E3 Ubiquitin Research Articles

Ndfip1 limits TH17 differentiation and proinflammatory cytokine production. (LYM3P.741)

Abstract Ubiquitination and subsequent degradation of pathway proteins is an important way of terminating an effector cell response. Three classes of enzymes: E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases, work sequentially to ubiquitinate selected substrates. Ndfip1 (Nedd4-family interacting protein 1) is a protein that activates the catalytic function of several related E3 ubiquitin ligases. Previous work from our lab showed that mice lacking Ndfip1 have increased numbers of TH17 cells and that Ndfip1 is expressed in T cells. Thus, we hypothesized that Ndfip1 has a T cell-intrinsic role in limiting TH17 cell differentiation. Using a mixed chimera model, we show that T cells that lack Ndfip1 are much more likely to produce IL-17A and GM-CSF, two proinflammatory cytokines produced by TH17 cells. Additionally, Ndfip1-deficient T cells produce more IL-17A and GM-CSF when cultured under TH17 polarizing conditions. These data may help to explain why mice that lack Ndfip1 are more susceptible to IL-17-mediated pathology in a DSS colitis model, compared to their Ndfip1-sufficient counterparts. This work reveals a biologically relevant role for Ndfip1 in limiting TH17 differentiation and GM-CSF production, as well as associated inflammatory pathology. We are now working to identify components of Ndfip1-mediated ubiquitin complexes and determine the underlying mechanism by which Ndfip1 limits proinflammatory cytokine production by TH17 cells.

Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules.

The activation of ubiquitin by the ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent modification of target proteins with ubiquitin. This activation is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to a catalytic cysteine residue of Uba1 and the subsequent adenylation of a second ubiquitin. Here, a ubiquitin E1 structure loaded with two ubiquitin molecules is presented for the first time. While one ubiquitin is bound in its adenylated form to the active adenylation domain of E1, the second ubiquitin represents the status after transfer and is covalently linked to the active-site cysteine. The covalently linked ubiquitin enables binding of the E2 enzyme without further modification of the ternary Uba1-ubiquitin2 arrangement. This doubly loaded E1 structure constitutes a missing link in the structural analysis of the ubiquitin-transfer cascade.

A site-directed mutagenesis study of the MdmX RING domain

Mdm2 and MdmX are important negative regulators of the tumor suppressor p53. Structurally homologous Mdm2 and MdmX inhibit p53 by directly blocking p53 transcriptional activation. Mdm2 also modifies and targets p53 for 26S proteasome dependent protein degradation through E3 ligase activity mediated by its C-terminal RING domain. However, MdmX lacks intrinsic E3 ligase activity and fails to catalyze ubiquitination of p53 despite containing a conserved RING domain. Thus, a comparative structural analysis between the Mdm2 and MdmX RING domains offers a unique way to elucidate the distinct functions of the two proteins in ubiquitination. We performed site-directed mutagenesis of the MdmX RING domain and found that the substitution of the residue N448 for cysteine and the substitution of the residue K478 for arginine granted MdmX RING domain ubiquitination activity. The structural analysis of the Mdm2 and MdmX RING domains revealed that the residue C449 of Mdm2 (structurally homologous to MdmX RING N448) located at the Mdm2 RING dimer interface is critical for the stability of the RING dimer structure, while the residue R479 (structurally homologous to MdmX RING K478) plays a role in recruiting and activating the ubiquitin E2 conjugating enzyme. This study provides new insight into the molecular mechanism of Mdm2 RING domain mediated ubiquitination.

