WD Repeat Research Articles

TBL2 Promotes Tumorigenesis via PRMT5/WDR77-Mediated AKT Activation in Breast Cancer.

Breast cancer (BC) is a common malignancy that affects women worldwide. Although transducing beta-like 2 (TBL2), a member of the WD40 repeat protein family, has been implicated in various intracellular signaling pathways, its precise function in BC remains unclear. The expression of TBL2 is analyzed using real-time PCR, western blotting, and immunohistochemistry in BC patient specimens. Kaplan-Meier survival analysis is employed to assess its prognostic significance. Proteomic analysis, immunoprecipitation tests, and protein immunoblotting are employed to examine the impact of TBL2 on AKT phosphorylation activation. The findings reveal selective overexpression of TBL2 in BC, correlating significantly with various clinicopathological characteristics and poor survival outcomes in patients with BC. Through in vivo and in vitro experiments, it is observed that TBL2 suppression inhibits BC cell proliferation, while TBL2 overexpression has the opposite effect. Mechanistically, TBL2 is identified as a scaffolding protein that promotes PRMT5 and WDR77 interaction. This interaction enhances the methyltransferase activity of PRMT5, leading to increased AKT phosphorylation activation and promotion of breast cancer cell proliferation. In conclusion, this study uncovers a novel function of TBL2 in the activation of AKT by PRMT5 and suggests TBL2 as a potential therapeutic target for BC treatment.

A Target Class Ligandability Evaluation of WD40 Repeat-Containing Proteins.

Target class-focused drug discovery has a strong track record in pharmaceutical research, yet public domain data indicate that many members of protein families remain unliganded. Here we present a systematic approach to scale up the discovery and characterization of small molecule ligands for the WD40 repeat (WDR) protein family. We developed a comprehensive suite of protocols for protein production, crystallography, and biophysical, biochemical, and cellular assays. A pilot hit-finding campaign using DNA-encoded chemical library selection followed by machine learning (DEL-ML) to predict ligands from virtual libraries yielded first-in-class, drug-like ligands for 7 of the 16 WDR domains screened, thus demonstrating the broader ligandability of WDRs. This study establishes a template for evaluation of protein family wide ligandability and provides an extensive resource of WDR protein biochemical and chemical tools, knowledge, and protocols to discover potential therapeutics for this highly disease-relevant, but underexplored target class.

F-Box and WD repeat domain containing 7 induces infectious osteomyelitis by regulating MYB stability and ubiquitination.

Osteomyelitis is a bone inflammation initiated by invading pathogens. Macrophages and inflammation play essential roles in osteomyelitis. F-Box and WD repeat domain containing 7 (Fbxw7) is a tumour suppressor and E3 ubiquitin ligase. In the present study, the potential roles of Fbxw7 in osteomyelitis were explored. The mRNA level of Fbxw7 was measured in bone marrow cells from patients with osteomyelitis and Staphylococcus aureus (S. aureus)-infected macrophages. The conditional knockout mice with Fbxw7 deficiency in myeloid cells were generated. The expression of interleukin (IL)-6, IL-23a and nitric oxide synthase 2 (Nos2) was measured in S. aureus-infected Fbxw7-deficient bone marrow-derived macrophages (BMDMs). The body weight loss, bacterial burden, bone loss and formation and serum level of IL-6, IL-23 and TNF-α were measured in S. aureus-infected Fbxw7 conditional KO mice. The interacting partners of Fbxw7 were predicted using STRING and the interaction were tested. Elevated expression of Fbxw7 was observed in bone marrow cells from patients with osteomyelitis and in S. aureus-infected macrophages. The expression of IL-6, IL-23a and Nos2 was remarkably suppressed in S. aureus-infected Fbxw7-deficient BMDMs. Fbxw7 conditional knockout mice had less body weight loss, higher bacterial burden, less bone loss and formation and decreased serum level of cytokines. Fbxw7 interacted with MYB. S. aureus-infected Fbxw7-deficient BMDMs had higher level of MYB and less ubiquitination of MYB. Fbxw7 promotes osteomyelitis symptoms by regulating ubiquitination and stability of MYB.

