Abstract
The reverse reaction of ubiquitylation is catalyzed by different classes of deubiquitylation enzymes (DUBs), including ovarian tumor domain (OTU)-containing DUBs; experiments using Homo sapiens proteins have demonstrated that OTU DUBs modulate various cellular processes. With the exception of OTLD1, plant OTU DUBs have not been characterized. We identified 12 Arabidopsis thaliana OTU loci and analyzed 11 of the encoded proteins in vitro to determine their preferences for the ubiquitin (UB) chains of M1, K48, and K63 linkages as well as the UB-/RUB-/SUMO-GST fusions. The A. thaliana OTU DUBs were shown to be cysteine proteases and classified into four groups with distinct linkage preferences: OTU1 (M1 = K48 > K63), OTU3/4/7/10 (K63 > K48 > M1), OTU2/9 (K48 = K63), and OTU5/11/12/OTLD1 (inactive). Five active OTU DUBs (OTU3/4/7/9/10) also cleaved RUB fusion. OTU1/3/4 cleaved M1 UB chains, suggesting a possible role for M1 chains in plant cellular signaling. The different substrate specificities of the various A. thaliana OTU DUBs indicate the involvement of distinct structural elements; for example, the OTU1 oxyanion residue D89 is essential for cleaving isopeptide bond-linked chains but dispensable for M1 chains. UB-binding activities were detected only for OTU2 and OTLD1, with distinct linkage preferences. These differences in biochemical properties support the involvement of A. thaliana OTU DUBs in different functions. Moreover, based on the established phylogenetic tree, plant- and H. sapiens-specific clades exist, which suggests that the proteins within these clades have taxa-specific functions. We also detected five OTU clades that are conserved across species, which suggests that the orthologs in different species within each clade are involved in conserved cellular processes, such as ERAD and DNA damage responses. However, different linkage preferences have been detected among potential cross-species OTU orthologs, indicating functional and mechanistic differentiation.
Highlights
THALIANA HAS AN ovarian tumor domain (OTU)-deubiquitylation enzymes (DUBs) FAMILY WITH 12 PHYLOGENETICALLY DISTINCT MEMBERS To characterize A. thaliana OTU-DUBs, 12 loci, OTU1-5, OTLD1, and OTU7-12, encoding OTU domain-containing proteins were identified through database searches using the OTU domain sequences for H. sapiens OTUB1 (NP_060140) and OTUB2 (NP_075601) as well as S. cerevisiae Otu1 (P43558) as the initial queries (Table 1)
Based on the splicing products detected, a single isoform exists for the OTU1-3, OTU9-10, and OTU12 loci; two isoforms exist for the OTU5, OTLD1, OTU7, and OTU11 loci; and five isoforms exist for OTU4 (OTU4a-e) (Table 1; Figure S1)
Because binding preferences for UB chains with different linkages may contribute to substrate selectivity or intracellular targeting, we examined the association between the A. thaliana OTU proteins and K48- and K63-linked UB chains in vitro through pull-down assays using the GST-fusion proteins (Table S1)
Summary
Activity, interaction, or subcellular localization of critical regulatory and mechanistic components, the covalent attachment and removal of ubiquitin (UB), i.e., ubiquitylation and deubiquitylation, are essential mechanistic and regulatory elements of numerous cellular processes, such as chromatin silencing, transcriptional activation, mRNA splicing and export, cell division, DNA damage response, intracellular trafficking, and signal transduction (Hershko and Ciechanover, 1998; Komander et al, 2009; Reyes-Turcu et al, 2009).The close connection between reversible ubiquitylation and almost all aspects of cellular processes and organismic functions is primarily due to the many components involved, which include substrates, conjugation enzymes, deconjugation enzymes, and diverse signals derived from the small but highly conserved UB proteins and their cognate binding partners, which decipher the signals (Smalle and Vierstra, 2004; Fu et al, 2010). Activity, interaction, or subcellular localization of critical regulatory and mechanistic components, the covalent attachment and removal of ubiquitin (UB), i.e., ubiquitylation and deubiquitylation, are essential mechanistic and regulatory elements of numerous cellular processes, such as chromatin silencing, transcriptional activation, mRNA splicing and export, cell division, DNA damage response, intracellular trafficking, and signal transduction (Hershko and Ciechanover, 1998; Komander et al, 2009; Reyes-Turcu et al, 2009). The numerous protein-interacting interfaces of UB and attachment as a monomer or polymers with distinct linkages enables UB assembly on substrates to yield diverse signals (Komander and Rape, 2012). While K48-linked UB chains target modified proteins for proteasomal degradation (Chau et al, 1989), K63-linked chains are critical for signaling complex assembly, endocytosis, and DNA damage responses (Chen and Sun, 2009). The cellular functions of the other atypical chains remain elusive (Chen and Sun, 2009; Kulathu and Komander, 2012)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
