Enzyme-mediated damage repair or mitigation, while common for nucleic acids, is rare for proteins. Examples of protein damage are elimination of phosphorylated Ser/Thr to dehydroalanine/dehydrobutyrine (Dha/ Dhb) in pathogenesis and aging. Bacterial LanC enzymes use Dha/Dhb to form carbon-sulfur linkages in antimicrobial peptides, but the functions of eukaryotic LanC-like (LanCL) counterparts are unknown. We show that LanCLs catalyze the addition of glutathione to Dha/Dhb in proteins, driving irreversible C-glutathionylation. Chemo-enzymatic methods were developed to site-selectively incorporate Dha/Dhb at phospho-regulated sites in kinases. In human MAPK-MEK1, such ''elimination damage'' generated aberrantly activated kinases, which were deactivated by LanCL-mediated C-glutathionylation. Surveys of endogenous proteins bearing damage from elimination (the eliminylome) also suggest it is a source of electrophilic reactivity. LanCLs thus remove these reactive electrophiles and their potentially dysregulatory effects from the proteome. As knockout of LanCL in mice can result in premature death, repair of this kind of protein damage appears important physiologically. ll
Through
a “tag-and-modify” protein chemical modification
strategy, we site-selectively phosphorylated the activation
loop of protein kinase p38α. Phosphorylation at natural
(180) and unnatural (172) sites created two pure phospho-forms. p38α
bearing only a single phosphocysteine (pCys) as a mimic of pThr at
180 was sufficient to switch the kinase to an active state, capable
of processing natural protein substrate ATF2; 172 site phosphorylation
did not. In this way, we chemically recapitulated triggering of a
relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric
phosphorylation events catalyzed by p38α and revealed
that site 180 is a sufficient activator alone and engenders dominant
mono-phosphorylation activity. Moreover, a survey of kinase
inhibition using inhibitors with different (Type I/II) modes (including
therapeutically relevant) revealed unambiguously that Type II inhibitors
inhibit phosphorylated p38α and allowed discovery of a
predictive kinetic analysis based on cooperativity to distinguish
Type I vs II.
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