RING-type E3 Ubiquitin Ligase Research Articles

PROTACs: A novel strategy for cancer drug discovery and development.

Proteolysis targeting chimera (PROTAC) technology has become a powerful strategy in drug discovery, especially for undruggable targets/proteins. A typical PROTAC degrader consists of three components: a small molecule that binds to a target protein, an E3 ligase ligand (consisting of an E3 ligase and its small molecule recruiter), and a chemical linker that hooks first two components together. In the past 20 years, we have witnessed advancement of multiple PROTAC degraders into the clinical trials for anticancer therapies. However, one of the major challenges of PROTAC technology is that only very limited number of E3 ligase recruiters are currently available as E3 ligand for targeted protein degradation (TPD), although human genome encodes more than 600 E3 ligases. Thus, there is an urgent need to identify additional effective E3 ligase recruiters for TPD applications. In this review, we summarized the existing RING-type E3 ubiquitin ligase and their small molecule recruiters that act as effective E3 ligands of PROTAC degraders and their application in anticancer drug discovery. We believe that this review could serve as a reference in future development of efficient E3 ligands of PROTAC technology for cancer drug discovery and development.

The E3 ubiquitin ligase OsRGLG5 targeted by the Magnaporthe oryzae effector AvrPi9 confers basal resistance against rice blast

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases of rice. During infection, M. oryzae secretes effectors to facilitate blast development. Among these effectors, the avirulence factor AvrPi9 is recognized by Pi9, a broad-spectrum blast resistance protein that triggers Pi9-mediated resistance in rice. However, little is known about the interaction between AvrPi9 and Pi9 and how AvrPi9 exerts virulence to promote infection. In this study, we found that ectopic expression of AvrPi9 in the Pi9-lacking cultivar TP309 suppressed basal resistance against M. oryzae. Furthermore, we identified an AvrPi9-interacting protein in rice, which we named OsRGLG5, encoding a functional RING-type E3 ubiquitin ligase. During infection, AvrPi9 was ubiquitinated and degraded by OsRGLG5. Meanwhile, AvrPi9 affected the stability of OsRGLG5. Infection assays revealed that OsRGLG5 is a positive regulator of basal resistance against M. oryzae, but it is not essential for Pi9-mediated blast resistance in rice. In conclusion, our results revealed that OsRGLG5 is targeted by the M. oryzae effector AvrPi9 and positively regulates basal resistance against rice blast.

A Novel Recognition by the E3 Ubiquitin Ligase of HSV-1 ICP0 Enhances the Degradation of PML Isoform I to Prevent ND10 Reformation in Late Infection.

Upon viral entry, components of ND10 nuclear bodies converge with incoming DNA to repress viral expression. The infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) contains a RING-type E3 ubiquitin ligase that targets the ND10 organizer, PML, for proteasomal degradation. Consequently, ND10 components are dispersed and viral genes are activated. Previously, we reported that ICP0 E3 differentiates two similar substrates, PML isoforms I and II, and demonstrated that SUMO-interaction has profound regulatory effects on PML II degradation. In the present study, we investigated elements that regulate the PML I degradation and found that: (i) two regions of ICP0 flanking the RING redundantly facilitate the degradation of PML I; (ii) downstream of the RING, the SUMO-interaction motif located at residues 362-364 (SIM362-364) targets the SUMOylated PML I in the same manner as that of PML II; (iii) upstream of the RING, the N-terminal residues 1-83 mediate PML I degradation regardless of its SUMOylation status or subcellular localization; (iv) the reposition of residues 1-83 to downstream of the RING does not affect its function in PML I degradation; and (v) the deletion of 1-83 allows the resurgence of PML I and reformation of ND10-like structures late in HSV-1 infection. Taken together, we identified a novel substrate recognition specific for PML I, by which ICP0 E3 enforces a continuous PML I degradation throughout the infection to prevent the ND10 reformation.

SUMOylation of RNF146 results in Axin degradation and activation of Wnt/β-catenin signaling to promote the progression of hepatocellular carcinoma.

Aberrant SUMOylation contributes to the progression of hepatocellular carcinoma (HCC), yet the molecular mechanisms have not been well elucidated. RING-type E3 ubiquitin ligase RNF146 is a key regulator of the Wnt/β-catenin signaling pathway, which is frequently hyperactivated in HCC. Here, it is identified that RNF146 can be modified by SUMO3. By mutating all lysines in RNF146, we found that K19, K61, K174 and K175 are the major sites for SUMOylation. UBC9/PIAS3/MMS21 and SENP1/2/6 mediated the conjugation and deconjugation of SUMO3, respectively. Furthermore, SUMOylation of RNF146 promoted its nuclear localization, while deSUMOylation induced its cytoplasmic localization. Importantly, SUMOylation promotes the association of RNF146 with Axin to accelerate the ubiquitination and degradation of Axin. Intriguingly, only UBC9/PIAS3 and SENP1 can act at K19/K175 in RNF146 and affect its role in regulating the stability of Axin. In addition, inhibiting RNF146 SUMOylation suppressed the progression of HCC both in vitro and in vivo. And, patients with higher expression of RNF146 and UBC9 have the worst prognosis. Taken together, we conclude that RNF146 SUMOylation at K19/K175 promotes its association with Axin and accelerates Axin degradation, thereby enhancing β-catenin signaling and contributing to cancer progression. Our findings reveal that RNF146 SUMOylation is a potential therapeutic target in HCC.

Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded proteins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Overexpression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3). Elevated Doa10 or Hrd1 (but not Asi1) RING activity at the ER membrane resulting from protein overexpression inhibits the degradation of specific Doa10 substrates. The ERAD E2 ubiquitin-conjugating enzyme Ubc6 becomes limiting under these conditions, and UBC6 overexpression restores Ubc6-mediated ERAD. Using a subset of the dominant-negative mutants, which contain the Doa10 RING domain but lack the E2-binding region, we show that they induce degradation of membrane tail-anchored Ubc6 independently of endogenous Doa10 and the other ERAD E3 complexes. This remains true even if the cells lack the Dfm1 rhomboid pseudoprotease, which is also a proposed retrotranslocon. Hence, rogue RING activity at the ER membrane elicits a highly specific off-pathway defect in the Doa10 pathway, and the data point to an additional ERAD E3-independent retrotranslocation mechanism.

JUL1, Ring-Type E3 Ubiquitin Ligase, Is Involved in Transcriptional Reprogramming for ERF15-Mediated Gene Regulation.

JAV1-associated ubiquitin ligase 1 (JUL1) is a RING-type E3 ubiquitin ligase that catalyzes ubiquitination of JAV1, a jasmonate signaling repressor, in Arabidopsis thaliana in response to herbivore attack. Here we present a new insight into the nature of JUL1 as a multi-targeting enzyme for not only JAV1 but also transcription factors (TFs) screened using in vitro and in vivo protein interaction assays. Reporter assays using protoplasts showed that the JUL1-interacting TFs (JiTFs), including ERF15, bZIP53 and ORA59, were involved in transcriptional activation of jasmonate-responsive PDF1.2 and abscisic acid-responsive GEA6. Likewise, assays using mutant plants suggested that the 3 JiTFs were indeed responsible for transcriptional regulation of PDF1.2 and/or GEA6, and ERF15 and ORA59 were substantially responsible for the anti-herbivore trait. In vitro protein ubiqutination assays showed that JUL1 catalyzed ubiqutination of JAV1 but not any of the TFs. This was in accord with the finding that JUL1 abolished JAV1's interference with ERF15 function, according to the reporter assay. Moreover, of great interest is our finding that ERF15 but not bZIP53 or ORA59 serves as a scaffold for the JAV1/JUL1 system, indicating that there is narrow selectivity of the transcriptional reprogramming by the JAV1/JUL1 system.

A grape VvHOS1-interacting HIPP protein (VvHIPP21) negatively regulates cold and drought stress

