Degron Motif Research Articles

SPOP-mediated RIPK3 destabilization desensitizes LPS/sMAC/zVAD-induced necroptotic cell death.

RIPK1/RIPK3-MLKL signaling molecules are fundamental in initiating necroptotic cell death, but their roles in the development of colon cancer are unclear. This study reports that RIPK3 interacted with SPOP, a component of the E3 ligase within the Cul3 complex. This interaction leads to K48-linked ubiquitination and subsequent proteasomal degradation of RIPK3. Two distinct degron motifs, PETST and SPTST, were identified within the linker domain of RIPK3 for SPOP. RIPK3 phosphorylations at Thr403 by PIM2 and at Thr412/Ser413 by ERK2 are essential to facilitate its interaction with SPOP. Computational docking studies and immunoprecipitation analyses showed that these PIM2 and ERK2 phosphorylations bolster the stability of the RIPK3-SPOP interaction. In particular, mutations of RIPK3 at the degron motifs extended the half-life of RIPK3 by preventing its phosphorylation and subsequent ubiquitination. The deletion of SPOP, which led to increased stability of the RIPK3 protein, intensified LPS/sMAC/zVAD-induced necroptotic cell death in colon cancer cells. These findings underscore the critical role of the SPOP-mediated RIPK3 stability regulation pathway in controlling necroptotic cell death.

A gain-of-function mutation in BnaIAA13 disrupts vascular tissue and lateral root development in Brassica napus.

Rapeseed (Brassica napus) is an important oilseed crop worldwide. Plant vascular tissues are responsible for long-distance transport of water and nutrients and for providing mechanical support. The lateral roots absorb water and nutrients. The genetic basis of vascular tissue and lateral root development in rapeseed remains unknown. This study characterized an ethyl methanesulfonate-mutagenized rapeseed mutant, T16, which showed dwarf stature, reduced lateral roots, and leaf wilting. SEM observations showed that the internode cells were shortened. Observations of tissue sections revealed defects in vascular bundle development in the stems and petioles. Genetic analysis revealed that the phenotypes of T16 were controlled by a single semi-dominant nuclear gene. Map-based cloning and genetic complementarity identified BnaA03.IAA13 as the functional gene; a G-to-A mutation in the second exon changed glycine at position 79 to glutamic acid, disrupting the conserved degron motif VGWPP. Transcriptome analysis in roots and stems showed that auxin and cytokinin signaling pathways were disordered in T16. Evolutionary analysis showed that AUXIN/INDOLE-3-ACETIC ACID is conserved during plant evolution. The heterozygote of T16 showed significantly reduced plant height while maintaining other agronomic traits. Our findings provide novel insights into the regulatory mechanisms of vascular tissue and lateral root development, and offer a new germplasm resource for rapeseed breeding.

Molecular insights into degron recognition by CRL5ASB7 ubiquitin ligase

The ankyrin (ANK) SOCS box (ASB) family, encompassing ASB1–18, is the largest group of substrate receptors of cullin 5 Ring E3 ubiquitin ligase. Nonetheless, the mechanism of substrate recognition by ASB family proteins has remained largely elusive. Here we present the crystal structure of ASB7-Elongin B-Elongin C ternary complex bound to a conserved helical degron. ASB7 employs its ANK3-6 to form an extended groove, effectively interacting with the internal α-helix-degron through a network of side-chain-mediated electrostatic and hydrophobic interactions. Our structural findings, combined with biochemical and cellular analyses, identify the key residues of the degron motif and ASB7 required for their recognition. This will facilitate the identification of additional physiological substrates of ASB7 by providing a defined degron motif for screening. Furthermore, the structural insights provide a basis for the rational design of compounds that can specifically target ASB7 by disrupting its interaction with its cognate degron.

Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

A novel mutation in IAA16 is associated with dicamba resistance in Chenopodium album.