Centrosomes at M phase act as a scaffold for the accumulation of intracellular ubiquitinated proteins

Centrosome size varies considerably during the cell cycle; it is greatest during metaphase, partly because of pericentriolar matrix recruitment and an increase in microtubule-organizing activity. However, the mechanism of centrosome maturation during M phase is poorly defined. In the present study, we identified and quantified centrosomal proteins during S and M phases using stable isotope labeling by amino acids in cell culture (SILAC) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS). We identified 991 proteins, of which 310 and 325 proteins were upregulated during S and M phases, respectively. Ubiquitinated proteins containing K48- and K63-linked polyubiquitin chains accumulated in the centrosomes during M phase, although 26S proteasome activity in the centrosomes did not markedly differ between S and M phases. Conversely, cytoplasmic dynein, which transports ubiquitinated proteins to the centrosomes, increased 2-fold in the centrosomes during M phase relative to S phase. Furthermore, PYR-41, a ubiquitin E1 inhibitor, reduced centrosome size during metaphase, causing increased aneuploidy. RNA interference suppression of Ecm29, which inhibits proteasome activity, decreased the accumulation of ubiquitinated proteins in the centrosomes. These results show that accumulation of ubiquitinated proteins promotes centrosome maturation during M phase and further suggest a novel function of centrosomes as a scaffold temporarily gathering intracellular ubiquitinated proteins.

The E3 Ubiquitin Ligase MARCH6 Degrades Squalene Monooxygenase and Affects 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase and the Cholesterol Synthesis Pathway

The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.

A Unique N-Terminal Sequence in the Carnation Italian ringspot virus p36 Replicase-Associated Protein Interacts with the Host Cell ESCRT-I Component Vps23

Like most positive-strand RNA viruses, infection by plant tombusviruses results in extensive rearrangement of specific host cell organelle membranes that serve as the sites of viral replication. The tombusvirus Tomato bushy stunt virus (TBSV) replicates within spherules derived from the peroxisomal boundary membrane, a process that involves the coordinated action of various viral and cellular factors, including constituents of the endosomal sorting complex required for transport (ESCRT). ESCRT is comprised of a series of protein subcomplexes (i.e., ESCRT-0 -I, -II, and -III) that normally participate in late endosome biogenesis and some of which are also hijacked by certain enveloped retroviruses (e.g., HIV) for viral budding from the plasma membrane. Here we show that the replication of Carnation Italian ringspot virus (CIRV), a tombusvirus that replicates at mitochondrial membranes also relies on ESCRT. In plant cells, CIRV recruits the ESCRT-I protein, Vps23, to mitochondria through an interaction that involves a unique region in the N terminus of the p36 replicase-associated protein that is not conserved in TBSV or other peroxisome-targeted tombusviruses. The interaction between p36 and Vps23 also involves the Vps23 C-terminal steadiness box domain and not its N-terminal ubiquitin E2 variant domain, which in the case of TBSV (and enveloped retroviruses) mediates the interaction with ESCRT. Overall, these results provide evidence that CIRV uses a unique N-terminal sequence for the recruitment of Vps23 that is distinct from those used by TBSV and certain mammalian viruses for ESCRT recruitment. Characterization of this novel interaction with Vps23 contributes to our understanding of how CIRV may have evolved to exploit key differences in the plant ESCRT machinery. Positive-strand RNA viruses replicate their genomes in association with specific host cell membranes. To accomplish this, cellular components responsible for membrane biogenesis and modeling are appropriated by viral proteins and redirected to assemble membrane-bound viral replicase complexes. The diverse pathways leading to the formation of these replication structures are poorly understood. We have determined that the cellular ESCRT system that is normally responsible for mediating late endosome biogenesis is also involved in the replication of the tombusvirus Carnation Italian ringspot virus (CIRV) at mitochondria. Notably, CIRV recruits ESCRT to the mitochondrial outer membrane via an interaction between a unique motif in the viral protein p36 and the ESCRT component Vps23. Our findings provide new insights into tombusvirus replication and the virus-induced remodeling of plant intracellular membranes, as well as normal ESCRT assembly in plants.

A SnapShot of Ubiquitin Chain Elongation: LYSINE 48-TETRA-UBIQUITIN SLOWS DOWN UBIQUITINATION

We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCF(βTrCP)) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF(βTrCP) formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using "elongation intermediates" containing β-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF(βTrCP)/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.