Heavy metal-associated isoprenylated plant proteins (HIPPs) are metallochaperones that contribute significantly to heavy metal homeostasis and detoxification processes. However, their specific regulatory roles in abiotic stresses are largely limited. Here, we show evidence that a HIPP21 from Vitis vinifera (named as VvHIPP21) is a novel functional component of the RING-type E3 ubiquitin ligase HOS1, which is recognized to be a key negative regulator of the cold signaling. VvHIPP21 gene is substantially suppressed in response to cold and ABA, but elevated by drought, and its product localized in both the cytoplasm and nucleus. Ectopic expression of 35 S:VvHIPP21 in Arabidopsis might alleviate the metal ion stress and the relative toxicity of Cu2+ and Cd2+, respectively. Notably, overexpression of VvHIPP21 alone or co-expression of VvHOS1 and VvHIPP21 greatly increase cold sensitivity in yeast, as evidenced by the survival rate of yeast cells under freezing stress conditions. Overexpression of VvHIPP21 in Arabidopsis resulted in cold sensitivity by down-regulating the cold-related genes of CBF-dependent pathway, relative to wild-type plants. In addition, VvHIPP21-overexpressing plants exhibited attenuated drought tolerance, as shown by an increased water loss rate, expanded stomatal aperture, accumulation of reactive oxygen species (ROS), and altered expression of ABA-dependent drought-responsive genes. Our results show that VvHIPP21 is favorably implicated in heavy metal homeostasis and detoxification, but adversely affects the cold signal pathway and ABA-mediated drought tolerance. Our findings shed light on how plants control growth and development in response to heavy metal and abiotic stresses.

Diverse functions of GmNLA1 members in controlling phosphorus homeostasis highlight coordinate response of soybean to nitrogen and phosphorus availability

Nitrogen (N) and phosphorus (P) are two essential mineral nutrients for plant growth, which are required in relative high amount in plants. Plants have evolved a series of strategies for coordinately acquiring and utilizing N and P. However, physiological and molecular mechanisms underlying of N and P interactions remain largely unclear in soybean (Glycine max). In this study, interactions of N and P were demonstrated in soybean as reflected by significant increases of phosphate (Pi) concentration in both leaves and roots by N deficiency under Pi sufficient conditions. A total of four nitrogen limitation adaptation (NLA), encoding RING-type E3 ubiquitin ligase were subsequently identified in soybean genome. Among them, transcription of GmNLA1-1 and GmNLA1-3 was decreased in soybean by N starvation under Pi sufficient conditions, not for GmNLA1-2. Suppression of all three GmNLA1 members was able to increase Pi concentration regardless of the P and N availability in the growth medium, but decrease fresh weight under normal conditions in soybean hairy roots. However, comparted to changes in control lines at two N levels, N deficiency only resulted in a relatively higher increase of Pi concentration in GmNLA1-1 or GmNLA1-3 suppression lines, strongly indicating that GmNLA1-1 and GmNLA1-3 might regulate P homeostasis in soybean response to N starvation. Taken together, our result suggest that redundant and diverse functions present in GmNLA1 members for soybean coordinate responses to P and N availability, which mediate P homeostasis.

RING-Type E3 Ubiquitin Ligases AtRDUF1 and AtRDUF2 Positively Regulate the Expression of PR1 Gene and Pattern-Triggered Immunity.

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae.

TRIM13 Is a Novel Cell Cycle Hub Regulated By CHAF1B to Promote Acute Myeloid Leukemogenesis