Resistance to dicamba in Chenopodium album was first documented over a decade ago, however, the molecular basis of dicamba resistance in this species has not been elucidated. In this research, the resistance mechanism in a dicamba-resistant C. album phenotype was investigated using a transcriptomics (RNA-sequence) approach. The dose-response assay showed that the resistant (R) phenotype was nearly 25-fold more resistant to dicamba than a susceptible (S) phenotype of C. album. Also, dicamba treatment significantly induced transcription of the known auxin-responsive genes, Gretchen Hagen 3 (GH3), small auxin-up RNAs (SAURs), and 1-aminocyclopropane-1-carboxylate synthase (ACS) genes in the susceptible phenotype. Comparing the transcripts of auxin TIR/AFB receptors and auxin/indole-3-acetic acid (AUX/IAA) proteins identified from C. album transcriptomic analysis revealed that the R phenotype contained a novel mutation at the first codon of the GWPPV degron motif of IAA16, resulting in an amino acid substitution of glycine (G) with aspartic acid (D). Sequencing the IAA16 gene in other R and S individuals further confirmed that all the R individuals contained the mutation. In this research, we describe the dicamba resistance mechanism in the only case of dicamba-resistant C. album reported to date. Prior work has shown that the dicamba resistance allele confers significant growth defects to the R phenotype investigated here, suggesting that dicamba-resistant C. album carrying this novel mutation in the IAA16 gene may not persist at high frequencies upon removal of dicamba application. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2′3′-cyclic GMP-AMP (cGAMP)1–7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8–15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin–RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.

Lysine Methylation-Dependent Proteolysis by the Malignant Brain Tumor (MBT) Domain Proteins.

Lysine methylation is a major post-translational protein modification that occurs in both histones and non-histone proteins. Emerging studies show that the methylated lysine residues in non-histone proteins provide a proteolytic signal for ubiquitin-dependent proteolysis. The SET7 (SETD7) methyltransferase specifically transfers a methyl group from S-Adenosyl methionine to a specific lysine residue located in a methylation degron motif of a protein substrate to mark the methylated protein for ubiquitin-dependent proteolysis. LSD1 (Kdm1a) serves as a demethylase to dynamically remove the methyl group from the modified protein. The methylated lysine residue is specifically recognized by L3MBTL3, a methyl-lysine reader that contains the malignant brain tumor domain, to target the methylated proteins for proteolysis by the CRL4DCAF5 ubiquitin ligase complex. The methylated lysine residues are also recognized by PHF20L1 to protect the methylated proteins from proteolysis. The lysine methylation-mediated proteolysis regulates embryonic development, maintains pluripotency and self-renewal of embryonic stem cells and other stem cells such as neural stem cells and hematopoietic stem cells, and controls other biological processes. Dysregulation of the lysine methylation-dependent proteolysis is associated with various diseases, including cancers. Characterization of lysine methylation should reveal novel insights into how development and related diseases are regulated.

Intrinsic disorder and conformational coexistence in auxin coreceptors

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Defining E3 ligase–substrate relationships through multiplex CRISPR screening

Specificity within the ubiquitin–proteasome system is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates—and in particular the molecular features (degrons) that they recognize—remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9/13 and Cul3KLHL15, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the ubiquitin–proteasome system.

Degron Pathways and Leishmaniasis: Debating Potential Roles of Leishmania spp. Proteases Activity on Guiding Hosts Immune Response and Their Relevance to the Development of Vaccines.

Degrons are short peptide sequences that signalize target sites for protein degradation by proteases. Herein, we bring forth the discussion on degrons present in proteins related to the immune system of Mus musculus that are potential targets for cysteine and serine proteases of Leishmania spp. and their possible roles on host immune regulation by parasites. The Merops database was used to identify protease substrates and proteases sequence motifs, while MAST/MEME Suite was applied to find degron motifs in murine cytokines (IFN-y, IL-4, IL-5, IL-13, IL-17) and transcription factors (NF-kappaB, STAT-1, AP-1, CREB, and BACH2). STRING tool was used to construct an interaction network for the immune factors and SWISS-MODEL server to generate three-dimensional models of proteins. In silico assays confirm the occurrence of degrons in the selected immune response factors. Further analyses were conducted only in those with resolved three-dimensional structures. The predicted interaction network of degron-containing M. musculus proteins shows the possibility that the specific activity of parasite proteases could interfere with the trend of Th1/Th2 immune responses. Data suggest that degrons may play a role in the immune responses in leishmaniases as targets for parasite proteases activity, directing the degradation of specific immune-related factors.

In silico analysis of the profilaggrin sequence indicates alterations in the stability, degradation route, and intracellular protein fate in filaggrin null mutation carriers.

