Site-specific, intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal.
Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue-residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.
†A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, ‡Department of Chemistry, and §Mass Spectrometry Research Core, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States.
Christopher D
CD
Aluise
†A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, ‡Department of Chemistry, and §Mass Spectrometry Research Core, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States.
Jeannie M
JM
Camarillo
†A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, ‡Department of Chemistry, and §Mass Spectrometry Research Core, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States.
Yuki
Y
Shimozu
†A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, ‡Department of Chemistry, and §Mass Spectrometry Research Core, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States.
James J
JJ
Galligan
†A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, ‡Department of Chemistry, and §Mass Spectrometry Research Core, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States.
Kristie L
KL
Rose
†A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, ‡Department of Chemistry, and §Mass Spectrometry Research Core, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States.
Keri A
KA
Tallman
†A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, ‡Department of Chemistry, and §Mass Spectrometry Research Core, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States.
Lawrence J
LJ
Marnett
D000447:Aldehydes / Q000737:chemistry*; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006801:Humans; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D018384:Oxidative Stress; D019696:Peptidylprolyl Isomerase / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C403894:4-oxo-2-nonenal; D000447:Aldehydes; D003432:Cross-Linking Reagents; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase