Search

Plant intracellular nucleotide-binding and leucine-rich repeat immune receptors (NLRs) play a key role in activating a strong pathogen defense response. Plant NLR proteins are tightly regulated and accumulate at very low levels in the absence of pathogen effectors. However, little is known about how this low level of NLR proteins is able to induce robust immune responses upon recognition of pathogen effectors. Here, we report that, in the absence of effector, the inactive form of the tomato NLR Sw-5b is targeted for ubiquitination by the E3 ligase SBP1. Interaction of SBP1 with Sw-5b via only its N-terminal domain leads to slow turnover. In contrast, in its auto-active state, Sw-5b is rapidly turned over as SBP1 is upregulated and interacts with both its N-terminal and NB-LRR domains. During infection with the tomato spotted wilt virus, the viral effector NSm interacts with Sw-5b and disrupts the interaction of Sw-5b with SBP1, thereby stabilizing the active Sw-5b and allowing it to induce a robust immune response. Synopsis: Plant immune receptors are expressed in low amounts in the absence of pathogen effectors, but accumulate rapidly after infection. This study shows that a viral pathogen effector competes with an E3 ligase from the host plant, blocking receptor turnover and triggering a robust immune response. In the absence of the viral pathogen effector, the inactive form of the Sw-5b NLR is targeted at its N-terminal domain by host E3 ligase SBP1 for ubiquitination and slow turnover. Sw-5b auto-activation leads to SBP1 level upregulation and the auto-active form of the Sw-5b NLR is targeted by SBP1 at both its N-terminal and LRR domains, leading to its rapid turnover. The viral effector NSm disrupts the interaction of Sw-5b with SBP1, thereby allowing the active Sw-5b to accumulate at sufficient levels to induce a robust immune response. A viral effector out-competes a host E3 ligase for intracellular NLR immune receptor binding, thus potentiating the receptor's response. [ABSTRACT FROM AUTHOR]

SUMMARY: Heat stress is an environmental factor that significantly threatens crop production worldwide. Nevertheless, the molecular mechanisms governing plant responses to heat stress are not fully understood. Plant zinc finger CCCH proteins have roles in stress responses as well as growth and development through protein–RNA, protein–DNA, and protein–protein interactions. Here, we reveal an integrated multi‐level regulation of plant thermotolerance that is mediated by the CCCH protein C3H15 in Arabidopsis. Heat stress rapidly suppressed C3H15 transcription, which attenuated C3H15‐inhibited expression of its target gene HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), a central regulator of heat stress response (HSR), thereby activating HEAT SHOCK COGNATE 70 (HSC70.3) expression. The RING‐type E3 ligase MED25‐BINDING RING‐H2 PROTEIN 2 (MBR2) was identified as an interacting partner of C3H15. The mbr2 mutant was susceptible to heat stress compared to wild‐type plants, whereas plants overexpressing MBR2 showed increased heat tolerance. MBR2‐dependent ubiquitination mediated the degradation of phosphorylated C3H15 protein in the cytoplasm, which was enhanced by heat stress. Consistently, heat sensitivities of C3H15 overexpression lines increased in MBR2 loss‐of‐function and decreased in MBR2 overexpression backgrounds. Heat stress‐induced accumulation of HSC70.3 promoted MBR2‐mediated degradation of C3H15 protein, implying that an auto‐regulatory loop involving C3H15, HSFA2, and HSC70.3 regulates HSR. Heat stress also led to the accumulation of C3H15 in stress granules (SGs), a kind of cytoplasmic RNA granule. This study advances our understanding of the mechanisms plants use to respond to heat stress, which will facilitate technologies to improve thermotolerance in crops. Significance Statement: Heat stress is a severe environmental factor that significantly inhibits plant growth and development, but the molecular mechanisms governing plant responses to heat stress are not fully understood. This study reveals a mechanism of multi‐level regulation for thermotolerance in Arabidopsis, which will facilitate creating heat‐tolerant crops. [ABSTRACT FROM AUTHOR]

Summary: Potato (Solanum tuberosum) is the fourth largest food crop in the world. Late blight, caused by oomycete Phytophthora infestans, is the most devastating disease threatening potato production. Previous research has shown that StRFP1, a potato Arabidopsis Tóxicos en Levadura (ATL) family protein, positively regulates late blight resistance via its E3 ligase activity. However, the underlying mechanism is unknown.Here, we reveal that StRFP1 is associated with the plasma membrane (PM) and undergoes constitutive endocytic trafficking. Its PM localization is essential for inhibiting P. infestans colonization. Through in vivo and in vitro assays, we investigated that StRFP1 interacts with two sugar transporters StSWEET10c and StSWEET11 at the PM.Overexpression (OE) of StSWEET10c or StSWEET11 enhances P. infestans colonization. Both StSWEET10c and StSWEET11 exhibit sucrose transport ability in yeast, and OE of StSWEET10c leads to an increased sucrose content in the apoplastic fluid of potato leaves. StRFP1 ubiquitinates StSWEET10c and StSWEET11 to promote their degradation.We illustrate a novel mechanism by which a potato ATL protein enhances disease resistance by degrading susceptibility (S) factors, such as Sugars Will Eventually be Exported Transporters (SWEETs). This offers a potential strategy for improving disease resistance by utilizing host positive immune regulators to neutralize S factors. [ABSTRACT FROM AUTHOR]

Cellular energy is primarily produced from glucose and fat through glycolysis and fatty acid oxidation (FAO) followed by the tricarboxylic acid cycle in mitochondria; energy homeostasis is carefully maintained via numerous feedback pathways. In this report, we uncovered a new master regulator of carbohydrate and lipid metabolism. When ubiquitin E3 ligase β‐TrCP2 was inducibly knocked out in β‐TrCP1 knockout adult mice, the resulting double knockout mice (DKO) lost fat mass rapidly. Biochemical analyses of the tissues and cells from β‐TrCP2 KO and DKO mice revealed that glycolysis, FAO, and lipolysis were dramatically upregulated. The absence of β‐TrCP2 increased the protein stability of metabolic rate‐limiting enzymes including 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB3), adipose triglyceride lipase (ATGL), carnitine palmitoyltransferase 1A (CPT1A), and carnitine/acylcarnitine translocase (CACT). Our data suggest that β‐TrCP is a potential regulator for total energy homeostasis by simultaneously controlling glucose and fatty acid metabolism and that targeting β‐TrCP could be an effective strategy to treat obesity and other metabolic disorders. [ABSTRACT FROM AUTHOR]

Alpha‐2‐Glycoprotein 1, Zinc‐binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co‐immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif‐containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA. [ABSTRACT FROM AUTHOR]

Our previous study identified an evolutionarily conserved C4HC3‐type E3 ligase, named microtubule‐associated E3 ligase (MEL), that regulates broad‐spectrum plant resistance against viral, fungal and bacterial pathogens in multiple plant species by mediating serine hydroxymethyltransferase (SHMT1) degradation via the 26S proteasome pathway. In the present study, we found that NS3 protein encoded by rice stripe virus could competitively bind to the MEL substrate recognition site, thereby inhibiting MEL interacting with and ubiquitinating SHMT1. This, in turn, leads to the accumulation of SHMT1 and the repression of downstream plant defence responses, including reactive oxygen species accumulation, mitogen‐activated protein kinase pathway activation, and the up‐regulation of disease‐related gene expression. Our findings shed light on the ongoing arms race between pathogens and demonstrate how a plant virus can counteract the plant defence response. [ABSTRACT FROM AUTHOR]

Summary: Ubiquitination is a fundamental mechanism regulating the stability of target proteins in eukaryotes; however, the regulatory mechanism in seed longevity remains unknown. Here, we report that an uncharacterized E3 ligase, ARABIDOPSIS TÓXICOS EN LEVADURA 5 (ATL5), positively regulates seed longevity by mediating the degradation of ACTIVATOR OF BASAL TRANSCRIPTION 1 (ABT1) in Arabidopsis.Seeds in which ATL5 was disrupted showed faster accelerated aging than the wild‐type, while expressing ATL5 in atl5‐2 basically restored the defective phenotype. ATL5 was highly expressed in the embryos of seeds, and its expression could be induced by accelerated aging.A yeast two‐hybrid screen identified ABT1 as an ATL5 interacting protein, which was further confirmed by bimolecular fluorescence complementary assay and co‐immunoprecipitation analysis. In vitro and in vivo assays showed that ATL5 functions as an E3 ligase and mediates the polyubiquitination and degradation of ABT1. Disruption of ATL5 diminished the degradation of translated ABT1, and the degradation could be induced by seed ageing and occurred in a proteasome‐dependent manner. Furthermore, disruption of ABT1 enhanced seed longevity.Taken together, our study reveals that ATL5 promotes the polyubiquitination and degradation of the ABT1 protein posttranslationally and positively regulates seed longevity in Arabidopsis. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusions TUB‐like protein 4 (TULP4) is one of the distant members of tubby family proteins, whose function remains largely unknown. In the present study, we intend to identify the role of TULP4 in schizophrenia from human samples and animal models.Whole‐exome sequencing was used to detect the four schizophrenia families collected. In different cell lines, the effects of identified variants in TULP4 gene on its expression and localization were analyzed. Knockdown models in utero and adult mice were employed to investigate the role of Tulp4 on neuronal migration and schizophrenia‐related behavior. Subsequently, co‐IP assays were used to search for proteins that interact with TULP4 and the effects of mutants on the molecular function of TULP4.For the first time, we identified five rare variants in TULP4 from schizophrenia families, of which three significantly reduced TULP4 protein expression. Knockdown the expression of Tulp4 delayed neuronal migration during embryological development and consequently triggered abnormal behaviors in adult mice, including impaired sensorimotor gating and cognitive dysfunction. Furthermore, we confirmed that TULP4 is involved in the formation of a novel E3 ligase through interaction with CUL5‐ELOB/C‐RNF7 and the three deleterious variants affected the binding amount of TULP4 and CUL5 to a certain extent.Together, we believe TULP4 plays an important role in neurodevelopment and subsequent schizophrenic‐related phenotypes through its E3 ubiquitin ligase function. [ABSTRACT FROM AUTHOR]

For many inflammatory cytokines, the response elicited is dependent on the recruitment of the tumour necrosis factor receptor‐associated factor (TRAF) family of adaptor proteins. All TRAF proteins have a trimeric C‐terminal TRAF domain, while at the N‐terminus most TRAFs have a RING domain that forms dimers. The symmetry mismatch of the N‐ and C‐terminal halves of TRAF proteins means that when receptors cluster, it is presumed that RING dimers connect TRAF trimers to form a network. Here, using purified TRAF6 proteins, we provide direct evidence in support of this model, and we show that TRAF6 trimers bind Lys63‐linked ubiquitin chains to promote their processive assembly. This study provides critical evidence in support of TRAF trimers as key players in signalling. [ABSTRACT FROM AUTHOR]

Summary: Apolygus lucorum (Meyer‐Dur; Heteroptera: Miridae) is a major agricultural pest infesting crops, vegetables, and fruit trees. During feeding, A. lucorum secretes a plethora of effectors into its hosts to promote infestation. However, the molecular mechanisms of these effectors manipulating plant immunity are largely unknown.Here, we investigated the molecular mechanism underlying the effector Al106 manipulation of plant–insect interaction by RNA interference, electrical penetration graph, insect and pathogen bioassays, protein–protein interaction studies, and protein ubiquitination experiment.Expression of Al106 in Nicotiana benthamiana inhibits pathogen‐associated molecular pattern‐induced cell death and reactive oxygen species burst, and promotes insect feeding and plant pathogen infection. In addition, peptidyl‐prolyl cis‐trans isomerase (PPIase) activity of Al106 is required for its function to inhibit PTI.Al106 interacts with a plant U‐box (PUB) protein, PUB33, from N. benthamiana and Arabidopsis thaliana. We also demonstrated that PUB33 is a positive regulator of plant immunity. Furthermore, an in vivo assay revealed that Al106 inhibits ubiquitination of NbPUB33 depending on PPIase activity.Our findings revealed that a novel cyclophilin effector may interact with plant PUB33 to suppress plant immunity and facilitate insect feeding in a PPIase activity‐dependent manner. [ABSTRACT FROM AUTHOR]

Proteolysis‐targeting chimeras (PROTACs) are novel therapeutics for the treatment of human disease. They exploit the enormous potential of the E3 ligases, a class of proteins that mark a target protein for degradation via the ubiquitin–proteasome system. Despite the existence of several E3 ligase‐related databases, the choice of the functioning ligase is limited to only 1.6% of those available, probably due to the fragmentary understanding of their structures and their known ligands; in fact, none of the existing databases report detailed studies covering their 3D structure or their pockets. Here, we report ELIOT (E3 LIgase pocketOme navigaTor), an accurate and complete platform containing the E3 ligase pocketome to enable navigation and selection of new E3 ligases and new ligands for the design of new PROTACs. All E3 ligase pockets were characterized with innovative 3D descriptors including their PROTAC‐ability score, and similarity analyses between E3 pockets are presented. Tissue specificity and their degree of involvement in patients with specific cancer types are also annotated for each E3 ligase, enabling appropriate selection for the design of a PROTAC with improved specificity. All data are available at https://eliot.moldiscovery.com. [ABSTRACT FROM AUTHOR]

Summary: Receptor‐like proteins (RLPs) lacking the cytoplasmic kinase domain play crucial roles in plant growth, development and immunity. However, what remains largely elusive is whether RLP protein levels are fine‐tuned by E3 ubiquitin ligases, which are employed by receptor‐like kinases for signaling attenuation. Nicotiana benthamiana NbEIX2 is a leucine‐rich repeat RLP (LRR‐RLP) that mediates fungal xylanase‐triggered immunity.Here we show that NbEIX2 associates with an F‐box protein NbPFB1, which promotes NbEIX2 degradation likely by forming an SCF E3 ubiquitin ligase complex, and negatively regulates NbEIX2‐mediated immune responses.NbEIX2 undergoes ubiquitination and proteasomal degradation in planta. Interestingly, NbEIX2 without its cytoplasmic tail is still associated with and destabilized by NbPFB1. In addition, NbPFB1 also associates with and destabilizes NbSOBIR1, a co‐receptor of LRR‐RLPs, and fails to promote NbEIX2 degradation in the sobir1 mutant.Our findings reveal a distinct model of NbEIX2 degradation, in which an F‐box protein destabilizes NbEIX2 indirectly in a SOBIR1‐dependent manner. [ABSTRACT FROM AUTHOR]

Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN‐γ) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN‐γ activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN‐γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock‐out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections. Synopsis: Interferon gamma activation of macrophages increases phagocytic uptake of particles, but delays phagosomal maturation via unclear mechanisms. Here, a phagosome‐localised E3 ubiquitin ligase, RNF115, is shown to control phagosomal maturation and cytokine responses to bacterial infection in vitro and in vivo. Quantitative proteomics reveal high levels of typical and atypical ubiquitin chain types on phagosomal proteins.RNF115 is enriched on phagosomes and ubiquitylates various proteins involved in immune responses and vesicle trafficking.RNF115 regulates Salmonella adaptation and replication within macrophages.Loss of RNF115 increases inflammatory responses in vitro and reduces tissue damage to infection in knock‐out mice. [ABSTRACT FROM AUTHOR]

Cullin‐based RING E3 ligases that use BTB/POZ‐MATH (BPM) proteins as substrate receptors have been established over the last decade as critical regulators in plant development and abiotic stress tolerance. As such they affect general aspects of shoot and root development, flowering time, embryo development, and different abiotic stress responses, such as heat, drought and salt stress. To generate tools that can help to understand the role of CRL3BPM E3 ligases in plants, we developed a novel system using two conserved protein‐binding motifs from BPM substrates to transiently block CRL3BPM activity. The work investigates in vitro and in planta this novel approach, and shows that it can affect stress tolerance in plants as well as developmental aspects. It thereby can serve as a new tool for studying this E3 ligase in plants. [ABSTRACT FROM AUTHOR]

Protein UFMylation, i.e., post‐translational modification with ubiquitin‐fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold‐type E3 ligase mechanism that activates the UFM1‐conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex. Synopsis: UFMylation is a ubiquitin‐like post‐translational modification important for endoplasmic reticulum homeostasis, but how UFM1 is conjugated to target proteins remains poorly understood. Here, in vitro reconstitutions reveal the molecular players, minimal requirements and biochemical principles of UFM1 attachment to substrates. UFL1 and UFBP1 together form an active E3 ligase for UFM1.The UFL1/UFBP1 complex is an atypical scaffold‐type ligase that activates aminolysis by the UFM1‐conjugating E2 enzyme.CDK5RAP3 regulates activity of the UFL1/UFBP1 E3 ligase complex and promotes mono‐UFMylation of ribosomes.Activity of the E2 UFC1 is regulated by an N‐terminal extension and a unique TAK motif. [ABSTRACT FROM AUTHOR]

Summary: Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in plants. A key regulatory system for protein stability is given by the CULLIN‐RING E3 ligases (CRLs).In this work, MYB43 is identified as a novel target of a CUL4‐DDB1‐PRL1 (PLEIOTROPIC REGULATORY LOCUS 1)‐RING E3 ligase (CRL4PRL1 E3 ligase). Its stability depends on the presence of PRL1, a WD40‐containing protein functioning as a substrate receptor of the CRL4 E3 ligases.Genetic studies have indicated that MYB43 is a negative regulator of cadmium (Cd) tolerance in Arabidopsis by transcriptional inhibition of important Cd transporters (HMA2, HMA3 and HMA4), while PRL1 and CUL4 positively regulate Cd tolerance. Expression of CUL4 and PRL1 was enhanced in response to Cd stress, and PRL1 can interact with and target MYB43 for degradation depending on assembly of CRL4PRL1 E3 ligase, and consequently increase the expression of HMA2, HMA3 and HMA4 through attenuating the transcriptional inhibition.HMA2 and HMA4 are shown to transport cadmium ion (Cd2+) from the roots of plants to the shoots through the xylem, ultimately increasing the plants' tolerance to Cd stress. [ABSTRACT FROM AUTHOR]

Plant hormone abscisic acid (ABA) plays an indispensable role in the control of leaf senescence, during which ABA signaling depends on its biosynthesis. Nevertheless, the role of ABA transport in leaf senescence remains unknown. Here, we identified two novel RING‐box protein‐encoding genes UBIQUITIN LIGASE of SENESCENCE 1 and 2 (ULS1 and ULS2) involved in leaf senescence. Lack of ULS1 and ULS2 accelerates leaf senescence, which is specifically promoted by ABA treatment. Furthermore, the expression of senescence‐related genes is significantly affected in mature leaves of uls1/uls2 double mutant (versus wild type (WT)) in an ABA‐dependent manner, and the ABA content is substantially increased. ULS1 and ULS2 are mainly expressed in the guard cells and aging leaves, and the expression is induced by ABA. Further RNA‐seq and quantitative proteomics of ubiquitination reveal that ABA transporter ABCG40 is highly expressed in uls1/uls2 mutant versus WT, though it is not the direct target of ULS1/2. Finally, we show that the acceleration of leaf senescence, the increase of leaf ABA content, and the promotion of stomatal closure in uls1/usl2 mutant are suppressed by abcg40 loss‐of‐function mutation. These results indicate that ULS1 and ULS2 function in feedback inhibition of ABCG40‐dependent ABA transport during ABA‐induced leaf senescence and stomatal closure. [ABSTRACT FROM AUTHOR]

Monoubiquitination of histone H2B at lysine 120 plays a vital role in active transcription and DNA damage response pathways. Ubiquitin protein ligase E3 component N‐recognin 7 (UBR7) has been recently identified as an H2BK120 monoubiquitin ligase. However, the molecular details of its ubiquitin transfer mechanism are not well understood. Here, we report that the plant homeodomain (PHD) finger of UBR7 is essential for its association with E2 UbcH6 and consequent ubiquitin transfer to its substrate histone H2B. We also identified the critical region of UbcH6 involved in this function and shown that the residues stretching from 114 to 125 of histone H2B C‐terminal tail are sufficient for UBR7/UbcH6‐mediated ubiquitin transfer. We also employed antibody‐independent mass spectrometry to confirm UBR7‐mediated ubiquitination of the H2B C‐terminal tail. We demonstrated that the PHD finger of UBR7 forms a dimer and this dimerization is essential for ubiquitination of histone H2B. We mapped the critical residues involved in the dimerization and mutation of these residues that abrogate E3 ligase activity and are associated with cancer. Furthermore, we compared the mode of ubiquitin discharge from UbcH6 mediated by UBR7 and RING finger protein 20 (RNF20) through a thioester hydrolysis assay. Interestingly, binding of substrate H2B to UBR7 induces a conformational change in the PHD finger, which triggers ubiquitin transfer from UbcH6. However, the RNF20 RING finger alone is sufficient to promote the release of ubiquitin from UbcH6. Overall, the mechanism of ubiquitin transfer by the newly identified E3 ubiquitin ligase UBR7 is markedly different from that of RNF20. [ABSTRACT FROM AUTHOR]

Summary: Plants use pattern recognition receptors (PRRs) to detect pathogen‐associated molecular patterns (PAMPs) and activate pattern‐triggered immunity (PTI). Precise regulation of information from PRRs to downstream signaling components is vital to mounting an appropriate immune response and requires dynamic interactions of these PTI components.We used transcriptome profiling, phenotypic analysis, molecular genetics, and protein–protein interaction analysis to understand the roles of the Arabidopsis plant U‐box (PUB) proteins PUB2 and PUB4 in disease resistance and PTI signaling.Loss of function of both PUB2 and PUB4 diminishes the PAMP‐triggered oxidative bursts and dampens mitogen‐activated protein kinase signaling, resulting in a severe compromise in resistance to not only pathogenic but also nonpathogenic strains of Pseudomonas syringae. Within PUB4, the E3 ligase activity is dispensable, but the armadillo repeat region is essential and sufficient for its function in immunity. PUB2 and PUB4 interact with PTI signaling components, including FLS2, BIK1, PBL27, and RbohD, and enhance FLS2‐BIK1 and BIK1‐RbohD interactions.Our study reveals that PUB2 and PUB4 are critical components of plant immunity and connect PTI components to positively regulate defense responses. [ABSTRACT FROM AUTHOR]

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide‐binding domain leucine‐rich repeat receptors (NLRs) and activate effector‐triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N1‐107) and RipAW E3‐ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N1‐107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW‐triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW‐triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW‐activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant–pathogen co‐evolution. [ABSTRACT FROM AUTHOR]

Repair of DNA double‐stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post‐translational modification of repair factors. In budding yeast, single‐stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA–RPA complexes are then SUMO‐modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single‐stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO‐RPA signal is amplified by successive rounds of Siz2‐dependent SUMO modification of RPA and dissociation of SUMO‐RPA at the junction between single‐ and double‐stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair. SYNOPSIS: DNA damage elicits protein group modification by SUMO of factors involved in mediating DNA repair and homologous recombination. The SUMO E3 ligase Siz2 is central to this response and is required for efficient SUMO modification of Replication Protein A (RPA) when bound to single stranded DNA. Asymmetric DNA with a 3' single‐stranded overhang specifically increases the rate of SUMO modification of RPA by Siz2.SUMO modification of RPA is dependent on interactions between the SAP domain of Siz2 and double‐stranded DNA and to lesser extent, the WH domain of RPA.SUMO‐modified RPA is more readily displaced from DNA compared to unmodified RPA, thus allowing additional molecules of RPA and other repair factors to be modified while Siz2 remains bound to duplex DNA. [ABSTRACT FROM AUTHOR]

The human GID (hGID) complex is a conserved E3 ubiquitin ligase regulating diverse biological processes, including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi‐subunit complex remain poorly understood. Using biochemical assays, cross‐linking mass spectrometry, and cryo‐electron microscopy, we show that hGID engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 and RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N‐end degron. GID4 substrate binding and ligase activity is regulated by ARMC8α, while the shorter ARMC8β isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo‐EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring‐like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non‐overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provides insights into the regulation of its E3 ligase activity. Synopsis: The human GID E3 ligase complex forms tetramers with two distinct substrate‐recruitment modules, namely WDR26‐RanBP9 and GID4‐ARMC8α. Although the shorter ARMC8β isoform stably assembles into the hGID complex, it lacks the ability to recruit the GID4 substrate‐receptor. The hGID complex engages two substrate‐recruitment modules, WDR26‐RanBP9 and GID4‐ARMC8α, which in turn target distinct substrates.The hGID complex assembles oligomeric complexes via the RING subunits and WDR26 as interfaces, respectively.The hGID complex stably recruits a specific isoform of the ARMC8 subunit, namely ARMC8β, which lacks a binding site for the GID4 substrate‐receptor. [ABSTRACT FROM AUTHOR]

The ubiquitin system is an important part of the host cellular defense program during bacterial infection. This is in particular evident for a number of bacteria including Salmonella Typhimurium and Mycobacterium tuberculosis which—inventively as part of their invasion strategy or accidentally upon rupture of seized host endomembranes—become exposed to the host cytosol. Ubiquitylation is involved in the detection and clearance of these bacteria as well as in the activation of innate immune and inflammatory signaling. Remarkably, all these defense responses seem to emanate from a dense layer of ubiquitin which coats the invading pathogens. In this review, we focus on the diverse group of host cell E3 ubiquitin ligases that help to tailor this ubiquitin coat. In particular, we address how the divergent ubiquitin conjugation mechanisms of these ligases contribute to the complexity of the anti‐bacterial coating and the recruitment of different ubiquitin‐binding effectors. We also discuss the activation and coordination of the different E3 ligases and which strategies bacteria evolved to evade the activities of the host ubiquitin system. [ABSTRACT FROM AUTHOR]

Nonavailability of glucose as a carbon source results in glyoxylate pathway activation, which metabolizes nonfermentable carbon for energy generation in Saccharomyces cerevisiae. Ucc1p of S. cerevisiae inhibits activation of the glyoxylate pathway by targeting Cit2p, a key glyoxylate enzyme for ubiquitin‐mediated proteasomal degradation when glucose is available as a carbon source. Sro9p, a La‐motif protein involved in RNA biogenesis, interacts physically with the messenger RNA of UCC1; however, its functional relevance is yet to be discovered. This study presents binary epistatic interaction between UCC1 and SRO9, with functional implication on the growth rate, response to genotoxic stress, resistance to apoptosis, and petite mutation. Cells with ucc1Δsro9Δ, as their genetic background, exhibit alteration in morphology, improvement in growth rate, resistance to apoptosis, and petite mutation. Moreover, the study indicates a cross‐link between ubiquitin‐proteasome system and RNA biogenesis and metabolism, with applications in industrial fermentation and screening for cancer therapeutics. [ABSTRACT FROM AUTHOR]

Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post‐translational regulation of the activities of transcription factors (TFs) is a dynamic and essential mechanism for plant growth and development. CRL3BPM E3 ligase is a CULLIN3‐based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over‐expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106‐1 mutants, including Flowering Locus T (FT) which is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays (EMSA), in vivo chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP‐qPCR) assays and dual luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3BPM E3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3BPM E3 ligases in Arabidopsis. [ABSTRACT FROM AUTHOR]

Summary: The unfoldase CDC48 (Cell Division Cycle 48) is highly conserved in eukaryotes, serving as an AAA + ATPase to extract ubiquitinated proteins from large protein complexes and membranes. Although its biochemical properties have been studied extensively in yeast and animal systems, the biological roles and regulations of the plant CDC48s have been explored only recently.Here we describe the identification of a novel E3 ligase from the SNIPER (snc1‐influencing plant E3 ligase reverse genetic) screen, which contributes to plant defense regulation by targeting CDC48A for degradation. SNIPER7 encodes an F‐box protein and its overexpression leads to autoimmunity.We identified CDC48s as interactors of SNIPER7 through immunoprecipitation followed by mass spectrometry proteomic analysis. SNIPER7 overexpression lines phenocopy the autoimmune mutant Atcdc48a‐4. Furthermore, CDC48A protein levels are reduced or stabilized when SNIPER7 is overexpressed or inhibited, respectively, suggesting that CDC48A is the ubiquitination substrate of SCFSNIPER7.Taken together, this study reveals a new mechanism where a SCFSNIPER7 complex regulates CDC48 unfoldase levels and modulates immune output. [ABSTRACT FROM AUTHOR]

Mitogen activated protein kinase kinase kinase 18 (MAPKKK18) mediated signaling cascade plays important roles in Arabidopsis drought stress tolerance. However, the post‐translational modulation patterns of MAPKKK18 are not characterized. In this study, we found that the protein level of MAPKKK18 was tightly controlled by the 26S proteasome. Ubiquitin ligases RGLG1 and RGLG2 ubiquitinated MAPKKK18 at lysine residue K32 and K154, and promoted its degradation. Deletion of RGLG1 and RGLG2 stabilized MAPKKK18 and further enhanced the drought stress tolerance of MAPKKK18‐overexpression plants. Our data demonstrate that RGLG1 and RGLG2 negatively regulate MAPKKK18‐mediated drought stress tolerance in Arabidopsis. [ABSTRACT FROM AUTHOR]

Both plant receptor‐like protein kinases (RLKs) and ubiquitin‐mediated proteolysis play crucial roles in plant responses to drought stress. However, the mechanism by which E3 ubiquitin ligases modulate RLKs is poorly understood. In this study, we showed that Arabidopsis PLANT U‐BOX PROTEIN 11 (PUB11), an E3 ubiquitin ligase, negatively regulates abscisic acid (ABA)‐mediated drought responses. PUB11 interacts with and ubiquitinates two receptor‐like protein kinases, LEUCINE RICH REPEAT PROTEIN 1 (LRR1) and KINASE 7 (KIN7), and mediates their degradation during plant responses to drought stress in vitro and in vivo. pub11 mutants were more tolerant, whereas lrr1 and kin7 mutants were more sensitive, to drought stress than the wild type. Genetic analyses show that the pub11 lrr1 kin7 triple mutant exhibited similar drought sensitivity as the lrr1 kin7 double mutant, placing PUB11 upstream of the two RLKs. Abscisic acid and drought treatment promoted the accumulation of PUB11, which likely accelerates LRR1 and KIN7 degradation. Together, our results reveal that PUB11 negatively regulates plant responses to drought stress by destabilizing the LRR1 and KIN7 RLKs. [ABSTRACT FROM AUTHOR]

Neur1 and Neur2, mouse homologs of the Drosophila neur gene, consist of two neuralized homology repeat domains and a RING domain. Both Neur1 and Neur2 are expressed in the whole adult brain and encode E3 ubiquitin ligases, which play a crucial role in the Notch signaling pathways. A previous study reported that overexpression of Neur1 enhances hippocampus‐dependent memory, whereas the role of Neur2 remains largely unknown. Here, we aimed to elucidate the respective roles of Neur1 and Neur2 in hippocampus‐dependent memory using three lines of genetically modified mice: Neur1 knock‐out, Neur2 knock‐out, and Neur1 and Neur2 double knock‐out (D‐KO). Our results showed that spatial memory was impaired when both Neur1 and Neur2 were deleted, but not in the individual knock‐out of either Neur1 or Neur2. In addition, basal synaptic properties estimated by input–output relationships and paired‐pulse facilitation did not change, but a form of long‐term potentiation that requires protein synthesis was specifically impaired in the D‐KO mice. These results collectively suggest that Neur1 and Neur2 are crucially involved in hippocampus‐dependent spatial memory and synaptic plasticity. [ABSTRACT FROM AUTHOR]

In both plants and animals, nucleotide‐binding leucine‐rich repeat (NLR) immune receptors perceive pathogen‐derived molecules to trigger immunity. Global NLR homeostasis must be tightly controlled to ensure sufficient and timely immune output while avoiding aberrant activation, the mechanisms of which are largely unclear. In a previous reverse genetic screen, we identified two novel E3 ligases, SNIPER1 and its homolog SNIPER2, both of which broadly control the levels of NLR immune receptors in Arabidopsis. Protein levels of sensor NLRs (sNLRs) are inversely correlated with SNIPER1 amount and the interactions between SNIPER1 and sNLRs seem to be through the common nucleotide‐binding (NB) domains of sNLRs. In support, SNIPER1 can ubiquitinate the NB domains of multiple sNLRs in vitro. Our study thus reveals a novel process of global turnover of sNLRs by two master E3 ligases for immediate attenuation of immune output to effectively avoid autoimmunity. Such unique mechanism can be utilized in the future for engineering broad‐spectrum resistance in crops to fend off pathogens that damage our food supply. Synopsis: In plants, intracellular pathogen effector molecules are recognized by NOD‐like receptors (sensor NLRs) to activate effector‐triggered immunity. Here, two RING‐domain E3 ubiquitin ligases are shown to suppress sensor NLR‐induced immune response to avoid autoimmunity. SNIPER1 and SNIPER2 are redundant regulators of NLR‐mediated plant immunity.Overexpression of SNIPER1 or SNIPER2 suppresses autoimmunity phenotypes of snc1 mutant plants.SNIPER1 ubiquitinates multiple sensor NLRs and targets them for degradation.SNIPER1 associates with the NB domains of sensor NLRs. [ABSTRACT FROM AUTHOR]

The anaphase‐promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of the APC/C in mitosis is restrained by the spindle assembly checkpoint (SAC), which coordinates chromosome segregation with the assembly of the mitotic spindle. The SAC effector is the mitotic checkpoint complex (MCC), which binds and inhibits the APC/C. It is incompletely understood how the APC/C switches substrate specificity in a cell cycle‐specific manner. For instance, it is unclear how in prometaphase, when APC/C activity towards cyclin B and securin is repressed by the MCC, the kinase Nek2A is ubiquitinated. Here, we combine biochemical and structural analysis with functional studies in cells to show that Nek2A is a conformational‐specific binder of the APC/C–MCC complex (APC/CMCC) and that, in contrast to cyclin A, Nek2A can be ubiquitinated efficiently by the APC/C in conjunction with both the E2 enzymes UbcH10 and UbcH5. We propose that these special features of Nek2A allow its prometaphase‐specific ubiquitination. Synopsis: The APC/C regulates mitosis by catalysing the sequential ubiquitination of specific substrates. This study shows how the APC/C ubiquitinates the Nek2A kinase in prometaphase when the APC/C is inhibited by the mitotic checkpoint complex (MCC). Nek2A binds preferentially the open conformation of the APC/CMCC complex where the APC/C catalytic site is liberated from the MCC inhibition.Nek2A, which exists as a dimer, binds through its C‐terminal MR tail dipeptide in two distinct sites on the APC/C which overlap with MCC binding sites.Contrary to the other prometaphase‐specific substrate cyclin A, which can preferentially be ubiquitinated via the UbcH10‐APC/C complex, Nek2A can be efficiently ubiquitinated by APC/C complexes with both UbcH10 and UbcH5. [ABSTRACT FROM AUTHOR]

Apoptosis is an organised ATP‐dependent programmed cell death that organisms have evolved to maintain homoeostatic cell numbers and eliminate unnecessary or unhealthy cells from the system. Dysregulation of apoptosis can have serious manifestations culminating into various diseases, especially cancer. Accurate control of apoptosis requires regulation of a wide range of growth enhancing as well as anti‐oncogenic factors. Appropriate regulation of magnitude and temporal expression of key proteins is vital to maintain functional apoptotic signalling. Controlled protein turnover is thus critical to the unhindered operation of the apoptotic machinery, disruption of which can have severe consequences, foremost being oncogenic transformation of cells. The ubiquitin proteasome system (UPS) is one such major cellular pathway that maintains homoeostatic protein levels. Recent studies have found interesting links between these two fundamental cellular processes, wherein UPS depending on the cue can either inhibit or promote apoptosis. A diverse range of E3 ligases are involved in regulating the turnover of key proteins of the apoptotic pathway. This review summarises an overview of key E3 ubiquitin ligases involved in the regulation of the fundamental proteins involved in apoptosis, linking UPS to apoptosis and attempts to emphasize the significance of this relationship in context of cancer. [ABSTRACT FROM AUTHOR]

Summary: Recent work has established a core ABA signaling pathway in which A‐type PP2C protein phosphatases act as central negative modulators. Although ABA signaling inhibits PP2C activity through ABA‐receptor complex, it remains unknown if other mechanisms exist to modulate the level of PP2Cs. Here, we identified a RING domain ubiquitin E3 ligase, PIR1 (PP2CA interacting RING finger protein 1), that interacted with PP2CA. Of the two splicing isoforms, PIR1.2 was isolated from leaf tissue. The PIR1.2 exhibited E3 ligase activity and determined PP2CA stability in the presence of ABA. Consistent with the conclusion that PIR1 promotes ABA signaling by removing PP2CA, a negative modulator, the pir1 knockout mutant displayed an ABA‐hyposensitive phenotype. We further showed that PIR2, the closest homologue of PIR1.2, also interacted with PP2CA. Although the pir2 knockout mutant did not display altered ABA response, the pir1‐1/pir2 double mutant became more insensitive to ABA than the wild‐type or pir1‐1 and pir2 single mutants. Using a cell‐free degradation assay, ABA promoted degradation of PP2CA, however, such degradation was delayed when incubated with protein extract prepared from the pir1‐1/pir2 double mutant. Our data suggest that PIR1 and PIR2 positively modulate ABA signaling by targeting PP2CA for degradation. Significance Statement: Several type 2C protein phosphatases (PP2Cs) act as negative modulators of ABA signaling in Arabidopsis. In this study, we isolated the RING‐type E3 ligases PIR1 and PIR2 that interact with and ubiquitinate PP2CA. We revealed that PIR1 and PIR2 play a positive role in ABA signaling via modulation of PP2CA stability. Our findings provide insight into modulation of PP2C in ABA signaling. [ABSTRACT FROM AUTHOR]

Summary: Ubiquitination is a eukaryotic protein modulation system for identifying and affecting proteins that are no longer needed in the cell. In a previous study, we elucidated the biological function of CaASRF1, which contains a RING finger domain and functions as an E3 ligase. We showed that CaASRF1 positively modulates abscisic acid (ABA) signalling and drought stress tolerance by modulating the stability of subgroup D bZIP transcription factor CaAIBZ1. We performed yeast two‐hybrid (Y2H) screening to identify an additional target protein of CaASRF1. In this study, we identified pepper CaATBZ1 (Capsicumannuum ASRF1 target bZIP transcription factor 1), which belongs to the subgroup A bZIP transcription factors. We investigated the biological function of this protein using virus‐induced gene silencing (VIGS) in pepper plants and by generating overexpressing transgenic Arabidopsis plants. Our loss‐of‐function and gain‐of‐function studies revealed that CaATBZ1 negatively modulates ABA signalling and drought stress response. Consistent with CaATBZ1‐silenced pepper plants, CaASRF1/CaATBZ1‐silenced pepper plants displayed drought‐tolerant phenotypes via ABA‐mediated signalling. Our results demonstrated that CaASRF1‐mediated ubiquitination plays a crucial role in regulating the stability of CaATBZ1. These findings provide valuable insight into the post‐translational regulation of transcriptional factors. Significance Statement: In a previous study, we elucidated that CaASRF1 plays a positive role in ABA and drought signaling via modulation of CaAIBZ1 stability. In the present study, we isolated CaASRF1 target bZIP transcription factor, CaATBZ1, which interacts with and is ubiquitinated by CaASRF1. CaATBZ1 functions as a negative modulator in ABA and drought signaling. These findings provide insight into ABA signaling‐mediated drought response. [ABSTRACT FROM AUTHOR]

Radiotherapy is an effective treatment for many cancer patients to eliminate malignant cells and increase survival rate. However, cancer cells can develop resistance in response to radiation through activation of signaling pathways that promote cell cycle progression, DNA damage response, cell survival, and inflammation. Various combination therapies are developed to sensitize radiotherapy by targeting key signaling proteins involved in radioresponse. The past decade has seen significant advances in the knowledge of ubiquitin signaling in cancer biology. Developing E3 ubiquitin ligase‐related molecules as novel strategies to cure cancer has become an emerging field. This review briefly discusses the potential of targeting diverse E3 ubiquitin ligases as promising strategies for radiosensitization in cancer. [ABSTRACT FROM AUTHOR]

Summary: Hormone‐ and stress‐induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone‐induced ubiquitination plays a crucial role to determine the half‐life of key negative regulators of hormone signaling. For ABAsignaling, the degradation of clade‐A PP2Cs, such as PP2CAor ABI1, is a complementary mechanism to PYR/PYL/RCAR‐mediated inhibition of PP2C activity. ABApromotes the degradation of PP2CAthrough the RGLG1 E3 ligase, although it is not known how ABAenhances the interaction of RGLG1 with PP2CAgiven that they are predominantly found in the plasma membrane and the nucleus, respectively. We demonstrate that ABAmodifies the subcellular localization of RGLG1 and promotes nuclear interaction with PP2CA. We found RGLG1 is myristoylated in vivo, which facilitates its attachment to the plasma membrane. ABAinhibits the myristoylation of RGLG1 through the downregulation of N‐myristoyltransferase 1(NMT1) and promotes nuclear translocation of RGLG1 in a cycloheximide‐insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP2CAprotein levels and the formation of RGLG1–receptor–phosphatase complexes. We show that RGLG1Gly2Alamutated at the N‐terminal myristoylation site shows constitutive nuclear localization and causes an enhanced response to ABAand salt or osmotic stress. RGLG1/5 can interact with certain monomeric ABAreceptors, which facilitates the formation of nuclear complexes such as RGLG1–PP2CA–PYL8. In summary, we provide evidence that an E3 ligase can dynamically relocalize in response to both ABAand increased levels of its target, which reveals a mechanism to explain how ABAenhances RGLG1–PP2CAinteraction and hence PP2CAdegradation. Significance Statement: The degradation of negative regulators is crucial for hormone signaling. ABA promotes the degradation of PP2CA through the RGLG1/5 E3 ligases and enhances the interaction of RGLG1/5 with PP2CA through an unknown mechanism. We show that ABA modifies the plasma membrane localization of RGLG1 by inhibiting its myristoylation and promoting cycloheximide‐insensitive nuclear translocation, which allows interaction with nuclear PP2CA‐receptor complexes. We explain how a hormone can modulate the interaction of an E3 ligase with its target. [ABSTRACT FROM AUTHOR]

Summary: Plants have evolved complex defense mechanisms to adapt and survive under adverse growth conditions. Abscisic acid (ABA) is a phytohormone that plays a pivotal role in the stress response, especially regulation of the stomatal aperture in response to drought. Here, we identified the pepper CaASRF1 (Capsicumannuum ABASensitive RINGFinger E3 ligase 1) gene, which modulates drought stress tolerance via ABA‐mediated signaling. CaASRF1 contains a C3H2C3‐type RING finger domain, which functions as an E3 ligase by attaching ubiquitins to the target proteins. CaASRF1 expression was enhanced after exposure to ABA, drought and NaCl. Loss‐of‐function in pepper plants and gain‐of‐function in Arabidopsis plants revealed that CaASRF1 positively modulates ABA signaling and the drought stress response. Moreover, CaASRF1 interacted with and was associated with degradation of the bZIP transcription factor CaAIBZ1 (Capsicumannuum ASRF1‐Interacting bZIP transcription factor 1). Contrary to CaASRF1 phenotypes, CaAIBZ1‐silenced pepper and CaAIBZ1‐overexpressing Arabidopsis exhibited drought‐tolerant and drought‐sensitive phenotypes, respectively. Taken together, our data indicate that CaASRF1 positively modulates ABA signaling and the drought stress response via modulation of CaAIBZ1 stability. Significance Statement: Several RING‐type E3 ligases and bZIP transcription factors act as positive or negative modulators of ABA and drought signaling; therefore, we isolated the RING‐type E3 ligase CaASRF1, which interacts with and ubiquitinates bZIP transcription factor CaAIBZ1 in pepper. CaASRF1 plays a positive role in ABA and drought signaling via regulation of CaAIBZ1 stability; our findings provide insight into ABA signaling and the drought stress response. [ABSTRACT FROM AUTHOR]

Summary: Intracellular nucleotide binding (NB) and leucine‐rich repeat (NLR) proteins function as immune receptors to recognize effectors from pathogens. They often guard host proteins that are the direct targets of those effectors. Recent findings have revealed that a typical NLR sometimes cooperates with another atypical NLR for effector recognition.Here, by using the CRISPR/Cas9 gene editing method, knockout analysis and biochemical assays, we uncovered differential pairings of typical Toll Interleukin1 receptor (TIR) type NLR (TNL) receptor SOC3 with atypical truncated TIR‐NB (TN) proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1.Overaccumulation of SAUL1 is monitored by the SOC3−TN2 pair, while SAUL1's disappearance is guarded by the SOC3−CHS1 pair. SOC3 forms a head‐to‐head genomic arrangement with CHS1 and TN2, indicative of transcriptional co‐regulation.Such intricate cooperative interactions can probably enlarge the recognition spectrum and increase the functional flexibility of NLRs, which can partly explain the overwhelming occurrence of NLR gene clustering in higher plants. [ABSTRACT FROM AUTHOR]

In addition to traditional drugs, such as enzyme inhibitors, receptor agonists/antagonists, and protein‐protein interaction inhibitors as well as genetic technology, such as RNA interference and the CRISPR/Cas9 system, protein knockdown approaches using proteolysis‐targeting chimeras (PROTACs) have attracted much attention. PROTACs, which induce selective degradation of their target protein via the ubiquitin‐proteasome system, are useful for the down‐regulation of various proteins, including disease‐related proteins and epigenetic proteins. Recent reports have shown that chemical protein knockdown is possible not only in cells, but also in vivo and this approach is expected to be used as the therapeutic strategy for several diseases. Thus, this approach may be a significant technique to complement traditional drugs and genetic ablation and will be more widely used for drug discovery and chemical biology studies in the future. In this personal account, a history of chemical protein knockdown is introduced, and its features, recent progress in the epigenetics field, and future outlooks are discussed. Chemical protein degradation approach by proteolysis‐targeting chimeras (PROTACs) can provide a brand‐new opportunity for drug discovery or chemical biology study. In this personal account, its history, features, and outlook are discussed as well as its recent application to epigenetic targets. [ABSTRACT FROM AUTHOR]

Summary: Abscisic acid (ABA) is a plant hormone that plays a key role in the environmental stress response, especially the induction of ABA‐responsive and stress‐responsive genes and modulation of the stomatal aperture in response to drought stress. Here, we identified CaDILZ1 (CapsicumannuumDrought‐Induced Leucine Zipper 1) belonging to subgroup D of the bZIP protein family; gene functions of this family in response to ABA and drought signaling still remain unknown. CaDILZ1 expression was significantly induced in pepper leaves after exposure to ABA, drought, and NaCl. The CaDILZ1 protein localized in the nucleus of plant cells. In response to drought stress, CaDILZ1‐silenced pepper and CaDILZ1‐overexpressing Arabidopsis plants exhibited drought‐sensitive and drought‐tolerant phenotypes, respectively, via altered ABA content, stomatal closure, and expression of ABA‐responsive and drought‐responsive marker genes. We isolated the RING finger protein CaDSR1 (CapsicumannuumDrought Sensitive RING finger protein 1), which interacted with CaDILZ1 in the nucleus. The CaDSR1 protein exhibited E3 ligase activity and promoted CaDILZ1 degradation via the 26S proteasome pathway. Under drought stress conditions, CaDSR1‐silenced pepper and CaDSR1‐overexpressing Arabidopsis plants exhibited contrasting phenotypes to those of CaDILZ1‐silenced pepper and CaDILZ1‐overexpressing Arabidopsis plants. Taken together, our data suggest that CaDSR1 and CaDILZ1 function in ABA‐mediated drought stress signaling in pepper plants. Significance Statement: The signal transduction occurring in response to drought stress is mediated from transcription to post‐translational modification. Here, the stress‐inducible pepper bZIP protein CaDILZ1 from subgroup D positively regulated ABA‐mediated drought responses, whereas RING‐type E3 ligase CaDSR1 played a contrasting role and triggered ubiquitination and degradation of CaDILZ1; our findings reveal a regulatory mechanism that links CaDILZ1 and CaDSR1 in the drought tolerance and ABA‐mediated responses in pepper plants. [ABSTRACT FROM AUTHOR]

Summary: Investigation of the endoplasmic reticulum‐associated degradation (ERAD) system in plants led to the identification of ERAD‐mediating RING finger protein (EMR) as a plant‐specific ERAD E3 ligase from Arabidopsis. EMR was significantly up‐regulated under endoplasmic reticulum (ER) stress conditions. The EMR protein purified from bacteria displayed high E3 ligase activity, and tobacco leaf‐produced EMR mediated mildew resistance locus O‐12 (MLO12) degradation in a proteasome‐dependent manner. Subcellular localization and coimmunoprecipitation analyses showed that EMR forms a complex with ubiquitin‐conjugating enzyme 32 (UBC32) as a cytosolic interaction partner. Mutation of EMR and RNA interference (RNAi) increased the tolerance of plants to ER stress. EMR RNAi in the bri1‐5 background led to partial recovery of the brassinosteroid (BR)‐insensitive phenotypes as compared with the original mutant plants and increased ER stress tolerance. The presented results suggest that EMR is involved in the plant ERAD system that affects BR signaling under ER stress conditions as a novel Arabidopsis ring finger E3 ligase mainly present in cytosol while the previously identified ERAD E3 components are typically membrane‐bound proteins. [ABSTRACT FROM AUTHOR]

ABSTRACT: As a prevalent post‐translational modification, ubiquitination is essential for many developmental processes. Once covalently attached to the small and conserved polypeptide ubiquitin (Ub), a substrate protein can be directed to perform specific biological functions via its Ub‐modified form. Three sequential catalytic reactions contribute to this process, among which E3 ligases serve to identify target substrates and promote the activated Ub to conjugate to substrate proteins. Ubiquitination has great plasticity, with diverse numbers, topologies and modifications of Ub chains conjugated at different substrate residues adding a layer of complexity that facilitates a huge range of cellular functions. Herein, we highlight key advances in the understanding of ubiquitination in epithelial morphogenesis, with an emphasis on the latest insights into its roles in cellular events involved in polarized epithelial tissue, including cell adhesion, asymmetric localization of polarity determinants and cytoskeletal organization. In addition, the physiological roles of ubiquitination are discussed for typical examples of epithelial morphogenesis, such as lung branching, vascular development and synaptic formation and plasticity. Our increased understanding of ubiquitination in epithelial morphogenesis may provide novel insights into the molecular mechanisms underlying epithelial regeneration and maintenance. [ABSTRACT FROM AUTHOR]