Background: Loss of function mutation in FLG is the major genetic risk factor for atopic dermatitis (AD) and other allergic manifestations. Presently, little is known about the cellular turnover and stability of profilaggrin, the protein encoded by FLG. Since ubiquitination directly regulates the cellular fate of numerous proteins, their degradation and trafficking, this process could influence the concentration of filaggrin in the skin. Objective: To determine the elements mediating the interaction of profilaggrin with the ubiquitin-proteasome system (i.e., degron motifs and ubiquitination sites), the features responsible for its stability, and the effect of nonsense and frameshift mutations on profilaggrin turnover. Methods: The effect of inhibition of proteasome and deubiquitinases on the level and modifications of profilaggrin and processed products was assessed by immunoblotting. Wild-type profilaggrin sequence and its mutated variants were analysed in silico using the DEGRONOPEDIA and Clustal Omega tool. Results: Inhibition of proteasome and deubiquitinases stabilizes profilaggrin and its high molecular weight of presumably ubiquitinated derivatives. In silico analysis of the sequence determined that profilaggrin contains 18 known degron motifs as well as multiple canonical and non-canonical ubiquitination-prone residues. FLG mutations generate products with increased stability scores, altered usage of the ubiquitination marks, and the frequent appearance of novel degrons, including those promoting C-terminus-mediated degradation routes. Conclusion: The proteasome is involved in the turnover of profilaggrin, which contains multiple degrons and ubiquitination-prone residues. FLG mutations alter those key elements, affecting the degradation routes and the mutated products' stability.

MYO10 regulates genome stability and cancer inflammation through mediating mitosis

Genomic instability can promote inflammation and tumor development. Previous research revealed an unexpected layer of regulation of genomic instability by a cytoplasmic protein MYO10; however, the underlying mechanism remained unclear. Here, we report a protein stability-mediated mitotic regulation of MYO10 in controlling genome stability. We characterized a degron motif and phosphorylation residues in the degron that mediate β-TrCP1-dependent MYO10 degradation. The level of phosphorylated MYO10 protein transiently increases during mitosis, which is accompanied by a spatiotemporal cellular localization change first accumulating at the centrosome then at the midbody. Depletion of MYO10 or expression of MYO10 degron mutants, including those found in cancer patients, disrupts mitosis, increases genomic instability and inflammation, and promotes tumor growth; however, they also increase the sensitivity of cancer cells to Taxol. Our studies demonstrate a critical role of MYO10 in mitosis progression, through which it regulates genome stability, cancer growth, and cellular response to mitotic toxins.

Three KINSSHIP syndrome patients with mosaic and germline AFF3 variants.

AFF3 at 2q11.2 encodes the nuclear transcriptional activator AF4/FMR2 Family Member 3. AFF3 constitutes super elongation complex like 3, which plays a role in promoting the expression of genes involved in neurogenesis and development. The degron motif in AFF3 with nine highly conserved amino acids is recognized by E3 ubiquitin ligase to induce protein degradation. Recently, AFF3 missense variants in this region and variants featuring deletion including this region were identified and shown to cause KINSSHIP syndrome. In this study, we identified two novel and one previously reported missense variants in the degron of AFF3 in three unrelated Japanese patients. Notably, two of these three variants exhibited mosaicism in the examined tissues. This study suggests that mosaic variants also cause KINSSHIP syndrome, showing various phenotypes.

MEKs/ERKs-mediated FBXO1/E2Fs interaction interference modulates G1/S cell cycle transition and cancer cell proliferation.

E2F 1, 2, and 3a, (refer to as E2Fs) are a subfamily of E2F transcription factor family that play essential roles in cell-cycle progression, DNA replication, DNA repair, apoptosis, and differentiation. Although the transcriptional regulation of E2Fs has focused on pocket protein retinoblastoma protein complex, recent studies indicate that post-translational modification and stability regulation of E2Fs play key roles in diverse cellular processes. In this study, we found that FBXO1, a component of S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) complex, is an E2Fs binding partner. Furthermore, FBXO1 to E2Fs binding induced K48 ubiquitination and subsequent proteasomal degradation of E2Fs. Binding domain analysis indicated that the Arg (R)/Ile (I) and R/Val (V) motifs, which are located in the dimerization domain of E2Fs, of E2F 1 and 3a and E2F2, respectively, acted as degron motifs (DMs) for FBXO1. Notably, RI/AA or RV/AA mutation in the DMs reduced FBXO1-mediated ubiquitination and prolonged the half-lives of E2Fs. Importantly, the stabilities of E2Fs were affected by phosphorylation of threonine residues located near RI and RV residues of DMs. Phosphorylation prediction database analysis and specific inhibitor analysis revealed that MEK/ERK signaling molecules play key roles in FBXO1/E2Fs' interaction and modulate E2F protein turnover. Moreover, both elevated E2Fs protein levels by knockdown of FBXO1 and decreased E2Fs protein levels by sh-E2F3a delayed G1/S cell cycle transition, resulting in inhibition of cancer cell proliferation. These results demonstrated that FBXO1-E2Fs axis-mediated precise E2Fs stability regulation plays a key role in cell proliferation via G1/S cell cycle transition.