Ubiquitin code assembly and disassembly

Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.

Oligomerization of the Nrdp1 E3 Ubiquitin Ligase Is Necessary for Efficient Autoubiquitination but Not ErbB3 Ubiquitination

Overexpression of the ErbB3 receptor tyrosine kinase protein in breast and other cancers contributes to tumor malignancy and therapeutic resistance. The RBCC/TRIM family RING finger E3 ubiquitin ligase Nrdp1 mediates the ubiquitination of ErbB3 in normal mammary epithelial cells to facilitate receptor degradation and suppress steady-state receptor levels. Post-transcriptional loss of Nrdp1 in patient breast tumors allows ErbB3 overexpression and receptor contribution to tumor progression, and elevated lability through autoubiquitination contributes to the observed loss of Nrdp1 in tumors relative to normal tissue. To begin to understand the mechanisms underlying Nrdp1 protein self-regulation through lability, we investigated the structural determinants required for efficient autoubiquitination and ErbB3 ubiquitination. Using mutagenesis, chemical cross-linking, size exclusion chromatography, and native polyacrylamide gel electrophoresis, we demonstrate that Nrdp1 self-associates into a stable oligomeric complex in cells. Deletion of its coiled-coil domain abrogates oligomerization but does not affect Nrdp1-mediated ErbB3 ubiquitination or degradation. On the other hand, the presence of the coiled-coil domain is necessary for efficient Nrdp1 autoubiquitination via a trans mechanism, indicating that Nrdp1 ubiquitination of its various targets is functionally separable. Finally, a GFP fusion of the coiled-coil domain stabilizes Nrdp1 and potentiates ErbB3 ubiquitination and degradation. These observations point to a model whereby the coiled-coil domain plays a key role in regulating Nrdp1 lability by promoting its assembly into an oligomeric complex, and raise the possibility that inhibition of ligase oligomerization via its coiled-coil domain could be of therapeutic benefit to breast cancer patients by restoring Nrdp1 protein.

Classification and interaction modes of 40 rice E2 ubiquitin-conjugating enzymes with 17 rice ARM-U-box E3 ubiquitin ligases

Rice, a monocot model crop, contains at least 48 putative E2 ubiquitin (Ub)-conjugating enzymes. Based on homology comparisons with 40 Arabidopsis E2 proteins and 35 human E2s, 48 rice E2s were classified into 15 different groups. Yeast two-hybrid analyses using the U-box-domain regions of armadillo (ARM)-U-box E3 Ub-ligases and the Ub-conjugating (UBC) domains of E2s showed that, among 40 rice E2s, 11 E2s accounted for 70% of the interactions with 17 ARM-U-box E3s. Thus, a single E2 could interact with multiple ARM-U-box E3s, suggesting the presence of E2 hubs for E2–E3 interactions in rice. Rice SPL11 ARM-U-box E3 displayed distinct self-ubiquitination patterns, including poly-ubiquitination, mono-ubiquitination, or no ubiquitination, depending on different E2 partners. This suggests that the mode of ubiquitination of SPL11 E3 is critically influenced by individual E2s.

An E4 Ligase Facilitates Polyubiquitination of Plant Immune Receptor Resistance Proteins in Arabidopsis

Proteins with nucleotide binding and leucine-rich repeat domains (NLRs) serve as immune receptors in animals and plants that recognize pathogens and activate downstream defense responses. As high accumulation of NLRs can result in unwarranted autoimmune responses, their cellular concentrations must be tightly regulated. However, the molecular mechanisms of this process are poorly detailed. The F-box protein Constitutive expressor of PR genes 1 (CPR1) was previously identified as a component of a Skp1, Cullin1, F-box protein E3 complex that targets NLRs, including Suppressor of NPR1, Constitutive 1 (SNC1) and Resistance to Pseudomonas syringae 2 (RPS2), for ubiquitination and further protein degradation. From a forward genetic screen, we identified Mutant, snc1-enhancing 3 (MUSE3), an E4 ubiquitin ligase involved in polyubiquitination of its protein targets. Knocking out MUSE3 in Arabidopsis thaliana results in increased levels of NLRs, including SNC1 and RPS2, whereas overexpressing MUSE3 together with CPR1 enhances polyubiquitination and protein degradation of these immune receptors. This report on the functional role of an E4 ligase in plants provides insight into the scarcely understood NLR degradation pathway.

