer from serious limitations.
The aim of this proposal is to establish a novel method for global profiling of proteolysis in biochemical mixtures that is sensitive, robust, and general. The basis for this method will be to detect newly formed protein N-termini. This will be accomplished using subtiligase, a variant of the protease subtilisin that has been engineered to function as an efficient peptide ligase. Subtiligase will be employed to selectively ligate a labeled peptide onto N-termini of proteolyzed proteins in complex biochemical mixtures. The label will allow affinity purification and enrichment of ligated proteins for subsequent identification by tandem mass spectrometry. Analysis of proteolysis in apoptosis will at first be used as a model system for method development. A matured version of the method will then be applied to: a) survey the diversity of proteolysis patterns in apoptosis elicited by different stimuli and in different cell types, and b) identify new targets of proteolysis in apoptosis, with particular emphasis on identification of new points for chemotherapeutic intervention. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM074458-02
Application #
7174230
Study Section
Special Emphasis Panel (ZRG1-F04B (20))
Program Officer
Fabian, Miles
Project Start
2006-02-01
Project End
2008-07-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
2
Fiscal Year
2007
Total Cost
$48,796
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Mahrus, Sami; Trinidad, Jonathan C; Barkan, David T et al. (2008) Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134:866-76