Chemoproteomic Strategy to Quantitatively Monitor Transnitrosation Uncovers Functionally Relevant S-Nitrosation Sites on Cathepsin D and HADH2.
S-Nitrosoglutathione (GSNO) is an endogenous transnitrosation donor involved in S-nitrosation of a variety of cellular proteins, thereby regulating diverse protein functions. Quantitative proteomic methods are necessary to establish which cysteine residues are most sensitive to GSNO-mediated transnitrosation. Here, a competitive cysteine-reactivity profiling strategy was implemented to quantitatively measure the sensitivity of >600 cysteine residues to transnitrosation by GSNO. This platform identified a subset of cysteine residues with a high propensity for GSNO-mediated transnitrosation. Functional characterization of previously unannotated S-nitrosation sites revealed that S-nitrosation of a cysteine residue distal to the 3-hydroxyacyl-CoA dehydrogenase type 2 (HADH2) active site impaired catalytic activity. Similarly, S-nitrosation of a non-catalytic cysteine residue in the lysosomal aspartyl protease cathepsin D (CTSD) inhibited proteolytic activation. Together, these studies revealed two previously uncharacterized cysteine residues that regulate protein function, and established a chemical-proteomic platform with capabilities to determine substrate specificity of other cellular transnitrosation agents.
Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA.
Yani
Y
Zhou
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Sarah L
SL
Wynia-Smith
Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA.
Shalise M
SM
Couvertier
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Kelsey S
KS
Kalous
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA; Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: marletta@berkeley.edu.
Michael A
MA
Marletta
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA. Electronic address: brismith@mcw.edu.
Brian C
BC
Smith
Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA. Electronic address: eranthie@bc.edu.
Eranthie
E
Weerapana
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002402:Cathepsin D / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D061986:MCF-7 Cells; D015538:Nitrosation; D040901:Proteomics
D015094:3-Hydroxyacyl CoA Dehydrogenases; C108634:HSD17B10 protein, human; C533922:CTSD protein, human; D002402:Cathepsin D