Construction of a mouse Aos1-Uba2 chimeric SUMO-E1 enzyme, mAU, and its expression in baculovirus-insect cells

Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme. The SUMO-E1 enzyme consists of two subunits, a heterodimer of activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2), which resembles the N- and C-terminal halves of ubiquitin E1 (Uba1). Herein, we describe the rational design of a single polypeptide version of SUMO-E1, a chimera of mouse Aos1 and Uba2 subunits, termed mAU, in which the functional domains appear to be arranged in a fashion similar to Uba1. We also describe the construction of a mAU plasmid for expression in a baculovirus-insect cell system and present an in situ SUMOylation assay using the recombinant mAU. Our results showed that mAU has SUMO-E1 activity, thereby indicating that mAU can be expressed in baculovirus-insect cells and represents a suitable source of SUMO-E1.

E3 ubiquitin-ligases and their target proteins during the regulation of plant innate immunity

Reversible protein ubiquitination plays a crucial role during the regulation of plant immune signaling. E3 ubiquitin (Ub)-ligase enzymes, which are classified into different families depending on their structural and functional features, confer the specificity of substrate and are the best characterized components of the ubiquitination cascade. E3 Ub-ligases of different families have been shown to be involved in all steps of plant immune responses. Indeed, they have been involved in the first steps of pathogen perception, as they appear to modulate perception of pathogen-associated molecular patterns by pattern-recognition receptors at the plasma membrane and to regulate the accumulation of nucleotide-binding leucine-rich repeat-type intracellular immune receptors. In addition, E3 Ub-ligase proteins are also involved in the regulation of the signaling responses downstream of pathogen perception through targeting vesicle trafficking components or nuclear transcription factors, for instance. Finally, we also discuss the case of microbial effector proteins that are able to target host E3 Ub-ligases, or to act themselves as E3 Ub-ligases, in their attempt to subvert the host proteasome to promote disease.

A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation.

The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of ∼45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.

The Active Form of E6-associated protein (E6AP)/UBE3A Ubiquitin Ligase Is an Oligomer

Employing 125I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508Å2 and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe(727) and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases kcat 62-fold but fails to affect E2 ubiquitin binding or subsequent formation of the Hect domain Cys(820) ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe(727)-dependent trimer formation and catalytic activity (Ki12 mM), as does a conserved-helical peptide corresponding to residues 474–490 of E6A Pisoform 1 (Ki22M) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe(727) intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (Kactivation 1.5 nM) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the kcat defect of the Phe(727) mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity.

Functional implications of K63-linked ubiquitination in the iron deficiency response of Arabidopsis roots

Iron is an essential micronutrient that plays important roles as a redox cofactor in a variety of processes, many of which are related to DNA metabolism. The E2 ubiquitin conjugase UBC13, the only E2 protein that is capable of catalyzing the formation of non-canonical K63-linked ubiquitin chains, has been associated with the DNA damage tolerance pathway in eukaryotes, critical for maintenance of genome stability and integrity. We previously showed that UBC13 and an interacting E3 ubiquitin ligase, RGLG, affect the differentiation of root epidermal cells in Arabidopsis. When grown on iron-free media, Arabidopsis plants develops root hairs that are branched at their base, a response that has been interpreted as an adaption to reduced iron availability. Mutations in UBC13A abolished the branched root hair phenotype. Unexpectedly, mutations in RGLG genes caused constitutive root hair branching. Based on recent results that link endocytotic turnover of plasma membrane-bound PIN transporters to K63-linked ubiquitination, we reinterpreted our results in a context that classifies the root hair phenotype of iron-deficient plants as a consequence of altered auxin distribution. We show here that UBC13A/B and RGLG1/2 are involved in DNA damage repair and hypothesize that UBC13 protein becomes limited under iron-deficient conditions to prioritize DNA metabolism. The data suggest that genes involved in combating detrimental effects on genome stability may represent essential components in the plant’s stress response.