The insulin/insulin‐like growth factor‐1 (IGF‐1) signaling (IIS) pathway is a pivotal genetic program regulating cell growth, tissue development, metabolic physiology, and longevity of multicellular organisms. IIS integrates a fine‐tuned cascade of signaling events induced by insulin/IGF‐1, which is precisely controlled by post‐translational modifications. The ubiquitin/proteasome‐system (UPS) influences the functionality of IIS through inducible ubiquitylation pathways that regulate internalization of the insulin/IGF‐1 receptor, the stability of downstream insulin/IGF‐1 signaling targets, and activity of nuclear receptors for control of gene expression. An age‐related decline in UPS activity is often associated with an impairment of IIS, contributing to pathologies such as cancer, diabetes, cardiovascular, and neurodegenerative disorders. Recent findings identified a key role of diverse ubiquitin modifications in insulin signaling decisions, which governs dynamic adaption upon environmental and physiological changes. In this review, we discuss the mutual crosstalk between ubiquitin and insulin signaling pathways in the context of cellular and organismal homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: Aim: Based upon a microarray assay, we have identified that triiodothyronine (T3) upregulates MDM2 gene expression in the rat skeletal muscle. As MDM2 protein is an E3 ligase, we hypothesized that this enzyme could play a role in T3 effects on skeletal muscle mass control. Methods: To test our hypothesis, male rats (2 months old) were randomly assigned into the following groups: intact controls, treated with 20 physiological doses of T3 for 0.5, 1 and 7 days, or with 5, 20 and 50 physiological doses of T3 for 7 days. For in vitro experiments, myotubes and C2C12 cells were treated with T3 for 3 days. Results: After validation of the microarray finding throughout RT‐PCR and confirmation that T3 induces increases in MDM2 protein expression in a dose‐dependent manner, we observed that MDM2 was upregulated by T3 exclusively in fibre type I. Moreover, detailed histological evaluation showed that MDM2 overexpression distributes punctiformily along the cross section of the fibre and also inside nuclei. MDM2 colocalizes with PAX7 in control muscle and T3 downregulates this myogenic factor. Pharmacological inhibition of MDM2 in cultured myotubes caused a severe decrease in their diameter (~35%, P < .001 vs Control), enhancing the effect of T3 (from ~12% to ~35%, P < .001) alone upon myotube diameter and mRNA levels of atrogenes. Finally, we observed that FOXO3 (MDM2 target) is kept outside the nucleus under T3 stimulation. Conclusion: Our results indicate that MDM2 might be involved in the pro‐trophic effects of T3 in skeletal muscle. [ABSTRACT FROM AUTHOR]

GATA3 is a key transcription factor in cell fate determination and its dysregulation has been implicated in various types of malignancies. However, how the abundance and function of GATA3 are regulated remains unclear. Here, we report that GATA3 is physically associated with FBXW7α, and FBXW7α destabilizes GATA3 through assembly of a SKP1-CUL1-F-box E3 ligase complex. Importantly, we showed that FBXW7α promotes GATA3 ubiquitination and degradation in a GSK3 dependent manner. Furthermore, we demonstrated that FBXW7α inhibits breast cancer cells survival through destabilizing GATA3, and the expression level of FBXW7α is negatively correlated with that of GATA3 in breast cancer samples. This study indicated that FBXW7α is a critical negative regulator of GATA3 and revealed a pathway for the maintenance of GATA3 abundance in breast cancer cells. [ABSTRACT FROM AUTHOR]

The RNA-binding protein, UPF1, is best known for its central role in the nonsense-mediated RNA decay (NMD) pathway. Feng et al. now report a new function for UPF1-it is an E3 ubiquitin ligase that specifically promotes the decay of a key pro-muscle transcription factor: MYOD. UPF1 achieves this through its RING-like domain, which confers ubiquitin E3 ligase activity. Feng et al. provide evidence that the ability of UPF1 to destabilize MYOD represses myogenesis. In the future, it will be important to define other protein substrates of UPF1-driven ubiquitination and to determine whether this biochemical activity is responsible for some of UPF1's previously defined biological functions, including in development and stress responses. The exciting findings presented by Feng et al. open up the possibility that protein turnover and RNA turnover are coupled processes. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. [ABSTRACT FROM AUTHOR]

Cilium, E3 ligase, proteasome, ubiquitin, signaling Keywords: cilium; E3 ligase; proteasome; ubiquitin; signaling EN cilium E3 ligase proteasome ubiquitin signaling 1 2 2 08/25/22 20220901 NES 220901 Primary cilia are microtubule-based, sensory organelles present in growth-arrested cells that play an important role in growth, metabolism, differentiation, and development. Components of the UPS, including E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases and deubiquitylating enzymes (DUBs) have been identified as resident of - or closely associated to - the ciliary compartment. [Extracted from the article]

In both plants and animals, intracellular nucleotide-binding leucine-rich repeat proteins ( NLRs; or Nod-like receptors) serve as immune receptors to recognize pathogen-derived molecules and mount effective immune responses against microbial infections. Plant NLRs often guard the presence or activity of other host proteins, which are the direct virulence targets of pathogen effectors. These guardees are sometimes immune-promoting components such as those in a mitogen-activated protein kinase cascade. Plant E3 ligases serve many roles in immune regulation, but it is unclear whether they can also be guarded by NLRs., Here, we report on an immune-regulating E3 ligase SAUL1, whose homeostasis is monitored by a Toll interleukin 1 receptor (TIR)-type NLR (TNL), SOC3., SOC3 can associate with SAUL1, and either loss or overexpression of SAUL1 triggers autoimmunity mediated by SOC3. By contrast, SAUL1 functions redundantly with its close homolog PUB43 to promote PAMP-triggered immunity ( PTI)., Taken together, the E3 ligase SAUL1 serves as a positive regulator of PTI and its homeostasis is monitored by the TNL SOC3. [ABSTRACT FROM AUTHOR]

Cellular differentiation, developmental processes, and environmental factors challenge the integrity of the proteome in every eukaryotic cell. The maintenance of protein homeostasis, or proteostasis, involves folding and degradation of damaged proteins, and is essential for cellular function, organismal growth, and viability . Misfolded proteins that cannot be refolded by chaperone machineries are degraded by specialized proteolytic systems. A major degradation pathway regulating cellular proteostasis is the ubiquitin (Ub)/proteasome system ( UPS), which regulates turnover of damaged proteins that accumulate upon stress and during aging. Despite a large number of structurally unrelated substrates, Ub conjugation is remarkably selective. Substrate selectivity is mainly provided by the group of E3 enzymes. Several observations indicate that numerous E3 Ub ligases intimately collaborate with molecular chaperones to maintain the cellular proteome. In this review, we provide an overview of specialized quality control E3 ligases playing a critical role in the degradation of damaged proteins. The process of substrate recognition and turnover, the type of chaperones they team up with, and the potential pathogeneses associated with their malfunction will be further discussed. [ABSTRACT FROM AUTHOR]