CHAF1B (Chromatin Assembly Factor 1B) functions as a histone chaperone within the DNA replication machinery. It is almost universally overexpressed in cancers, and we have previously shown that this upregulation is necessary to enforce the differentiation block found in acute myeloid leukemia (AML) through CHAF1B's chromatin binding function. In this study, we found that CHAF1B creates a pro-leukemic state in hematopoietic cells by repressing the expression of the E3 ubiquitin ligase TRIM13. Our data suggest that TRIM13 is a strong promoter of entry into the cell cycle functioning through novel nuclear localization and activating ubiquitination of key cell cycle genes. We propose that repression of TRIM13 by CHAF1B is a critical event for leukemogenesis because unchecked entry into cell cycle leads to loss of self-renewal and eventual exhaustion of the tumor. First, we measured the expression levels of CHAF1B and TRIM13 in several bone marrow aspirates from primary pediatric AML patients using immunohistochemistry. TRIM13 was expressed in healthy marrow and almost undetectable upon leukemic progression. CHAF1B, on the other hand, had low to moderate expression in healthy tissues and substantially higher expression upon leukemic progression and relapse. We found restoration of healthy CHAF1B and TRIM13 expression levels upon resolution of the tumor in matched patient aspirates upon remission. To determine if CHAF1B is capable of directly repressing TRIM13 expression, first we observed CHAF1B occupancy at the TRIM13 promoter by ChIPseq in multiple AML cell lines. Then we found loss of CHAF1B by shRNA resulted in increased TRIM13 transcription, and overexpression of CHAF1B by dox-inducible lentivirus drove TRIM13 transcriptional repression. Overexpression of a CHAF1B chromatin binding deficient mutant (CHAF1BRRAA) was unable to repress TRIM13, suggesting that CHAF1B chromatin binding is necessary for TRIM13 repression. This led us to hypothesize that TRIM13 may be a novel tumor suppressor in AML. To test this hypothesis, we overexpressed TRIM13 in AML cell lines and several patient-derived xenografts (PDXs) spanning many subtypes of AML and observed consistent repression of colony formation, increase in apoptosis, and reduction of leukemic progression in vivo. Conversely, loss of TRIM13 expression by shRNA led to an increase in colony formation in vitro and enhanced leukemogenicity of PDXs and AML cell lines in vivo. To better understand the mechanistic contribution of TRIM13 to myeloid leukemogenesis, we started by identifying potential catalytic targets of TRIM13. Since TRIM13 is a RING-type E3 ubiquitin ligase, we used CRISPR to generate in-frame RING domain deleted alleles of TRIM13 (T13RINGdel) and performed ubiquitin proteomics comparing our T13RINGdel and TRIM13 WT clones. We expected that loss of the catalytic function would lead to stabilization of pro-leukemic proteins as has been shown in solid tumor models. We did not find that, but we did observe a profound reduction in ubiquitination of proteins associated with the cell cycle, including CCNA1, CCNA2, CCNB2, and CDK1. Additionally, the total levels of these cell cycle-promoting proteins was reduced in T13RINGdel cells suggesting the ubiquitination event leads to their stability and activity. We confirmed that our T13RINGdel clones of AML cell lines had reduced entry and progression through cell cycle. Using imaging cytometry, we found a novel nuclear localization of TRIM13 and an expression pattern characterized by increased TRIM13 expression as cell cycle progressed. We confirmed the stabilization/activation relationship between TRIM13 and CCNA1 by restoring CCNA1 expression and confirming loss of the colony formation benefit in T13RINGdel AML cell lines. This study is the first to link CHAF1B, a cell cycle gene, with transcriptional repression of genes that promote cell cycle progression. We believe this relationship is critical for AML cells to support high levels of CHAF1B expression, thereby promoting an immature leukemic state throughout replication, without premature entry into multiple damaging rounds of proliferation.

Comparative structural analyses of the NHL domains from the human E3 ligase TRIM-NHL family.

Tripartite motif (TRIM) proteins constitute one of the largest subfamilies of the RING-type E3 ubiquitin ligases that play a role in diverse processes from homeostasis and immune response to viral restriction. While TRIM proteins typically harbor an N-terminal RING finger, a B-box and a coiled-coil domain, a high degree of diversity lies in their C termini that contain diverse protein interaction modules, most of which, both structures and their roles in intermolecular interactions, remain unknown. Here, high-resolution crystal structures of the NHL domains of three of the four human TRIM-NHL proteins, namely TRIM2, TRIM3 and TRIM71, are presented. Comparative structural analyses revealed that, despite sharing an evolutionarily conserved six-bladed β-propeller architecture, the low sequence identities resulted in distinct properties of these interaction domains at their putative binding sites for macromolecules. Interestingly, residues lining the binding cavities represent a hotspot for genetic mutations linked to several diseases. Thus, high sequence diversity within the conserved NHL domains might be essential for differentiating binding partners among TRIM-NHL proteins.

PpMYB52 negatively regulates peach bud break through the gibberellin pathway and through interactions with PpMIEL1.

