Mechanism of Ubiquitin Chain Synthesis Employed by a HECT Ubiquitin Ligase [Protein Synthesis and Degradation]

Homologous to E6AP Carboxyl Terminus (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing K63, K48, and K11 linkages (K63 > K48 > K11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addi-tion mechanism, in which Ub monomers are trans-ferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the pres-ence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and linked exclusively through K63 of Ub. In the second phase, chains are elongated in a multidirec-tional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for under-standing how this family of E3s generates Ub sig-nals that function in proteasome-independent and proteasome-dependent pathways.

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