2 research outputs found
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 <i>cis</i>/<i>trans</i> 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. <i>In vitro</i> 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
Quantitative Analysis and Discovery of Lysine and Arginine Modifications
Post-translational
modifications (PTMs) affect protein function,
localization, and stability, yet very little is known about the ratios
of these modifications. Here, we describe a novel method to quantitate
and assess the relative stoichiometry of Lys and Arg modifications
(QuARKMod) in complex biological settings. We demonstrate the versatility
of this platform in monitoring recombinant protein modification of
peptide substrates, PTMs of individual histones, and the relative
abundance of these PTMs as a function of subcellular location. Lastly,
we describe a product ion scanning technique that offers the potential
to discover unexpected and possibly novel Lys and Arg modifications.
In summary, this approach yields accurate quantitation and discovery
of protein PTMs in complex biological systems without the requirement
of high mass accuracy instrumentation