Bud dormancy, which enables damage from cold temperatures to be avoided during winter and early spring, is an important adaptive mechanism of deciduous fruit trees to cope with seasonal environmental changes and temperate climates. Understanding the regulatory mechanism of bud break in fruit trees is highly important for the artificial control of bud break and the prevention of spring frost damage. However, the molecular mechanism underlying the involvement of MYB TFs during the bud break of peach is still unclear. In this study, we isolated and identified the PpMYB52 (Prupe.5G240000.1) gene from peach; this gene is downregulated in the process of bud break, upregulated in response to ABA and downregulated in response to GA. Overexpression of PpMYB52 suppresses the germination of transgenic tomato seeds. In addition, Y2H, Bimolecular fluorescence complementation (BiFC) assays verified that PpMYB52 interacts with a RING-type E3 ubiquitin ligase, PpMIEL1, which is upregulated during bud break may positively regulate peach bud break by ubiquitination-mediated degradation of PpMYB52. Our findings are the first to characterize the molecular mechanisms underlying the involvement of MYB TFs in peach bud break, increasing awareness of dormancy-related molecules to avoid bud damage in perennial deciduous fruit trees.

Grass Carp Mex3A Promotes Ubiquitination and Degradation of RIG-I to Inhibit Innate Immune Response.

As one of the Mex3 family members, Mex3A is crucial in cell proliferation, migration, and apoptosis in mammals. In this study, a novel gene homologous to mammalian Mex3A (named CiMex3A, MW368974) was cloned and identified in grass carp, which is 1,521 bp in length encoding a putative polypeptide of 506 amino acids. In CIK cells, CiMex3A is upregulated after stimulation with LPS, Z-DNA, and especially with intracellular poly(I:C). CiMex3A overexpression reduces the expressions of IFN1, ISG15, and pro-inflammatory factors IL8 and TNFα; likewise, Mex3A inhibits IRF3 phosphorylation upon treatment with poly(I:C). A screening test to identify potential targets suggested that CiMex3A interacts with RIG-I exclusively. Co-localization analysis showed that Mex3A and RIG-I are simultaneously located in the endoplasmic reticulum, while they rarely appear in the endosome, mitochondria, or lysosome after exposure to poly(I:C). However, RIG-I is mainly located in the early endosome and then transferred to the late endosome following stimulation with poly(I:C). Moreover, we investigated the molecular mechanism underlying CiMex3A-mediated suppression of RIG-I ubiquitination. The results demonstrated that Mex3A truncation mutant (deletion in the RING domain) can still interact physically with RIG-I, but fail to degrade it, suggesting that Mex3A also acts as a RING-type E3 ubiquitin ligase. Taken together, this study showed that grass carp Mex3A can interact with RIG-I in the endoplasmic reticulum following poly(I:C) stimulation, and then Mex3A facilitates the ubiquitination and degradation of RIG-I to inhibit IRF3-mediated innate antiviral immune response.

RNF216 Alleviates Radiation-Induced Apoptosis and DNA Damage Through Regulating Ubiquitination-Mediated Degradation of p53 in Glioblastoma

Glioblastoma (GBM) is the most common and lethal subtype of glioma, characterized by uncontrolled cancer cell proliferation, extensive infiltration, and therapeutic resistance. Ring finger protein 216 (RNF216) is a RING-type E3 ubiquitin ligase aberrantly expressed in multiple human cancers. Tumor protein 53 (p53) is a transcription factor that acts as a tumor suppressor. This study aimed to compare the RNF216 expression in GBM tissues and normal peritumoral tissues and to examine the effects of RNF216 overexpression/knockdown on tumorigenesis, radioresistance, and the p53 pathway in GBM. The results showed that RNF216 was overexpressed in GBM tissues and cell lines, and high RNF216 expression was related to a poor prognosis. RNF216 overexpression promoted GBM cell growth and inhibited apoptosis, while RNF216 knockdown impaired GBM cell growth and enhanced cell death. RNF216 was also highly expressed in recurrent GBM tissues compared with paired primary tumors. The upregulation of RNF216 not only facilitated GBM cell growth but also protected cells against X-ray irradiation-induced apoptosis and DNA damage, while RNF216 knockdown exerted opposite effects. Moreover, the implantation of GBM cells with RNF216 silencing suppressed tumorigenesis and increased radiosensitivity of mice bearing GBM xenografts. Further analysis revealed that RNF216 overexpression reduced the stability of p53 protein via ubiquitination and negatively regulated the p53 pathway, while RNF216 knockdown preserved the p53 protein. In conclusion, RNF216 effectively attenuated radiation-induced apoptosis and DNA damage in GBM via inducing ubiquitination-mediated degradation of p53. These findings suggest the potential therapeutic use of RNF216 inhibition for tumorigenesis and therapeutic resistance in GBM.