E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin.

Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities.

UBE2Q1 expression in human colorectal tumors and cell lines

Colorectal cancer is the third most common cancer in the world. Ubiquitin-proteasome system has shown to be activated in colorectal and other malignancies. UBE2Q1 is a novel human gene that encodes a putative E2 ubiquitin conjugating enzyme. Here, we investigated the expression pattern of UBE2Q1 gene in cell lines and tissues from human colorectal tumors. Quantitative (q) RT-PCR were employed to evaluate the expression levels of the mRNA for UBE2Q1 in colorectal cancer cell lines (HT29/219, LS180, SW742, Caco2, HTC116, SW48, SW480 and SW1116). Expression of UBE2Q1 at the protein levels were assessed by Western blotting in cell lines as well as in 43 human colorectal tumor tissues. All cell lines tested expressed UBE2Q1 gene at the level of both mRNA and protein, with the SW1116 line representing the lowest level of expression. The cell lines HT29/219 and SW742 showed the highest levels of UBE2Q1 protein and mRNA respectively. When compared to corresponding normal tissues, malignant parts of colorectal tumors showed increased levels of UBE2Q1 immunoreactivity in 32 (74.42%) of cases. These data suggest that UBE2Q1 is differentially expressed in colorectal cell lines and shows overexpression in colorectal tumors.

The UbL protein UBTD1 stably interacts with the UBE2D family of E2 ubiquitin conjugating enzymes

UBTD1 is a previously uncharacterized ubiquitin-like (UbL) domain containing protein with high homology to the mitochondrial Dc-UbP/UBTD2 protein. Here we show that UBTD1 and UBTD2 belong to a family of proteins that is conserved through evolution and found in metazoa, funghi, and plants. To gain further insight into the function of UBTD1, we screened for interacting proteins. In a yeast-2-hybrid (Y2H) screen, we identified several proteins involved in the ubiquitylation pathway, including the UBE2D family of E2 ubiquitin conjugating enzymes. An affinity capture screen for UBTD1 interacting proteins in whole cell extracts also identified members of the UBE2D family. Biochemical characterization of recombinant UBTD1 and UBE2D demonstrated that the two proteins form a stable, stoichiometric complex that can be purified to near homogeneity. We discuss the implications of these findings in light of the ubiquitin proteasome system (UPS).

The fate of tandemly duplicated genes assessed by the expression analysis of a group of Arabidopsis thaliana RING-H2 ubiquitin ligase genes of the ATL family

Gene duplication events exert key functions on gene innovations during the evolution of the eukaryotic genomes. A large portion of the total gene content in plants arose from tandem duplications events, which often result in paralog genes with high sequence identity. Ubiquitin ligases or E3 enzymes are components of the ubiquitin proteasome system that function during the transfer of the ubiquitin molecule to the substrate. In plants, several E3s have expanded in their genomes as multigene families. To gain insight into the consequences of gene duplications on the expansion and diversification of E3s, we examined the evolutionary basis of a cluster of six genes, duplC-ATLs, which arose from segmental and tandem duplication events in Brassicaceae. The assessment of the expression suggested two patterns that are supported by lineage. While retention of expression domains was observed, an apparent absence or reduction of expression was also inferred. We found that two duplC-ATL genes underwent pseudogenization and that, in one case, gene expression is probably regained. Our findings provide insights into the evolution of gene families in plants, defining key events on the expansion of the Arabidopsis Tóxicos en Levadura family of E3 ligases.