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Mutations in the ‘Fingers’ subdomain of the deubiquitinase USP 1 modulate its function and activity
Author(s) -
OlazabalHerrero Anne,
GarcíaSantisteban Iraia,
Rodríguez Jose Antonio
Publication year - 2016
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13648
Subject(s) - deubiquitinating enzyme , ubiquitin , histone , dna damage , small interfering rna , chemistry , microbiology and biotechnology , biology , dna , biochemistry , rna , gene
Ubiquitin‐specific protease ( USP )1 is a member of the USP family of deubiquitinating enzymes. Efficient USP 1 activity requires binding to its cofactor USP 1‐associated factor 1 ( UAF 1), and the USP 1– UAF 1 deubiquitinase complex has important roles in regulating DNA damage‐related processes. USP s show common folding of their catalytic domain, with three subdomains termed Thumb, Palm, and Fingers. The Fingers subdomain appears to be the primary site for ubiquitin binding. In USP 1, the Fingers subdomain also mediates its interaction with UAF 1, and thus represents a crucial, but poorly characterized, motif in USP 1. To explore the role of USP 1– UAF 1 in ubiquitin‐dependent nuclear processes, we tested the effect of modulating USP 1– UAF 1 activity on the level and/or localization of conjugated ubiquitin and the DNA damage‐related proteins phosphorylated histone H2 AX , Lys56‐acetylated histone H3, and p53‐binding protein 1 (53 BP 1). Small interfering RNA ‐mediated USP 1 knockdown or treatment with the novel USP 1– UAF 1 inhibitor ML 323 increased the recruitment of conjugated ubiquitin and 53 BP 1 into nuclear foci. Strikingly, ectopic coexpression of USP 1 and UAF 1 depleted conjugated ubiquitin in the nucleus and blocked the recruitment of 53 BP 1 to DNA damage foci. In a direct comparison with other overexpressed USP s, USP 1– UAF 1 behaved as a relatively promiscuous deubiquitinase. Experimental and cancer‐related mutations in the USP 1 The Fingers subdomain abrogated substrate deubiquitination without interfering with other USP 1 activities, such as UAF 1 binding or autocleavage. These results provide new insights into the function and regulation of the USP 1– UAF 1 complex.