RING-type E3 Ubiquitin Ligase Research Articles

Transcription factors PuNAC37/PuWRKY74 and E3 ubiquitin ligase PuRDUF2 inhibit volatile ester synthesis in 'Nanguo' pear.

As major contributors to flavor in many fruit species, volatile esters are useful for investigating the regulation of the biosynthesis pathways of volatile aroma compounds in plants. Ethylene promotes the biosynthesis of volatile esters during fruit storage while accelerating fruit ripening; thus, the ethylene perception inhibitor 1-methylcyclopropene (1-MCP) is employed to prolong the shelf life of fruits. However, the mechanisms by which 1-MCP regulates volatiles synthesis remain unclear. In this study, we analyzed the pathway of 1-MCP-mediated volatile ester synthesis in 'Nanguo' pear (Pyrus ussuriensis). 1-MCP significantly decreased volatile ester synthesis during storage. Comparative transcriptome analysis showed that the genes encoding two transcription factors (PuNAC37 and PuWRKY74) and a RING-type E3 ubiquitin ligase that have a domain of unknown function (PuRDUF2) were expressed at high levels, whereas ALCOHOL ACYLTRANSFERASE 1 (PuAAT1), encoding an enzyme responsible for volatile ester synthesis, was expressed at low levels in 1-MCP-treated fruit. Moreover, PuNAC37 inhibited the expression of PuWRKY74 via transcriptional regulation, whereas PuNAC37 and PuWRKY74, after directly binding to the promoter of PuAAT1, synergistically inhibited its expression in 1-MCP-treated fruit. In addition, in vitro and in vivo ubiquitination experiments revealed that PuRDUF2 functions as an E3 ubiquitin ligase that ubiquitinates PuAAT1, causing its degradation via the 26S proteasome pathway following 1-MCP treatment. Subsequent PuAAT1 degradation resulted in a reduction of volatile esters during fruit storage. Our findings provide insights into the complex transcriptional regulation of volatile ester formation in 'Nanguo' pears and contribute to future research on AAT protein ubiquitination in other species.

Genome-wide association study and transcriptome analysis reveal the genetic basis underlying the environmental adaptation of plant height in a woody plant

Studies on plant height have been conducted in several crops. However, the underlying genetic mechanisms in woody plants remain unclear. To improve the genetic understanding of plant height, the genome-wide association study (GWAS) was conducted on the 298 individuals of paper mulberry (Broussonetia papyrifera), and the individuals with the highest and lowest plant heights were selected for comparative transcriptome analysis. The analysis of phenotypic data showed that plant height decreased from low latitude (N: 24°30′) to high latitude (N: 41°00′), ranging from 372 to 150 cm. Furthermore, the plant height of paper mulberry was significantly correlated with environmental factors, such as latitude, frost-free period, hours of sunshine and so on, indicating adaptive phenotypic divergence across environmental gradients. A total of 228 candidate genes were identified through the GWAS, including three genes (Bp10g0547, Bp10g0551 and Bp10g0817) that contained nonsynonymous SNP variations significantly affecting plant height. A total of 2554 differentially expressed genes (DEGs) were identified through RNA sequencing (RNA-seq) analysis, including 28, 5, 3, 20 and 138 DEGs involved in auxin, gibberellin, cytokinin, ubiquitylation and transcription factors, respectively. Besides, there were 13 common genes identified by integrating GWAS and RNA-seq analysis, including Bp10g0817, which encodes COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1) and belongs to the RING type E3 ubiquitin ligase gene family. Collectively, this study provides valuable insights into the genetic mechanisms underlying plant height and adaptation of woody plants to diverse environments.

RLIM-specific activity reporters define variant pathogenicity in Tonne-Kalscheuer syndrome

Tonne-Kalscheuer syndrome (TOKAS; MIM: 300978) is an X-linked recessive disorder with devastating consequences for patients, such as intellectual disability, developmental delay, and multiple congenital abnormalities. TOKAS is associated with hemizygous variants in the RLIM gene, which encodes a RING-type E3 ubiquitin ligase. The current sustained increase in reported RLIM variants of uncertain significance creates an urgent need to develop assays that can screen these variants and experimentally determine their pathogenicity and disease association. Here, we engineered flow cytometry-based RLIM-specific reporters to measure RLIM activity in TOKAS. This paper describes the design and use of RLIM-specific reporters to determine the pathogenicity of a TOKAS RLIM gene variant. Our data demonstrate that RLIM-specific flow cytometry reporters based on either the full length or a degron region of the substrate REX1 measure RLIM activity in cells. Further, we describe the TOKAS variant RLIM p.Asn581Lys and, using reporter assays, determine that it disrupts RLIM catalytic activity. These data reveal how the p.Asn581Lys variant impairs RLIM function and suggests pathogenic mechanisms. The use of RLIM-specific reporters will greatly accelerate the resolution of variants of uncertain significance and disease association in TOKAS.

The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin.

Polypeptides arising from interrupted translation undergo proteasomal degradation by the ribosome-associated quality control (RQC) pathway. The ASC-1 complex splits stalled ribosomes into 40S subunits and nascent chain-tRNA-associated 60S subunits (60S RNCs). 60S RNCs associate with NEMF that promotes recruitment of the RING-type E3 ubiquitin (Ub) ligase Listerin (Ltn1 in yeast), which ubiquitinates nascent chains. RING-type E3s mediate the transfer of Ub directly from the E2~Ub conjugate, implying that the specificity of Ub linkage is determined by the given E2. Listerin is most efficient when it is paired with promiscuous Ube2D E2s. We previously found that TCF25 (Rqc1 in yeast) can impose K48-specificity on Listerin paired with Ube2D E2s. To determine the mechanism of TCF25's action, we combined functional biochemical studies and AlphaFold3 modeling and now report that TCF25 specifically interacts with the RING domain of Listerin and the acceptor ubiquitin (UbA) and imposes K48-specificity by orienting UbA such that its K48 is directly positioned to attack the thioester bond of the Ube2D1~Ub conjugate. We also found that TCF25 itself undergoes K48-specific ubiquitination by Listerin suggesting a mechanism for the reported upregulation of Rqc1 in the absence of Ltn1 and the observed degradation of TCF25 by the proteasome in vivo.

The ubiquitin E3 ligase RZFP1 affects drought tolerance in poplar by mediating the degradation of the protein phosphatase PP2C-9.

Abscisic acid (ABA) signaling has been implicated in plant responses to water deficit-induced osmotic stress. However, the underlying molecular mechanism remains unelucidated. This study identified the RING-type E3 ubiquitin ligase RING ZINC FINGER PROTEIN1 (PtrRZFP1) in poplar (Populus trichocarpa), a woody model plant. PtrRZFP1 encodes an ubiquitin E3 ligase that participates in protein ubiquitination. PtrRZFP1 mainly functions in the nucleus and endoplasmic reticulum and is activated by drought and ABA. PtrRZFP1-overexpressing transgenic poplars (35S:PtrRZFP1) showed greater tolerance to drought, whereas PtrRZFP1-knockdown lines (KD-PtrRZFP1) showed greater sensitivity to drought. Under treatment with polyethylene glycol and ABA, PtrRZFP1 promoted the production of nitric oxide and hydrogen peroxide in stomatal guard cells, ultimately enhancing stomatal closure and improving drought tolerance. Additionally, PtrRZFP1 physically interacted with the clade A Protein Phosphatase 2C protein PtrPP2C-9, a core regulator of ABA signaling, and mediated its ubiquitination and eventual degradation through the ubiquitination-26S proteasome system, indicating that PtrRZFP1 positively regulates the ABA signaling pathway. Furthermore, the PtrPP2C-9-overexpression line was insensitive to ABA and more sensitive to drought than the wild-type plants, whereas the opposite phenotype was observed in 35S:PtrRZFP1 plants. In general, PtrRZFP1 negatively regulates the stability of PtrPP2C-9 to mediate poplar drought tolerance. The results of this study provide a theoretical framework for the targeted breeding of drought-tolerant traits in perennial woody plants.

Genome-wide identification of SINA gene family in Medicago truncatula and functional analysis of MtSINAL7.

Ubiquitination is an essential biological process that is vital for maintaining cellular activity and plays a critical role in precisely regulating protein levels within cells. The SINA (seven in absentia) protein belongs to the RING-type E3 ubiquitin ligase, which is one of the key enzymes involved in the process of ubiquitination. However, there have been few reports on the genome-wide identification of SINA gene family and the functional analysis of its specific genes, particularly in leguminous plants. In this study, a total of 20 MtSINA genes were identified from the genomes of Medicago truncatula, and classified into three subfamilies. These genes are distributed on 7 of 8 chromosomes with chromosome preference. The gene structures of most MtSINA genes are quite similar, and all MtSINA proteins contain conserved RING and SINA functional domains. Moreover, various cis-regulatory elements related to abiotic stress and hormone signals were found in the promoters of MtSINA genes. The expression profile indicates that a majority of MtSINA genes exhibit a significant response to abiotic stress. Furthermore, the study characterized the function of MtSINAL7 in plants and discovered its pivotal role in improving plant stress resistance. In summary, this study provides a new insight into the potential functions of MtSINA genes in Medicago truncatula.

GL5.2, a Quantitative Trait Locus for Rice Grain Shape, Encodes a RING-Type E3 Ubiquitin Ligase.

Grain weight and grain shape are important traits that determine rice grain yield and quality. Mining more quantitative trait loci (QTLs) that control grain weight and shape will help to further improve the molecular regulatory network of rice grain development and provide gene resources for high-yield and high-quality rice varieties. In the present study, a QTL for grain length (GL) and grain width (GW), qGL5.2, was firstly fine-mapped into a 21.4 kb region using two sets of near-isogenic lines (NILs) derived from the indica rice cross Teqing (TQ) and IRBB52. In the NIL populations, the GL and ratio of grain length to grain width (RLW) of the IRBB52 homozygous lines increased by 0.16-0.20% and 0.27-0.39% compared with the TQ homozygous lines, but GW decreased by 0.19-0.75%. Then, by analyzing the grain weight and grain shape of the knock-out mutant, it was determined that the annotation gene Os05g0551000 encoded a RING-type E3 ubiquitin ligase, which was the cause gene of qGL5.2. The results show that GL and RLW increased by 2.44-5.48% and 4.19-10.70%, but GW decreased by 1.69-4.70% compared with the recipient. Based on the parental sequence analysis and haplotype analysis, one InDel variation located at -1489 in the promoter region was likely to be the functional site of qGL5.2. In addition, we also found that the Hap 5 (IRBB52-type) increased significantly in grain length and grain weight compared with other haplotypes, indicating that the Hap 5 can potentially be used in rice breeding to improve grain yield and quality.

The RING-Type E3 Ubiquitin Ligase Gene GhDIRP1 Negatively Regulates Verticillium dahliae Resistance in Cotton (Gossypium hirsutum).

Cotton is one of the world's most important economic crops. Verticillium wilt is a devastating cotton disease caused by Verticillium dahliae, significantly impacting cotton yield and quality. E3 ubiquitin ligases are essential components of the ubiquitin-mediated 26S proteasome system, responsible for recognizing ubiquitinated target proteins and promoting their degradation, which play a crucial regulatory role in plant immune responses. In this study, on the basis of the confirmation of differential expression of GhDIRP1, a RING-type E3 ubiquitin ligase encoding gene, in two cotton varieties resistant (Zhongzhimian 2) or susceptible (Jimian 11) to V. dahliae, we demonstrated that GhDIRP1 is a negative regulator of V. dahliae resistance because silencing GhDIRP1 in cotton and heterogeneously overexpressing the gene in Arabidopsis enhanced and compromised resistance to V. dahliae, respectively. The GhDIRP1-mediated immune response seemed to be realized through multiple physiological pathways, including hormone signaling, reactive oxygen species, and lignin biosynthesis. Based on the sequences of GhDIRP1 isolated from Zhongzhimian 2 and Jimian 11, we found that GhDIRP1 had identical coding but different promoter sequences in the two varieties, with the promoter of Zhongzhimian 2 being more active than that of Jimian 11 because the former drove a stronger expression of GUS and LUC reporter genes. The results link the ubiquitination pathway to multiple physiological pathways acting in the cotton immune response and provide a candidate gene for breeding cotton varieties resistant to V. dahliae.

Role of MARCH E3 ubiquitin ligases in cancer development.

Membrane-associated RING-CH (MARCH) E3 ubiquitin ligases, a family of RING-type E3 ubiquitin ligases, have garnered increased attention for their indispensable roles in immune regulation, inflammation, mitochondrial dynamics, and lipid metabolism. The MARCH E3 ligase family consists of eleven distinct members, and the dysregulation of many of these members has been documented in several human malignancies. Over the past two decades, extensive research has revealed that MARCH E3 ligases play pivotal roles in cancer progression by ubiquitinating key oncogenes and tumor suppressors and orchestrating various signaling pathways. Some MARCH E3s act as oncogenes, while others act as tumor suppressors, and the majority of MARCH E3s play both oncogenic and tumor suppressive roles in a context-dependent manner. Notably, there is special emphasis on the sole mitochondrial MARCH E3 ligase MARCH5, which regulates mitochondrial homeostasis within cancer cells. In this review, we delve into the diverse functions of MARCH E3 ligases across different cancer types, shedding light on the underlying molecular mechanisms mediating their effects, their regulatory effects on cancer and their potential as therapeutic targets.

Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

CsMIEL1 effectively inhibits the accumulation of anthocyanins under low temperatures in tea plants (Camelliasinensis)

Tea is one of the most prevalent non-alcoholic beverages. The leaves of tea plants hyperaccumulate anthocyanins under cold stress, resulting in enhanced bitterness. Previously, we determined that the RING-type E3 ubiquitin ligase CsMIEL1 from the tea plant (Camellia sinensis (L.) O. Kuntze) is involved in the response to stress conditions. This study aimed to determine the role of CsMIEL1 in anthocyanin accumulation at the post-translational modification level. The results showed that the heterologous expression of CsMIEL1 led to an 86% decrease in anthocyanin levels, resulting in a significant decrease in the mRNA levels of related genes in Arabidopsis at low temperatures but no significant differences in other phenotypes. Furthermore, multi-omics analysis and yeast two-hybrid library screening were performed to identify potential downstream targets of CsMIEL1. The results showed that the overexpression of CsMIEL1 resulted in 45% (448) of proteins being differentially expressed, of which 8% (36) were downregulated in A.thaliana, and most of these differentially expressed proteins (DEPs) were clustered in the plant growth and secondary metabolic pathways. Among the 71 potential targets that may interact with CsMIEL1, CsMYB90 and CsGSTa, which are related to anthocyanin accumulation, were selected. In subsequent analyses, these two proteins were verified to interact with CsMIEL1 via yeast two-hybrid (Y2H) and pull-down analyses in vitro. In summary, we explored the potential mechanism by which the E3 ligase relieves anthocyanin hyperaccumulation at low temperatures in tea plants. These results provide a new perspective on the mechanisms of anthocyanin regulation and the molecular breeding of tea plants.

TRIM13 reduces cholesterol efflux and increases oxidized LDL uptake leading to foam cell formation and atherosclerosis

Impaired cholesterol efflux and/or uptake can influence arterial lipid accumulation leading to atherosclerosis. Here, we report that tripartite motif-containing protein 13 (TRIM13), a RING-type E3 ubiquitin ligase, plays a role in arterial lipid accumulation leading to atherosclerosis. Using molecular approaches and KO mouse model, we found that TRIM13 expression was induced both in the aorta and peritoneal macrophages (pMφ) of ApoE-/- mice in response to Western diet (WD) invivo. Furthermore, proatherogenic cytokine interleukin-1β also induced TRIM13 expression both in pMφ and vascular smooth muscle cells. Furthermore, we found that TRIM13 via ubiquitination and degradation of liver X receptor (LXR)α/β downregulates the expression of their target genes ABCA1/G1 and thereby inhibits cholesterol efflux. In addition, TRIM13 by ubiquitinating and degrading suppressor of cytokine signaling 1/3 (SOCS1/3) mediates signal transducer and activator of transcription 1 (STAT1) activation, CD36 expression, and foam cell formation. In line with these observations, genetic deletion of TRIM13 by rescuing cholesterol efflux and inhibiting foam cell formation protects against diet-induced atherosclerosis. We also found that while TRIM13 and CD36 levels were increased, LXRα/β, ABCA1/G1, and SOCS3 levels were decreased both in Mφ and smooth muscle cells of stenotic human coronary arteries as compared to nonstenotic arteries. More intriguingly, the expression levels of TRIM13 and its downstream signaling molecules were correlated with the severity of stenotic lesions. Together, these observations reveal for the first time that TRIM13 plays a crucial role in diet-induced atherosclerosis, and that it could be a potential drug target against this vascular lesion.

E3 Ligases Regulate Organelle Inheritance in Yeast.

Saccharomyces cerevisiae proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin-myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.

IbNIEL-mediated degradation of IbNAC087 regulates jasmonic acid-dependent salt and drought tolerance in sweet potato.

Sweet potato (Ipomoea batatas [L.] Lam.) is a crucial staple and bioenergy crop. Its abiotic stress tolerance holds significant importance in fully utilizing marginal lands. Transcriptional processes regulate abiotic stress responses, yet the molecular regulatory mechanisms in sweet potato remain unclear. In this study, a NAC (NAM, ATAF1/2, and CUC2) transcription factor, IbNAC087, was identified, which is commonly upregulated in salt- and drought-tolerant germplasms. Overexpression of IbNAC087 increased salt and drought tolerance by increasing jasmonic acid (JA) accumulation and activating reactive oxygen species (ROS) scavenging, whereas silencing this gene resulted in opposite phenotypes. JA-rich IbNAC087-OE (overexpression) plants exhibited more stomatal closure than wild-type (WT) and IbNAC087-Ri plants under NaCl, polyethylene glycol, and methyl jasmonate treatments. IbNAC087 functions as a nuclear transcriptional activator and directly activates the expression of the key JA biosynthesis-related genes lipoxygenase (IbLOX) and allene oxide synthase (IbAOS). Moreover, IbNAC087 physically interacted with a RING-type E3 ubiquitin ligase NAC087-INTERACTING E3 LIGASE (IbNIEL), negatively regulating salt and drought tolerance in sweet potato. IbNIEL ubiquitinated IbNAC087 to promote 26S proteasome degradation, which weakened its activation on IbLOX and IbAOS. The findings provide insights into the mechanism underlying the IbNIEL-IbNAC087 module regulation of JA-dependent salt and drought response in sweet potato and provide candidate genes for improving abiotic stress tolerance in crops.

MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner

DNA topoisomerase II (TOP2) is an enzyme that performs a critical function in manipulating DNA topology during replication, transcription, and chromosomal compaction by forming a vital intermediate known as the TOP2-DNA cleavage complex (TOP2cc). Although the TOP2cc is often transient, stabilization can be achieved by TOP2 poisons, a family of anti-cancer chemotherapeutic agents targeting TOP2, such as etoposide (VP-16), and then induce double-strand breaks (DSBs) in cellular DNA. TOP2cc first needs to be proteolyzed before it can be processed by TDP2 for the removal of these protein adducts and to produce clean DNA ends necessary for proper repair. However, the mechanism by which TOP2βcc is proteolyzed has not been thoroughly studied. In this study, we report that after exposure to VP-16, MDM2, a RING-type E3 ubiquitin ligase, attaches to TOP2β and initiates polyubiquitination and proteasomal degradation. Mechanistically, during exposure to VP-16, TOP2β binds to DNA to form TOP2βcc, which promotes MDM2 binding and subsequent TOP2β ubiquitination and degradation, and results in a decrease in TOP2βcc levels. Biologically, MDM2 inactivation abrogates TOP2β degradation, stabilizes TOP2βcc, and subsequently increases the number of TOP2β-concealed DSBs, resulting in the rapid death of cancer cells via the apoptotic process. Furthermore, we demonstrate the combination activity of VP-16 and RG7112, an MDM2 inhibitor, in the xenograft tumor model and in situ lung cancer mouse model. Taken together, the results of our research reveal an underlying mechanism by which MDM2 promotes cancer cell survival in the presence of TOP2 poisons by activating proteolysis of TOP2βcc in a p53-independent manner, and provides a rationale for the combination of MDM2 inhibitors with TOP2 poisons for cancer therapy.

RNF43 Inactivation Enhances the B-RAF/MEK Signaling and Creates a Combinatory Therapeutic Target in Cancer Cells.

RING finger 43 (RNF43), a RING-type E3 ubiquitin ligase, is a key regulator of WNT signaling and is mutated in 6-10% of pancreatic tumors. However, RNF43-mediated effects remain unclear, as only a few in vivo substrates of RNF43 are identified. Here, it is found that RNF43-mutated pancreatic cancer cells exhibit elevated B-RAF/MEK activity and are highly sensitive to MEK inhibitors. The depletion of RNF43 in normal pancreatic ductal cells also enhances MEK activation, suggesting that it is a physiologically regulated process. It is confirmed that RNF43 ubiquitinates B-RAF at K499 to promote proteasome-dependent degradation, resulting in reduced MEK activity and proliferative ability in cancer cells. In addition, phosphorylation of B-RAF at T491 suppresses B-RAF ubiquitination by decreasing the interaction between RNF43 and B-RAF. Mutations at K499 in B-RAF are identified in various cancer types. MEK and WNT inhibitors synergistically suppress the growth of RNF43-mutated pancreatic cancer cells in vitro and in vivo. Collectively, the research reveals a novel mechanism by which RNF43 inhibits B-RAF/MEK signaling to suppress tumor growth and provide a new strategy for the treatment of RNF43-inactivated pancreatic cancer.

The co-crystal structure of Cbl-b and a small-molecule inhibitor reveals the mechanism of Cbl-b inhibition

Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment. A Cbl-b inhibitor, Nx-1607, is currently in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirm potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing the compound’s interaction with both the TKBD and LHR, but not the RING domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.

TRAIP suppressed apoptosis and cell cycle to promote prostate cancer proliferation via TRAF2-PI3K-AKT pathway activation.

