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Protocol for Biochemical Analysis and Structure Determination of the ZZ Domain of the E3 Ubiquitin Ligase HERC2.

ABSTRACT: Since its discovery, several ligands of the ZZ domain have been identified; however, molecular and structural information underlying binding of these ligands remains limited. Here, we describe a protocol for biochemical and structural analysis of the ZZ domain of human E3 ubiquitin ligase HERC2 (HERC2_ZZ) and its interaction with its ligands: the N-terminal tails of histone H3 and SUMO1. This methodology could be applied for characterization of binding activities of other histone readers. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).

SUBMITTER: Liu J

PROVIDER: S-EPMC7757301 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CUL5-ARIH2 E3-E3 ubiquitin ligase structure reveals cullin-specific NEDD8 activation.

Project description:An emerging mechanism of ubiquitylation involves partnering of two distinct E3 ligases. In the best-characterized E3-E3 pathways, ARIH-family RING-between-RING (RBR) E3s ligate ubiquitin to substrates of neddylated cullin-RING E3s. The E3 ARIH2 has been implicated in ubiquitylation of substrates of neddylated CUL5-RBX2-based E3s, including APOBEC3-family substrates of the host E3 hijacked by HIV-1 virion infectivity factor (Vif). However, the structural mechanisms remained elusive. Here structural and biochemical analyses reveal distinctive ARIH2 autoinhibition, and activation on assembly with neddylated CUL5-RBX2. Comparison to structures of E3-E3 assemblies comprising ARIH1 and neddylated CUL1-RBX1-based E3s shows cullin-specific regulation by NEDD8. Whereas CUL1-linked NEDD8 directly recruits ARIH1, CUL5-linked NEDD8 does not bind ARIH2. Instead, the data reveal an allosteric mechanism. NEDD8 uniquely contacts covalently linked CUL5, and elicits structural rearrangements that unveil cryptic ARIH2-binding sites. The data reveal how a ubiquitin-like protein induces protein-protein interactions indirectly, through allostery. Allosteric specificity of ubiquitin-like protein modifications may offer opportunities for therapeutic targeting.

| S-EPMC8460447 | biostudies-literature

E3 Ubiquitin Ligase TRIP12: Regulation, Structure, and Physiopathological Functions.

Project description:The Thyroid hormone Receptor Interacting Protein 12 (TRIP12) protein belongs to the 28-member Homologous to the E6-AP C-Terminus (HECT) E3 ubiquitin ligase family. First described as an interactor of the thyroid hormone receptor, TRIP12's biological importance was revealed by the embryonic lethality of a murine model bearing an inactivating mutation in the TRIP12 gene. Further studies showed the participation of TRIP12 in the regulation of major biological processes such as cell cycle progression, DNA damage repair, chromatin remodeling, and cell differentiation by an ubiquitination-mediated degradation of key protein substrates. Moreover, alterations of TRIP12 expression have been reported in cancers that can serve as predictive markers of therapeutic response. The TRIP12 gene is also referenced as a causative gene associated to intellectual disorders such as Clark-Baraitser syndrome and is clearly implicated in Autism Spectrum Disorder. The aim of the review is to provide an exhaustive and integrated overview of the different aspects of TRIP12 ranging from its regulation, molecular functions and physio-pathological implications.

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Biochemical properties and in planta effects of NopM, a rhizobial E3 ubiquitin ligase.

Project description:Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

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HECT domain-containing E3 ubiquitin ligase Nedd4 interacts with and ubiquitinates Sprouty2.

Project description:Sprouty (Spry) proteins are important regulators of receptor tyrosine kinase signaling in development and disease. Alterations in cellular Spry content have been associated with certain forms of cancers and also in cardiovascular diseases. Thus, understanding the mechanisms that regulate cellular Spry levels are important. Herein, we demonstrate that Spry1 and Spry2, but not Spry3 or Spry4, associate with the HECT domain family E3 ubiquitin ligase, Nedd4. The Spry2/Nedd4 association involves the WW domains of Nedd4 and requires phosphorylation of the Mnk2 kinase sites, Ser(112) and Ser(121), on Spry2. The phospho-Ser(112/121) region on Spry2 that binds WW domains of Nedd4 is a novel non-canonical WW domain binding region that does not contain Pro residues after phospho-Ser. Endogenous and overexpressed Nedd4 polyubiquitinate Spry2 via Lys(48) on ubiquitin and decrease its stability. Silencing of endogenous Nedd4 increased the cellular Spry2 content and attenuated fibroblast growth factor-elicited ERK1/2 activation that was reversed when elevations in Spry2 levels were prevented by Spry2-specific small interfering RNA. Mnk2 silencing decreased Spry2-Nedd4 interactions and also augmented the ability of Spry2 to inhibit fibroblast growth factor signaling. This is the first report demonstrating the regulation of cellular Spry content and its ability to modulate receptor tyrosine kinase signaling by a HECT domain-containing E3 ubiquitin ligase.

| S-EPMC2804172 | biostudies-literature

HACE1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1)

Project description: Not available

| S-EPMC8547717 | biostudies-literature

Regulation of the MDM2-p53 pathway by the ubiquitin ligase HERC2.

Project description:The p53 tumor suppressor protein is a transcription factor that plays a prominent role in protecting cells from malignant transformation. Protein levels of p53 and its transcriptional activity are tightly regulated by the ubiquitin E3 ligase MDM2, the gene expression of which is transcriptionally regulated by p53 in a negative feedback loop. The p53 protein is transcriptionally active as a tetramer, and this oligomerization state is modulated by a complex formed by NEURL4 and the ubiquitin E3 ligase HERC2. Here, we report that MDM2 forms a complex with oligomeric p53, HERC2, and NEURL4. HERC2 knockdown results in a decline in MDM2 protein levels without affecting its protein stability, as it reduces its mRNA expression by inhibition of its promoter activation. DNA damage induced by bleomycin dissociates MDM2 from the p53/HERC2/NEURL4 complex and increases the phosphorylation and acetylation of oligomeric p53 bound to HERC2 and NEURL4. Moreover, the MDM2 promoter, which contains p53-response elements, competes with HERC2 for binding of oligomeric, phosphorylated and acetylated p53. We integrate these findings in a model showing the pivotal role of HERC2 in p53-MDM2 loop regulation. Altogether, these new insights in p53 pathway regulation are of great interest in cancer and may provide new therapeutic targets.

| S-EPMC6944118 | biostudies-literature

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase.

Project description:Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2-ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

| S-EPMC5638660 | biostudies-literature

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Project description:Cellular homeostasis is governed by the precise expression of genes that control the translation, localization, and termination of proteins. Oftentimes, environmental and biological factors can introduce mutations into the genetic framework of cells during their growth and division, and these genetic abnormalities can result in malignant transformations caused by protein malfunction. For example, p53 is a prominent tumor suppressor protein that is capable of undergoing more than 300 posttranslational modifications (PTMs) and is involved with controlling apoptotic signaling, transcription, and the DNA damage response (DDR). In this review, we focus on the molecular mechanisms and interactions that occur between p53, the HECT E3 ubiquitin ligases WWP1, SMURF1, HECW1 and HERC2, and other oncogenic proteins in the cell to explore how irregular HECT-p53 interactions can induce tumorigenesis.

| S-EPMC8044464 | biostudies-literature

Cryo-EM structure of the chain-elongating E3 ubiquitin ligase UBR5

Project description: Not available

| EMPIAR-11501 | biostudies-other

Structural Diversity of Ubiquitin E3 Ligase

Project description:The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases.

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