Building yeast libraries to dissect terminal degrons with fluorescent timers.

Selective degradation of unnecessary or abnormal proteins by the ubiquitin-proteasome system is an essential part of proteostasis. Ubiquitin ligases recognize substrates of selective protein degradation and modify them with polyubiquitin chains, which mark them for proteasomal degradation. Substrate recognition by ubiquitin ligases often involves degradation signals or degrons, which are typically short linear motifs found in intrinsically disordered regions, e.g., at protein termini. However, specificity in selective protein degradation is generally not well understood, as for most ubiquitin ligases no degrons have been identified thus far. To address this limitation, high-throughput mutagenesis approaches, such as multiplexed protein stability (MPS) profiling, have been developed, enabling systematic surveys of degrons in vivo or allowing to define degron motifs recognized by different ubiquitin ligases. In MPS profiling, thousands of short peptides can be assessed in parallel for their ability to trigger degradation of a fluorescent timer reporter. Here, we describe common types of libraries used to identify and dissect degrons located at protein termini using MPS profiling in budding yeast, and provide protocols for their construction.

Identification and characterization of a curly-leaf locus CL1 encoding an IAA2 protein in Brassica napus

The leaf is the main organ for rapeseed photosynthesis, and its morphology influences photosynthetic efficiency and supports increased planting density and yield. However, the molecular regulatory mechanism of leaf morphology in Brassica napus is poorly understood, restricting progress in breeding for the trait. We describe a novel dominant mutation, curly leaf 1 (cl1), which confers uneven dorsal–ventral axis development, irregular cellular structure and influenced gravitropic response in the seedling stage. The CL1 locus was mapped to a 1.573-Mb interval on chromosome A05 using simple sequence repeat (SSR) markers, and co-segregated with the phenotype of plants in the curly F2 population. A substitution (P62S) was identified in the highly conserved degron motif (GWSPV) of the IAA2 protein in the cl1 mutant, and the P62S substitution impaired the interaction between IAA2 and TIR1 in the presence of auxin, influencing auxin signaling. The P62S substitution-induced curly leaf phenotype was verified by ectopic expression of BnaA05.iaa2 in Arabidopsis and B. napus. Our findings explain the function of IAA2 in rapeseed, providing a foundation for future investigation of auxin signaling and the mechanisms underlying leaf development in B. napus.

Revisiting degron motifs in human AURKA required for its targeting by APC/CFZR1.

Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the anaphase-promoting complex (APC/CFZR1) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the "A-box" in addition to a reported canonical D-box degron in the C-terminus. Here, we find that the reported C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, the N-terminal intrinsically disordered region of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL short linear interacting motif of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA also depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA.

Novel Protease-Mediated Double Antigen Recognizing Chimeric Antigen Receptor (CAR) Enhances Directionality of CAR-T Cell Activity and Improves Target Cell Specificity

