Insights into the distinct membrane targeting mechanisms of WDR91 family proteins.

WDR91 and SORF1, members of the WD repeat-containing protein 91 family, control phosphoinositide conversion by inhibiting phosphatidylinositol 3-kinase activity on endosomes, which promotes endosome maturation. Here, we report the crystal structure of the human WDR91 WD40 domain complexed with Rab7 that has an unusual interface at the C-terminus of the Rab7 switch II region. WDR91 is highly selective for Rab7 among the tested GTPases. A LIS1 homology (LisH) motif within the WDR91 N-terminal domain (NTD) mediates self-association and may contribute partly to the augmented interaction between full-length WDR91 and Rab7. Both the Rab7 binding site and the LisH motif are indispensable for WDR91 function in endocytic trafficking. For the WDR91 orthologue SORF1 lacking the C-terminal WD40 domain, a C-terminal amphipathic helix (AH) mediates strong interactions with liposomes containing acidic lipids. During evolution the human WDR91 ancestor gene might have acquired a WD40 domain to replace the AH for endosomal membrane targeting.

The Innovative Role of Nuclear Receptor Interaction Protein in Orchestrating Invadosome Formation for Myoblast Fusion.

BACKGROUND: Nuclear receptor interaction protein (NRIP) is versatile and engages with various proteins to execute its diverse biological function. NRIP deficiency was reported to cause small myofibre size in adult muscle regeneration, indicating a crucial role of NRIP in myoblast fusion. METHODS: The colocalization and interaction of NRIP with actin were investigated by immunofluorescence and immunoprecipitation assay, respectively. The participation of NRIP in myoblast fusion was demonstrated by cell fusion assay and time-lapse microscopy. The NRIP mutants were generated for mechanism study in NRIP-null C2C12 (termed KO19) cells and muscle-specific NRIP knockout (NRIP cKO) mice. A GEO profile database was used to analyse NRIP expression in Duchenne muscular dystrophy (DMD) patients. RESULTS: In this study, we found that NRIP directly and reciprocally interacted with actin both in vitro and in cells. Immunofluorescence microscopy showed that the endogenous NRIP colocalized with components of invadosome, such as actin, Tks5, and cortactin, at the tips of cells during C2C12 differentiation. The KO19 cells were generated and exhibited a significant deficit in myoblast fusion compared with wild-type C2C12 cells (3.16% vs. 33.67%, p < 0.005). Overexpressed NRIP in KO19 cells could rescue myotube formation compared with control (3.37% vs. 1.00%, p < 0.01). We further confirmed that NRIP directly participated in cell fusion by using a cell-cell fusion assay. We investigated the mechanism of invadosome formation for myoblast fusion, which depends on NRIP-actin interaction, by analysing NRIP mutants in NRIP-null cells. Loss of actin-binding of NRIP reduced invadosome (enrichment ratio, 1.00 vs. 2.54, p < 0.01) and myotube formation (21.82% vs. 35.71%, p < 0.05) in KO19 cells and forced NRIP expression in KO19 cells and muscle-specific NRIP knockout (NRIP cKO) mice increased myofibre size compared with controls (over 1500 µm2, 61.01% vs. 20.57%, p < 0.001). We also found that the NRIP mRNA level was decreased in DMD patients compared with healthy controls (18 072 vs. 28 289, p < 0.001, N = 10 for both groups). CONCLUSIONS: NRIP is a novel actin-binding protein for invadosome formation to induce myoblast fusion.

Genome-wide identification of CaWD40 proteins reveals the involvement of a novel complex (CaAN1-CaDYT1-CaWD40-91) in anthocyanin biosynthesis and genic male sterility in Capsicum annuum.

BACKGROUND: The WD40 domain, one of the most abundant in eukaryotic genomes, is widely involved in plant growth and development, secondary metabolic biosynthesis, and mediating responses to biotic and abiotic stresses. WD40 repeat (WD40) protein has been systematically studied in several model plants but has not been reported in the Capsicum annuum (pepper) genome. RESULTS: Herein, 269, 237, and 257 CaWD40 genes were identified in the Zunla, CM334, and Zhangshugang genomes, respectively. CaWD40 sequences from the Zunla genome were selected for subsequent analysis, including chromosomal localization, phylogenetic relationships, sequence characteristics, motif compositions, and expression profiling. CaWD40 proteins were unevenly distributed on 12 chromosomes, encompassing 19 tandem duplicate gene pairs. The 269 CaWD40s were divided into six main branches (A to F) with 17 different types of domain distribution. The CaWD40 gene family exhibited diverse expression patterns, and several genes were specifically expressed in flowers and seeds. Yeast two-hybrid (Y2H) and dual-luciferase assay indicated that CaWD40-91 could interact with CaAN1 and CaDYT1, suggesting its involvement in anthocyanin biosynthesis and male sterility in pepper. CONCLUSIONS: In summary, we systematically characterized the phylogeny, classification, structure, and expression of the CaWD40 gene family in pepper. Our findings provide a valuable foundation for further functional investigations on WD40 genes in pepper.

Disturbed function of TBL1X has a differential effect on T3-regulated gene expression in two human liver cell models.

Background: Mutations in TBL1X, part of the NCOR1/SMRT corepressor complex, were identified in patients with hereditary X-linked central congenital hypothyroidism and associated hearing loss. The role of TBL1X in thyroid hormone (TH) action, however, is incompletely understood. The aim of the present study was to investigate the role of TBL1X on T3-regulated gene expression in two human liver cell models. Methods: A human hepatoma cell line (HepG2) wherein TBL1X was downregulated using siRNAs, and human-induced pluripotent stem cell-derived hepatocytes (iHeps) generated from individuals with a TBL1X N365Y mutation. Both cell types were treated with increasing concentrations of T3. The expression of T3-regulated genes was measured by qPCR. Results: KLF9, CPT1A, and PCK1 mRNA expression were higher upon T3 stimulation in the HepG2 cells with decreased TBL1X expression compared to controls, while DIO1 mRNA expression was lower. Hemizygous TBL1X N365Y iHeps exhibited decreased expression of CPT1A, G6PC1, PCK1, FBP1, and ELOVL2 compared to cells with the heterozygous TBL1X N365Y allele, but KLF9 and HMGCS2 expression was unaltered. Conclusion: Downregulation of TBL1X in HepG2 cells and the TBL1X N365Y variant in iHeps have differential effects on T3-regulated gene expression. This suggests that TBL1X may play a gene context role in TH action.

Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication.

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus-host interactions and potential antiviral strategies.

Seed color patterns in domesticated common bean are regulated by MYB-bHLH-WD40 transcription factors and temperature.

Seed colors and color patterns are critical for the survival of wild plants and the consumer appeal of crops. In common bean, a major global staple, these patterns are also essential in determining market classes, yet the genetic and environmental control of many pigmentation patterns remains unresolved. In this study, we genetically mapped variation for several important seed pattern loci, including T, Bip, phbw, and Z, which co-segregated with candidate genes PvTTG1, PvMYC1, PvTT8, and PvTT2, respectively. Proteins encoded by these genes are predicted to work together in MYB-bHLH-WD40 (MBW) complexes, propagating flavonoid biosynthesis across the seed coat as observed in Arabidopsis. Whole-genome sequencing of 37 accessions identified mutations, including seven unique parallel mutations in T (PvTTG1) and non-synonymous SNPs in highly conserved residues in bipana (PvMYC1) and z (PvTT2). A 612 bp intron deletion in phbw (PvTT8) eliminated motifs conserved since the Papilionoideae origin and corresponded to a 20-fold reduction in transcript abundance. In multi-location field trials of seven varieties with partial seed coat pigmentation patterning, the pigmented seed coat area correlated positively with ambient temperature, with up to 11-fold increases in the pigmented area from the coolest to the warmest environments. In controlled growth chamber conditions, an increase of 4°C was sufficient to cause pigmentation on an average additional 21% of the seed coat area. Our results shed light on key steps of flavonoid biosynthesis in common bean. They will inform breeding efforts for seed coat color/patterning to improve consumer appeal in this nutritious staple crop.

Comprehensive analysis of the Spartina alterniflora WD40 gene family reveals the regulatory role of SaTTG1 in plant development.

Introduction: The WD40 gene family, prevalent in eukaryotes, assumes diverse roles in cellular processes. Spartina alterniflora, a halophyte with exceptional salt tolerance, flood tolerance, reproduction, and diffusion ability, offers great potential for industrial applications and crop breeding analysis. The exploration of growth and development-related genes in this species offers immense potential for enhancing crop yield and environmental adaptability, particularly in industrialized plantations. However, the understanding of their role in regulating plant growth and development remains limited. Methods: In this study, we conducted a comprehensive analysis of WD40 genes in S. alterniflora at the whole-genome level, delving into their characteristics such as physicochemical properties, phylogenetic relationships, gene architecture, and expression patterns. Additionally, we cloned the TTG1 gene, a gene in plant growth and development across diverse species. Results: We identified a total of 582 WD40 proteins in the S. alterniflora genome, exhibiting an uneven distribution across chromosomes. Through phylogenetic analysis, we categorized the 582 SaWD40 proteins into 12 distinct clades. Examining the duplication patterns of SaWD40 genes, we observed a predominant role of segmental duplication in their expansion. A substantial proportion of SaWD40 gene duplication pairs underwent purifying selection through evolution. To explore the functional aspects, we selected SaTTG1, a homolog of Arabidopsis TTG1, for overexpression in Arabidopsis. Subcellular localization analysis revealed that the SaTTG1 protein localized in the nucleus and plasma membrane, exhibiting transcriptional activation in yeast cells. The overexpression of SaTTG1 in Arabidopsis resulted in early flowering and increased seed size. Discussion: These outcomes significantly contribute to our understanding of WD40 gene functions in halophyte species. The findings not only serve as a valuable foundation for further investigations into WD40 genes in halophyte but also offer insights into the molecular mechanisms governing plant development, offering potential avenues in molecular breeding.

A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.

Genetic analysis of PALB2 gene WD40 domain in canine mammary tumour patients.

BACKGROUND: DNA repair mechanisms are essential for tumorigenesis and disruption of HR mechanism is an important predisposing factor of human breast cancers (BC). PALB2 is an important part of the HR. There are similarities between canine mammary tumours (CMT) and BCs. As its human counterpart, PALB2 mutations could be a predisposing factor of CMT. OBJECTIVES: In this study, we aimed to investigate the impacts of PALB2 variants on tumorigenesis and canine mammary tumor (CMT) malignancy. METHODS: We performed Sanger sequencing to detect germline mutations in the WD40 domain of the canine PALB2 gene in CMT patients. We conducted in silico analysis to investigate the variants, and compared the germline PALB2 mutations in humans that cause breast cancer (BC) with the variants detected in dogs with CMT. RESULTS: We identified an intronic (c.3096+8C>G) variant, two exonic (p.A1050V and p.R1354R) variants, and a 3' UTR variant (c.4071T>C). Of these, p.R1354R and c.4071T>C novel variants were identified for the first time in this study. We found that the p.A1050V mutation had a significant effect. However, we could not determine sufficient similarity due to the differences in nucleotide/amino acid sequences between two species. Nonetheless, possible variants of human sequences in the exact location as their dog counterparts are associated with several cancer types, implying that the variants could be crucial for tumorigenesis in dogs. Our results did not show any effect of the variants on tumor malignancy. CONCLUSIONS: The current project is the first study investigating the relationship between the PALB2 gene WD40 domain and CMTs. Our findings will contribute to a better understanding of the pathogenic mechanism of the PALB2 gene in CMTs. In humans, variant positions in canines have been linked to cancer-related phenotypes such as familial BC, endometrial tumor, and hereditary cancer predisposition syndrome. The results of bioinformatics analyses should be investigated through functional tests or case-control studies.

Chemical inhibitors targeting histone methylation readers.

Histone methylation reader domains are protein modules that recognize specific histone methylation marks, such as methylated or unmethylated lysine or arginine residues on histones. These reader proteins play crucial roles in the epigenetic regulation of gene expression, chromatin structure, and DNA damage repair. Dysregulation of these proteins has been linked to various diseases, including cancer, neurodegenerative diseases, and developmental disorders. Therefore, targeting these proteins with chemical inhibitors has emerged as an attractive approach for therapeutic intervention, and significant progress has been made in this area. In this review, we will summarize the development of inhibitors targeting histone methylation readers, including MBT domains, chromodomains, Tudor domains, PWWP domains, PHD fingers, and WD40 repeat domains. For each domain, we will briefly discuss its identification and biological/biochemical functions, and then focus on the discovery of inhibitors tailored to target this domain, summarizing the property and potential application of most inhibitors. We will also discuss the structural basis for the potency and selectivity of these inhibitors, which will aid in further lead generation and optimization. Finally, we will also address the challenges and strategies involved in the development of these inhibitors. It should facilitate the rational design and development of novel chemical scaffolds and new targeting strategies for histone methylation reader domains with the help of this body of data.

Transcriptome Analysis Reveals Coexpression Networks and Hub Genes Involved in Papillae Development in Lilium auratum.

Lilium is a genus of important ornamental plants with many colouring pattern variations. Lilium auratum is the parent of Oriental hybrid lilies. A typical feature of L. auratum is the presence of red-orange special raised spots named papillae on the interior tepals. Unlike the usual raised spots, the papillae are slightly rounded or connected into sheets and usually have hairy tips. To elucidate the potential genes regulating papillae development in L. auratum, we performed high-throughput sequencing of its tepals at different stages. Genes involved in the flavonoid biosynthesis pathway were significantly enriched during the colouration of the papillae, and CHS, F3H, F3'H, FLS, DFR, ANS, and UFGT were significantly upregulated. To identify the key genes involved in the papillae development of L. auratum, we performed weighted gene coexpression network analysis (WGCNA) and further analysed four modules. In total, 51, 24, 1, and 6 hub genes were identified in four WGCNA modules, MEbrown, MEyellow, MEpurple, and MEred, respectively. Then, the coexpression networks were constructed, and important genes involved in trichome development and coexpressed with anthocyanin biosynthesis genes, such as TT8, TTG1, and GEM, were identified. These results indicated that the papillae are essentially trichomes that accumulate anthocyanins. Finally, we randomly selected 12 hub genes for qRT-PCR analysis to verify the accuracy of our RNA-Seq analysis. Our results provide new insights into the papillae development in L. auratum flowers.

Exploring promising natural compounds for breast cancer treatment: in silico molecular docking targeting WDR5-MYC protein interaction.

Cancer is an aberrant differentiation of normal cells, characterized by uncontrolled growth and the potential to acquire invasive and aggressive properties that ultimately lead to metastasis. In the realm of scientific exploration, a multitude of pathways has been investigated and targeted by researchers, among which one specific pathway is recognized as WDR5-MYC. Continuous investigations and research show that WDR5-MYC is a therapeutic target protein. Hence, the discovery of naturally occurring compounds with anticancer properties has been suggested as a rapid and efficient alternative for the development of anticancerous therapeutics. A virtual screening approach was used to identify the most potent compounds from the NP-lib database at the MTiOpenScreen webserver against WDR5-MYC. This process yielded a total of 304 identified compounds. Subsequently, after screening, four potent compounds, namely Estrone (ZINC000003869899), Ethyl-1,2-benzanthracene (ZINC000003157052), Strychnine (ZINC000000119434) and 7H-DIBENZO [C, G] CARBAZOLE (ZINC000001562130), along with a cocrystallized 5-[4-(trifluoromethyl) phenyl]-1H-tetrazole inhibitor (QBP) as a reference ligand, were considered for stringent molecular docking. Thus, each compound exhibited significant docking energy between -8.2 and -7.7 kcal/mol and molecular contacts with essential residue Asn225, Lys250, Ser267 and Lys272 in the active pocket of WDR5-MYC against the QBP inhibitor (the native ligand QBP serves as a reference in the comparative analysis of docked complexes). The results support the potent compounds for drug-likeness and strong binding affinity with WDR5-MYC protein. Further, the stability of the selected compounds was predicted by molecular dynamics simulation (100 ns) contributed by intermolecular hydrogen bonds and hydrophobic interactions. This demonstrates the potential of the selected compounds to be used against breast cancer treatment.Communicated by Ramaswamy H. Sarma.

Genome-wide identification and analysis of WD40 proteins reveal that NtTTG1 enhances drought tolerance in tobacco (Nicotiana tabacum).

BACKGROUND: WD40 proteins, which are highly prevalent in eukaryotes, play important roles in plant development and stress responses. However, systematic identification and exploration of WD40 proteins in tobacco have not yet been conducted. RESULTS: In this study, a total of 399 WD40 regulatory genes were identified in common tobacco (Nicotiana tabacum). Gene structure and motif analysis revealed structural and functional diversity among different clades of tobacco WD40 regulatory genes. The expansion of tobacco WD40 regulatory genes was mainly driven by segmental duplication and purifying selection. A potential regulatory network of NtWD40s suggested that NtWD40s might be regulated by miRNAs and transcription factors in various biological processes. Expression pattern analysis via transcriptome analysis and qRT-PCR revealed that many NtWD40s exhibited tissue-specific expression patterns and might be involved in various biotic and abiotic stresses. Furthermore, we have validated the critical role of NtTTG1, which was located in the nuclei of trichome cells, in enhancing the drought tolerance of tobacco plants. CONCLUSIONS: Our study provides comprehensive information to better understand the evolution of WD40 regulatory genes and their roles in different stress responses in tobacco.

APRF1 Interactome Reveals HSP90 as a New Player in the Complex That Epigenetically Regulates Flowering Time in Arabidopsis thaliana.

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana, such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 (aprf1) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

Genome-Wide Identification, Evolution, and Expression Analysis of the WD40 Subfamily in Oryza Genus.

The WD40 superfamily is widely found in eukaryotes and has essential subunits that serve as scaffolds for protein complexes. WD40 proteins play important regulatory roles in plant development and physiological processes, such as transcription regulation and signal transduction; it is also involved in anthocyanin biosynthesis. In rice, only OsTTG1 was found to be associated with anthocyanin biosynthesis, and evolutionary analysis of the WD40 gene family in multiple species is less studied. Here, a genome-wide analysis of the subfamily belonging to WD40-TTG1 was performed in nine AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, Oryza glumaepatula, Oryza nivara, and Oryza longistaminata. In this study, 383 WD40 genes in the Oryza genus were identified, and they were classified into four groups by phylogenetic analysis, with most members in group C and group D. They were found to be unevenly distributed across 12 chromosomes. A total of 39 collinear gene pairs were identified in the Oryza genus, and all were segmental duplications. WD40s had similar expansion patterns in the Oryza genus. Ka/Ks analyses indicated that they had undergone mainly purifying selection during evolution. Furthermore, WD40s in the Oryza genus have similar evolutionary patterns, so Oryza sativa ssp. indica was used as a model species for further analysis. The cis-acting elements analysis showed that many genes were related to jasmonic acid and light response. Among them, OsiWD40-26/37/42 contained elements of flavonoid synthesis, and OsiWD40-15 had MYB binding sites, indicating that they might be related to anthocyanin synthesis. The expression profile analysis at different stages revealed that most OsiWD40s were expressed in leaves, roots, and panicles. The expression of OsiWD40s was further analyzed by qRT-PCR in 9311 (indica) under various hormone treatments and abiotic stresses. OsiWD40-24 was found to be responsive to both phytohormones and abiotic stresses, suggesting that it might play an important role in plant stress resistance. And many OsiWD40s might be more involved in cold stress tolerance. These findings contribute to a better understanding of the evolution of the WD40 subfamily. The analyzed candidate genes can be used for the exploration of practical applications in rice, such as cultivar culture for colored rice, stress tolerance varieties, and morphological marker development.