TRAF-interacting protein (TRAIP) is a RING-type E3 ubiquitin ligase, which has been implicated in various cellular processes and participated in various cancers as an oncogene. However, the function and potential mechanism of TRAIP in prostate cancer (PCa) have not been investigated so far. Public TGCA data were used to evaluate the expression profile of TRAIP in prostatic tumors. The relative expression of TRAIP and TRAF2 in PCa tissues and tumor cell lines was detected by qPCR, western blot, and IHC staining. Next, TRAIP knockdown and overexpression plasmids were constructed and transfected into PCa cell lines. Moreover, cell proliferation, invasion, migration, and apoptosis were measured by colony formation, Transwell, wound healing, and flow cytometry assays. Subsequently, cell cycle and signaling pathway-related proteins were tested by western blot. Finally, the effect of TRAIP on PCa was measured based on the nude mouse xenograft model. TRAIP was significantly upregulated in PCa tissues and tumor cell lines. In addition, TRAIP promoted cell proliferation, invasion, and migration of PCa cell lines. Such an oncogenic property was mediated by the cell cycle arrest and the inhibition of apoptosis, as indicated by different functional assays and the expression of cell cycle and apoptosis regulatory proteins in cultured cells. Moreover, TRAIP combined with TRAF2 to activate PI3K/AKT pathway. Finally, TRAIP depletion suppressed the growth of tumors and cell proliferation in vivo. Our study first revealed that TRAIP promoted tumor progression and identified it as a potential therapeutic target for PCa patients in the future.

E3 ubiquitin ligase MaRZF1 modulates high temperature-induced green ripening of banana by degrading MaSGR1.

High temperatures (>24°C) prevent the development of a yellow peel on bananas called green ripening, owing to the inhibition of chlorophyll degradation. This phenomenon greatly reduces the marketability of banana fruit, but the mechanisms underlining high temperature-repressed chlorophyll catabolism need to be elucidated. Herein, we found that the protein accumulation of chlorophyll catabolic enzyme MaSGR1 (STAY-GREEN 1) was reduced when bananas ripened at high temperature. Transiently expressing MaSGR1 in banana peel showed its positive involvement in promoting chlorophyll degradation under high temperature, thereby weakening green ripening phenotype. Using yeast two-hybrid screening, we identified a RING-type E3 ubiquitin ligase, MaRZF1 (RING Zinc Finger 1), as a putative MaSGR1-interacting protein. MaRZF1 interacts with and targets MaSGR1 for ubiquitination and degradation via the proteasome pathway. Moreover, upregulating MaRZF1 inhibited chlorophyll degradation, and attenuated MaSGR1-promoted chlorophyll degradation in bananas during green ripening, indicating that MaRZF1 negatively regulates chlorophyll catabolism via the degradation of MaSGR1. Taken together, MaRZF1 and MaSGR1 form a regulatory module to mediate chlorophyll degradation associated with high temperature-induced green ripening in bananas. Therefore, our findings expand the understanding of posttranslational regulatory mechanisms of temperature stress-caused fruit quality deterioration.

ZmTDM1 encodes a tetratricopeptide repeat domain protein and is required for meiotic exit in maize.

Elaborate cell-cycle control must be adopted to ensure the continuity of the meiotic second division and termination after that. Despite its importance, however, the genetic controls underlying the meiotic cell cycle have not been reported in maize. Here, we characterized a meiotic cell-cycle controller ZmTDM1, which is a homolog of Arabidopsis TDM1 and encodes a canonical tetratricopeptide repeat domain protein in maize. The Zmtdm1 homozygous plants exhibited complete male sterility and severe female abortion. In Zmtdm1 mutants, cell-cycle progression was almost identical to that of wild type from leptotene to anaphase II. However, chromosomes in the tetrad failed meiotic termination at the end of the second division and underwent additional divisions in succession without DNA replication, reducing the ploidy to less than haploid in the product. In addition, two ZmTDM1-like homologs (ZmTDML1 and ZmTDML2) were not functional in meiotic cell-cycle control. Moreover, ZmTDM1 interacted with RING-type E3 ubiquitin ligase, revealing that it acts as a subunit of the APC/C E3 ubiquitin ligase complex. Overall, our results identified a regulator of meiotic cell cycle in maize and demonstrated that ZmTDM1 is essential for meiotic exit after meiosis II.