ADP-ribosylation proceeds from nicotinamide adenine dinucleotide (NAD +) as a donor of the ADP-ribosyl (ADPr), which is attached to a protein substrate via the 1′ carbon of the adenine-distal ribose (hitherto called C1″), accompanied by simultaneous departure of the activating nicotinamide moiety ( Fig. Ubiquitylation is in close cross-talk with other PTMs, including protein adenosine diphosphate (ADP)–ribosylation ( 16, 17). In addition, we determined the hydrolase specificity profile of this modification, identifying human and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enzymes that could reverse it in cells. Comparative analysis of known hydroxyl-ubiquitylating active sites points to the recurring use of a catalytic histidine residue, which, in DELTEX E3s, is potentiated by a glutamate in a catalytic triad-like manner. However, DELTEXes follow a previously unidentified paradigm for RING E3s, whereby the ligase not only forms a scaffold but also provides catalytic residues to activate the acceptor. Unlike other known hydroxyl-specific E3s, which proceed via a covalent E3~ubiqutin intermediate, DELTEX enzymes are RING E3s that stimulate a direct ubiquitin transfer from E2~ubiquitin onto a substrate.
Here, we show that DELTEX E3 ligases specifically target the 3′ hydroxyl of the adenosine diphosphate (ADP)–ribosyl moiety that can be linked to a protein, thus generating a hybrid ADP-ribosyl-ubiquitin modification. Ubiquitylation had been considered limited to protein lysine residues, but other substrates have recently emerged.
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