Recent Progress in Modulation of WD40-Repeat Domain 5 Protein (WDR5): Inhibitors and Degraders.

WD40-repeat (WDR) domain proteins play a crucial role in mediating protein-protein interactions that sustain oncogenesis in human cancers. One prominent example is the interaction between the transcription factor MYC and its chromatin co-factor, WD40-repeat domain protein 5 (WDR5), which is essential for oncogenic processes. The MYC family of proteins is frequently overexpressed in various cancers and has been validated as a promising target for anticancer therapies. The recruitment of MYC to chromatin is facilitated by WDR5, highlighting the significance of their interaction. Consequently, inhibiting the MYC-WDR5 interaction has been shown to induce the regression of malignant tumors, offering an alternative approach to targeting MYC in the development of anticancer drugs. WDR5 has two protein interaction sites, the "WDR5-binding motif" (WBM) site for MYC interaction and the histone methyltransferases SET1 recognition motif "WDR5-interacting" (WIN) site forming MLL complex. Significant efforts have been dedicated to the discovery of inhibitors that target the WDR5 protein. More recently, the successful application of targeted protein degradation technology has enabled the removal of WDR5. This breakthrough has opened up new avenues for inhibiting the interaction between WDR5 and the binding partners. In this review, we address the recent progress made in targeting WDR5 to inhibit MDR5-MYC and MDR5-MLL1 interactions, including its targeted protein degradation and their potential impact on anticancer drug discovery.

Strategies for rapid production of crystallization quality coatomer WD40 domains.

The vesicular secretion of soluble cargo proteins from the endoplasmic reticulum (ER) is accompanied by the export of ER-resident membrane proteins that are co-packaged in secretory vesicles. The cytosolic coatomer protein complex I (COPI) utilizes the N-terminal WD40 domains of α-COPI and ß'-COPI subunits to bind these membrane protein "clients" for ER retrieval. These "αWD40" and "ß'WD40" domains are structural homologs that demonstrate distinct selectivity for client proteins. However, elucidation of the atomic-level principles of coatomer-client interactions has been challenging due to the tendency of αWD40 domain to undergo aggregation during expression and purification. Here we describe a rapid recombinant production strategy from E. coli, which substantially enhances the quality of the purified αWD40 domain. The αWD40 purification and crystallization are completed within one day, which minimizes aggregation losses and yields a 1.9 Å resolution crystal structure. We demonstrate the versatility of this strategy by applying it to purify the ß'WD40 domain, which yields crystal structures in the 1.2-1.3 Å resolution range. As an alternate recombinant production system, we develop a cost-effective strategy for αWD40 production in human Expi293 cells. Finally, we suggest a roadmap to simplify these protocols further, which is of significance for the production of WD40 mutants prone to rapid aggregation. The WD40 production strategies presented here are likely to have broad applications because the WD40 domain represents one of the largest families of biomolecular interaction modules in the eukaryotic proteome and is critical for trafficking of host as well as viral proteins such as the SARS-CoV-2 spike protein.