The ABI3 Transcription Factor Interaction and Antagonism with Ubiquitin E3 Ligase ScPRT1 in Syntrichia caninervis.

The ubiquitination pathway has been found to regulate plant responses to environmental stress. However, the role of E3 ubiquitin ligase in desiccation tolerant moss has not yet been elucidated. Previous research has shown that the abscisic acid (ABA) signaling factor ScABI3 can significantly increase desiccation tolerance and reduce ABA sensitivity in the desert moss Syntrichia caninervis. In this study, we identified a RING-type E3 ubiquitin ligase, ScPRT1, and showed that ScABI3 can directly interact with ScPRT1 in vitro and in vivo. Furthermore, we found that the high expression of ScPRT1 can interfere with the transcription of ScABI3 under ABA treatment. Therefore, we speculate that ScPRT1 may degrade ScABI3 through the ubiquitin-26S proteasome system and participate in ABA-dependent signaling in response to ABA-insensitivity or desiccation tolerance in S. caninervis. The findings from our study may enrich our knowledge of the role of E3 ubiquitin ligase in desiccation tolerance and lay a theoretical foundation for an in-depth study of the relationship between ubiquitination modification and ABA signal transduction under environmental stress.

Silencing an E3 Ubiquitin Ligase Gene OsJMJ715 Enhances the Resistance of Rice to a Piercing-Sucking Herbivore by Activating ABA and JA Signaling Pathways.

The RING-type E3 ubiquitin ligases play an important role in plant growth, development, and defense responses to abiotic stresses and pathogens. However, their roles in the resistance of plants to herbivorous insects remain largely unknown. In this study, we isolated the rice gene OsJMJ715, which encodes a RING-domain containing protein, and investigated its role in rice resistance to brown planthopper (BPH, Nilaparvata lugens). OsJMJ715 is a nucleus-localized E3 ligase whose mRNA levels were upregulated by the infestation of gravid BPH females, mechanical wounding, and treatment with JA or ABA. Silencing OsJMJ715 enhanced BPH-elicited levels of ABA, JA, and JA-Ile as well as the amount of callose deposition in plants, which in turn increased the resistance of rice to BPH by reducing the feeding of BPH and the hatching rate of BPH eggs. These findings suggest that OsJMJ715 negative regulates the BPH-induced biosynthesis of ABA, JA, and JA-Ile and that BPH benefits by enhancing the expression of OsJMJ715.

CRISPR-Cas9 mediated mutation in GRAIN WIDTH and WEIGHT2 (GW2) locus improves aleurone layer and grain nutritional quality in rice

Enhancing crop productivity and their nutritional quality are the key components and primary focus of crop improvement strategy for fulfilling future food demand and improving human health. Grain filling and endosperm development are the key determinants of grain yield and nutritional quality. GRAIN WIDTH and WEIGHT2 (GW2) gene encodes a RING-type E3 ubiquitin ligase and determines the grain weight in cereal crops. Here we report GW2 knockout (KO) mutants in Indica (var. MTU1010) through CRISPR/Cas9 genome editing. The endosperm of GW2-KO mutant seed displays a thick aleurone layer with enhanced grain protein content. Further the loss of function of OsGW2 results in improved accumulation of essential dietary minerals (Fe, Zn, K, P, Ca) in the endosperm of rice grain. Additionally, the mutants displayed an early growth vigour phenotype with an improved root and shoot architecture. The hull morphology of GW2-KO lines also showed improved, grain filling thereby promoting larger grain architecture. Together, our findings indicate that GW2 may serve as a key regulator of improved grain architecture, grain nutritional quality and an important modulator of plant morphology. The study offers a strategy for the development of improved rice cultivars with enriched nutritional quality and its possible implementation in other cereals as well.

The ARRE RING-Type E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis thaliana by Controlling ECERIFERUM1 and ECERIFERUM3 Protein Levels.

