Abstract
The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.
Highlights
Ubiquitination is one of the common post-translational modifications of proteins in eukaryotic organisms [1]
Combined with mass spectrometry (LC-MS/MS) analysis and database searches (Fig. S1), we revealed two important enzymes, ubiquitin-activating enzyme E1 (UbE1) and ubiquitin-specific protease 5 (USP5), in the bands around 250 kDa (Fig. 1A)
All proteins identified in the 250-kDa bands are involved in or related to the ubiquitination processes. These proteins could be detected with corresponding antibodies, and all these three antibodies displayed positive results to the high molecular-weight species (Fig. 1B), implying that DCUbP interacts with Ub-activating enzyme E1 (UbE1) or USP5 and Ub plays a role in their associations
Summary
Ubiquitination is one of the common post-translational modifications of proteins in eukaryotic organisms [1]. By working as a versatile regulatory signal controlling protein stability, cellular localization and biological function, ubiquitination plays very important roles in gene regulation, cell cycle, cellular protein level, cell signaling and so on [2,3,4]. In these processes, ubiquitin is covalently attached to a target protein with the cascade participation of three enzymes, Ub-activating enzyme E1 (UbE1), Ub-conjugating enzyme E2 (UbE2) and Ub E3 ligase (UbE3) [1,5]. USP typically cleaves Ub conjugates from ubiquitinated protein substrates or unanchored Ub chains
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