Genome-wide identification and bioinformatics analysis of the WD40 transcription factor family and candidate gene screening for anthocyanin biosynthesis in Rhododendron simsii.

WD40 transcription factors (TFs) constitute a large gene family in eukaryotes, playing diverse roles in cellular processes. However, their functions in the major ornamental plant, Rhododendron simsii, remain poorly understood. In this study, we identified 258 WD40 proteins in the R. simsii genome, which exhibited an uneven distribution across chromosomes. Based on domain compositions and phylogenetic analysis, we classified these 258 RsWD40 proteins into 42 subfamilies and 47 clusters. Comparative genomic analysis suggested that the expansion of the WD40 gene family predates the divergence of green algae and higher plants, indicating an ancient origin. Furthermore, by analyzing the duplication patterns of RsWD40 genes, we found that transposed duplication played a major role in their expansion. Notably, the majority of RsWD40 gene duplication pairs underwent purifying selection during evolution. Synteny analysis identified significant orthologous gene pairs between R. simsii and Arabidopsis thaliana, Oryza sativa, Vitis vinifera, and Malus domestica. We also investigated potential candidate genes involved in anthocyanin biosynthesis during different flower development stages in R. simsii using RNA-seq data. Specifically, we identified 10 candidate genes during the bud stage and 7 candidate genes during the full bloom stage. GO enrichment analysis of these candidate genes revealed the potential involvement of the ubiquitination process in anthocyanin biosynthesis. Overall, our findings provide a valuable foundation for further investigation and functional analysis of WD40 genes, as well as research on the molecular mechanisms underlying anthocyanin biosynthesis in Rhododendron species.

CCNB1IP1 prevents ubiquitination-mediated destabilization of MYCN and potentiates tumourigenesis of MYCN-amplificated neuroblastoma.

BACKGROUND: MYCN amplification as a common genetic alteration that correlates with a poor prognosis for neuroblastoma (NB) patients. However, given the challenge of directly targeting MYCN, indirect strategies to modulate MYCN by interfering with its cofactors are attractive in NB treatment. Although cyclin B1 interacting protein 1 (CCNB1IP1) has been found to be upregulated in MYCN-driven mouse NB tissues, its regulation with MYCN and collaboration in driving the biological behaviour of NB remains unknown. METHODS: To evaluate the expression and clinical significance of CCNB1IP1 in NB patients, public datasets, clinical NB samples and cell lines were explored. MTT, EdU incorporation, colony and tumour sphere formation assays, and a mouse xenograft tumour model were utilized to examine the biological function of CCNB1IP1. The reciprocal manipulation of CCNB1IP1 and MYCN and the underlying mechanisms involved were investigated by gain- and loss-of-function approaches, dual-luciferase assay, chromatin immunoprecipitation (CHIP) and co-immunoprecipitation (Co-IP) experiments. RESULTS: CCNB1IP1 was upregulated in MYCN-amplified (MYCN-AM) NB cell lines and patients-derived tumour tissues, which was associated with poor prognosis. Phenotypic studies revealed that CCNB1IP1 facilitated the proliferation and tumourigenicity of NB cells in cooperation with MYCN in vitro and in vivo. Mechanistically, MYCN directly mediates the transcription of CCNB1IP1, which in turn attenuated the ubiquitination and degradation of MYCN protein, thus enhancing CCNB1IP1-MYCN cooperativity. Moreover, CCNB1IP1 competed with F box/WD-40 domain protein 7 (FBXW7) for MYCN binding and enabled MYCN-mediated tumourigenesis in a C-terminal domain-dependent manner. CONCLUSIONS: Our study revealed a previously uncharacterized mechanism of CCNB1IP1-mediated MYCN protein stability and will provide new prospects for precise treatment of MYCN-AM NB based on MYCN-CCNB1IP1 interaction.