Structural basis and specificity of human otubain 1-mediated deubiquitination

Abstract

OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its close homologue, OTUB2, suggesting variations in regulatory mechanisms and substrate specificity. Biochemical analysis reveals that OTUB1 has a preference for cleaving Lys(48)-linked polyubiquitin chains over Lys(63)-linked polyubiquitin chains, and it is capable of cleaving NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), but not SUMO (small ubiquitin-related modifier) 1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates. A functional comparison of OTUB1 and OTUB2 indicated a differential reactivity towards ubiquitin-based active-site probes carrying a vinyl methyl ester, a 2-chloroethyl or a 2-bromoethyl group at the C-terminus. Mutational analysis suggested that a narrow P1' site, as observed in OTUB1, correlates with its ability to preferentially cleave Lys(48)-linked ubiquitin chains. Analysis of cellular interaction partners of OTUB1 by co-immunoprecipitation and MS/MS (tandem mass spectrometry) experiments demonstrated that FUS [fusion involved in t(12;6) in malignant liposarcoma; also known as TLS (translocation in liposarcoma) or CHOP (CCAAT/enhancer-binding protein homologous protein)] and RACK1 [receptor for activated kinase 1; also known as GNB2L1 (guanine-nucleotide-binding protein beta polypeptide 2-like 1)] are part of OTUB1-containing complexes, pointing towards a molecular function of this deubiquitinating enzyme in RNA processing and cell adhesion/morphology.

Activated Protein C Ameliorates Renal Ischemia-Reperfusion Injury by Restricting Y-Box Binding Protein-1 Ubiquitination

“…[41][42][43] Although our immunoprecipitation experiments demonstrate that OTUB1 and YB-1 act in close proximity to each other, we currently cannot differentiate which of these two alternative mechanisms inhibit ubiquitination of YB-1. Interestingly, OTUB1 has been proposed to preferentially inhibit K48-linked ubiquitination, 40,44 which may specifically avert proteasomal degradation of target proteins, such as YB-1, while still allowing other ubiquitinationdependent processes. Future studies are required to decipher the concise molecular nature of the OTUB1 and YB-1 interaction and the consequences for YB-1 abundance, localization, and activity.…”

PTEN regulates RPA1 and protects DNA replication forks

“…For modeling of the PTEN/RPA and PTEN/OTUB1 complexes, we separately submitted the crystal structure of PTEN (PDB:1D5R) as a receptor and RPA RPA1(439-616)/RPA2(43-171)/RPA3 [23] or OTUB1 (PDB: 2ZFY) [28] as ligand to the web-based ClusPro2.0 docking server (http://cluspro.bu.edu/). This docking program filters docked conformations with good surface and charge complementarity and ranks them based on their clustering properties [25].…”

“…To confirm the structural linkage of these molecules, in silico analysis was used to evaluate docking of elements of the OTUB1 structure [28] with the PTEN crystal structure [24]. In the PTEN/OTUB1 complex, OTUB1 binds to the PTEN C2 domain through complimentary electrostatic charge distributions to within a distance of 3.0 Å (Supplementary information, Figure S4F; PTEN is in green and OTUB1 in yellow).…”

Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser 16 to trigger its nuclear localization

“…Therefore, it is likely that their activity, target recognition as well as subcellular localization are tightly regulated. OTUB1 protein is detected ubiquitously in tissues and recent reports have shed light into the molecular functions of OTUB1 in deubiquitylating K48-linked ubiquitin chains as well as inhibiting the action of E2 ubiquitin-conjugating enzymes (1,(3)(4)(5)(6)(7)(8)(9)(10). OTUB1 has been reported to target many proteins for deubiquitylation, including TNF receptor-associated factors 3/6 (TRAF3/6) (11), estrogen receptor α (ERα) (12), the tumor suppressor protein p53 (13), and the cellular inhibitor of apoptosis c-IAP1 (14).…”