Recently, Chimeric Antigen Receptor (CAR)-T cell therapies show excellent therapeutic effect against various hematologic malignancies, including B-cell leukemia, B-cell lymphoma, and multiple myeloma. Expansion of target diseases of this attractive immunotherapy is one of the most important issues. To identify the novel specific tumor antigen as CAR-T cell target is the standard way to accomplish this goal. However, recent CAR-T cells recognize single tumor antigen, so normal tissues expressing the target antigen are possible to be attacked by CAR-T cells. For example, anti-CD19 CAR-T cells against B-cell malignancies kill not only tumor cells but also normal B-cells, so patients receiving the CAR-T cells showed severe immune deficiency. Improvement of target cell recognition system of CAR-T cells minimizes this adverse effect and contributes to expand target diseases. To improve target-cell-specificity of CAR-T cell, we established a protease-mediated regulatory CAR, named "Scissors-CAR" (panel A). This regulatory CAR is constituted with extracellular antigen recognition portion and cytoplasmic Human Immunodeficiency Virus protease (HIVPR) catalytic domain. When CAR-T cells expressing both anti-A Scissors-CAR and anti-B Counterpart-CAR that has HIVPR recognition polypeptide contact with target tumor expressing both antigen-A and B, these two CARs interact on cell membrane. This interaction induces Scissors-CAR-mediated cleavage of target polypeptide and structural change of Counterpart-CAR. Previously, we reported double antigen recognition system using Scissors-CAR. Previous Counterpart-CAR named "Signal-CAR" had HIVPR recognition polypeptide upstream of CD3-zeta, so Scissors-CAR-mediated cleavage broke the CAR protein structure and suppressed CAR-T cell activity (panel B, named "Negative system"). This system allowed CAR-T cells to directional attack against target cells expressing only antigen-A and protect the cells expressing both antigen-A and B. Here, we established another double-antigen recognition system using Scissors-CAR. In many cases including both hematologic malignancies and solid tumors, additional ectopic antigens are expressed on malignant cells. If CAR-T cells empowered to directional attack against target cells expressing both antigen-A and B, much more tumors can be treated with this tumor immunotherapy (panel C, named "Positive system"). To establish this directional CAR-T cells, we constructed novel Counterpart-CAR named as "Activating-CAR" constituted with target recognition portion, co-stimulatory domain, CD3-zeta, and protein destabilization motif known as degron. Between CD3-zeta and degron motif, HIVPR recognition polypeptide was inserted. In vitro analysis revealed that this degron motif destabilized Activating-CAR and HIVPR-mediated cleavage and that release of degron motif increased Activating-CAR stability. Furthermore, cell proliferation of Jurkat cells expressing both anti-CD19 Activating-CAR and anti-HER2 Scissors-CAR was enhanced co-cultivated with target cells expressing both CD19 and HER2. From these results, combined use of Activating-CAR and Scissors-CAR allowed CAR-T cells to directionally activate against target cells expressing two antigens recognized by each CAR. By choosing the Counterpart-CAR from "Signal-CAR" or "Activating-CAR", this protease-mediated double antigen recognition system could optimize target-cell-specificity of CAR-T cells for the individual tumor and contribute to expand the target tumors of CAR-T cell therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification and structural basis of C-terminal cyclic imides as natural degrons for cereblon

Cereblon (CRBN) is a ubiquitously expressed E3 ligase substrate receptor and a key player in pharmaceutical targeted protein degradation. Despite substantial insight gained into its chemical ligand space that is exploited in small-molecule protein degraders, its cellular role and native mechanism of substrate recognition remained elusive so far. In this communication, we report the discovery of C-terminal aspartimide and aminoglutarimide residues as natural degron motifs that are recognized by CRBN with high specificity. These C-terminal cyclic imides are known to form in ageing proteins as a result of spontaneous chain breaks after an attack of an asparagine or glutamine side chain amide on the adjacent peptide bond, and thereby mark potentially malfunctional protein fragments. In crystal structures, we uncover that these C-terminal cyclic imides are bound in the same fashion as small-molecule CRBN modulators, and that the residues preceding the cyclic terminus contribute to the interaction with a sequence-unspecific backbone hydrogen bonding pattern with strictly conserved residues in CRBN. We postulate that C-terminal aspartimide and aminoglutarimide residues resulting from chain breaks are largely underappreciated protein damages and represent the native degrons of CRBN.

Integrated genetic mapping and transcriptome analysis reveal the BnaA03.IAA7 protein regulates plant architecture and gibberellin signaling in Brassica napus L.

A novel mutation in the BnaA03.IAA7 protein reduces plant height and enhances gibberellin signaling in Brassica napus L. Rapeseed (Brassica napus) is an excellent and important source for vegetable oil production, but its production is severely affected by lodging. Lodging hinders mechanization and decreases yield, and an ideal solution is semidwarf breeding. Limited by germplasm resources, semidwarf breeding developed slowly in rapeseed. In the current study, a mutant called sdA03 was isolated from EMS-mutagenized lines of Zhongshuang 11 (ZS11). The inheritance analysis showed that phenotypes of sdA03 were controlled by a single semidominant gene. Genetic mapping, RNA-seq and candidate gene analysis identified BnaA03.IAA7 as a candidate gene, and a function test confirmed that the mutated BnaA03.iaa7 regulates plant architecture in a dose-dependent manner. Yeast two-hybrid and transient expression experiments illustrated the P87L substitution in the GWPPV/I degron motif of BnaA03.iaa7 impaired the interaction between BnaA03.IAA7 and TIR1 proteins, and BnaA03.iaa7 prevented ARF from activating the auxin signaling pathway.The gibberellin (GA) content was higher in sdA03 hypocotyls than in those of ZS11. Further expression analysis showed more active gibberellin signaling in hypocotyl and richer expression of GA synthetic genes in root and cotyledon of sdA03 seedlings. Finally, a marker was developed based on the SNP found in BnaA03.iaa7 and used in molecular breeding. The study enriched our understanding of the architectural regulation of rapeseed and provided germplasm resources for breeding.