The outer epidermal cell walls of plant shoots are covered with a cuticle, a continuous lipid structure that provides protection from desiccation, UV light, pathogens, and insects. The cuticle is mostly composed of cutin and cuticular wax. Cuticular wax synthesis is synchronized with surface area expansion during plant development and is associated with plant responses to biotic and abiotic stresses. Cuticular wax deposition is tightly regulated by well-established transcriptional and post-transcriptional regulatory mechanisms, as well as post-translationally via the ubiquitin-26S proteasome system (UPS). The UPS is highly conserved in eukaryotes and involves the covalent attachment of polyubiquitin chains to the target protein by an E3 ligase, followed by the degradation of the modified protein by the 26S proteasome. A large number of E3 ligases are encoded in the Arabidopsis genome, but only a few have been implicated in the regulation of cuticular wax deposition. In this study, we have conducted an E3 ligase reverse genetic screen and identified a novel RING-type E3 ubiquitin ligase, AtARRE, which negatively regulates wax biosynthesis in Arabidopsis. Arabidopsis plants overexpressing AtARRE exhibit glossy stems and siliques, reduced fertility and fusion between aerial organs. Wax load and wax compositional analyses of AtARRE overexpressors showed that the alkane-forming branch of the wax biosynthetic pathway is affected. Co-expression of AtARRE and candidate target proteins involved in alkane formation in both Nicotiana benthamiana and stable Arabidopsis transgenic lines demonstrated that AtARRE controls the levels of wax biosynthetic enzymes ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3). CER1 has also been confirmed to be a ubiquitination substrate of the AtARRE E3 ligase by an in vivo ubiquitination assay using a reconstituted Escherichia coli system. The AtARRE gene is expressed throughout the plant, with the highest expression detected in fully expanded rosette leaves and oldest stem internodes. AtARRE gene expression can also be induced by exposure to pathogens. These findings reveal that wax biosynthesis in mature plant tissues and in response to pathogen infection is controlled post-translationally.

The Emerging Roles of Tripartite Motif Proteins (TRIMs) in Acute Lung Injury.

Acute lung injury (ALI) is an inflammatory disorder of the lung that causes high mortality and lacks any pharmacological intervention. Ubiquitination plays a critical role in the pathogenesis of ALI as it regulates the alveolocapillary barrier and the inflammatory response. Tripartite motif (TRIM) proteins are one of the subfamilies of the RING-type E3 ubiquitin ligases, which contains more than 80 distinct members in humans involved in a broad range of biological processes including antivirus innate immunity, development, and tumorigenesis. Recently, some studies have shown that several members of TRIM family proteins play important regulatory roles in inflammation and ALI. Herein, we integrate emerging evidence regarding the roles of TRIMs in ALI. Articles were selected from the searches of PubMed database that had the terms “acute lung injury,” “ubiquitin ligases,” “tripartite motif protein,” “inflammation,” and “ubiquitination” using both MeSH terms and keywords. Better understanding of these mechanisms may ultimately lead to novel therapeutic approaches by targeting TRIMs for ALI treatment.

The BRCA1/BARD1 ubiquitin ligase and its substrates.

Mutations in breast cancer type 1 susceptibility protein (BRCA1) and its heterodimeric binding partner BARD1 confer a high risk for the development of breast and ovarian cancers. The sole enzymatic function of the BRCA1/BARD1 complex is as a RING-type E3 ubiquitin (Ub) ligase, leading to the deposition of Ub signals onto a variety of substrate proteins. Distinct types of Ub signals deposited by BRCA1/BARD1 (i.e. degradative vs. non-degradative; mono-Ub vs. poly-Ub chains) on substrate proteins mediate aspects of its function in DNA double-stranded break repair, cell-cycle regulation, and transcriptional regulation. While cancer-predisposing mutations in both subunits lead to the inactivation of BRCA1/BARD1 ligase activity, controversy remains as to whether its Ub ligase activity directly inhibits tumorigenesis. Investigation of BRCA1/BARD1 substrates using rigorous, well-validated mutants and experimental systems will ultimately clarify the role of its ligase activity in cancer and possibly establish prognostic and diagnostic metrics for patients with mutations. In this review, we discuss the Ub ligase function of BRCA1/BARD1, highlighting experimental approaches, mechanistic considerations, and reagents that are useful in the study of substrate ubiquitylation. We also discuss the current understanding of two well-established BRCA1/BARD1 substrates (nucleosomal H2A and estrogen receptor α) and several recently discovered substrates (p50, NF2, Oct1, and LARP7). Lessons from the current body of work should provide a road map to researchers examining novel substrates and biological functions attributed to BRCA1/BARD1 Ub ligase activity.