Identification of metabolism related biomarkers in obesity based on adipose bioinformatics and machine learning

BackgroundObesity has emerged as a growing global public health concern over recent decades. Obesity prevalence exhibits substantial global variation, ranging from less than 5% in regions like China, Japan, and Africa to rates exceeding 75% in urban areas of Samoa.AimTo examine the involvement of metabolism-related genes.MethodsGene expression datasets GSE110729 and GSE205668 were accessed from the GEO database. DEGs between obese and lean groups were identified through DESeq2. Metabolism-related genes and pathways were detected using enrichment analysis, WGCNA, Random Forest, and XGBoost. The identified signature genes were validated by real-time quantitative PCR (qRT-PCR) in mouse models.ResultsA total of 389 genes exhibiting differential expression were discovered, showing significant enrichment in metabolic pathways, particularly in the propanoate metabolism pathway. The orangered4 module, which exhibited the highest correlation with propanoate metabolism, was identified using Weighted Correlation Network Analysis (WGCNA). By integrating the DEGs, WGCNA results, and machine learning methods, the identification of two metabolism-related genes, Storkhead Box 1 (STOX1), NACHT and WD repeat domain-containing protein 2(NWD2) was achieved. These signature genes successfully distinguished between obese and lean individuals. qRT-PCR analysis confirmed the downregulation of STOX1 and NWD2 in mouse models of obesity.ConclusionThis study has analyzed the available GEO dataset in order to identify novel factors associated with obesity metabolism and found that STOX1 and NWD2 may serve as diagnostic biomarkers.

WDR45 variants as a major cause for a clinically variable intellectual disability syndrome from early infancy in females.

Pathogenic variants of WD repeat domain 45 (WDR45) cause neurodegeneration with brain iron accumulation 5 (NBIA5), which is characterised by progressive neurological regression and brain iron accumulation in adulthood. Early diagnosis of NBIA5 patients is difficult because they often show only a non-specific developmental delay in childhood, but it is essential for lifelong medical management. We investigated 32 females with developmental delays for coding variants of WDR45 using Sanger sequencing. Whole-genome sequencing (WGS) and X chromosome inactivation (XCI) analysis were also performed. We identified two disease-causing variants, one of which was a novel stop-loss variant, c.1051delG p.(Val351CysfsTer60), in a female with severe developmental delay from early infancy with epileptic spasms. The XCI analysis (which we originally developed) suggested a random pattern in white blood cells. WGS did not reveal any other pathogenic variants, including those in two iron transporter genes. Together with our previous findings in the WGS study, WDR45 variants accounted for 12% (6/51) of the females with developmental delay, suggesting that WDR45 is a major gene in females with developmental delay. Pathogenic variants of WDR45 result in various phenotypes that do not necessarily correlate with variant types or XCI skewing patterns.

WDR20 prevents hepatocellular carcinoma senescence by orchestrating the simultaneous USP12/46-mediated deubiquitination of c-Myc

The dysfunction of the ubiquitin-proteasome system (UPS) facilitates the malignant progression of hepatocellular carcinoma (HCC). While targeting the UPS for HCC therapy has been proposed, identifying effective targets has been challenging. In this study, we conducted a focused screen of siRNA libraries targeting UPS-related WD40 repeat (WDR) proteins and found that silencing WDR20, a deubiquitinating enzyme activating factor, selectively inhibited the proliferation of HCC cells without affecting normal hepatocytes. Moreover, the downregulation of WDR20 expression induced HCC cellular senescence and suppressed tumor progression in xenograft, sleeping beauty transposon/transposase, and hydrodynamic tail vein injection-induced HCC models, and Alb-Cre+/MYC+ HCC transgenic mouse models. Mechanistically, we found that WDR20 silencing disturbed the protein stability of c-Myc, orchestrating the simultaneous USP12/46-mediated deubiquitination of c-Myc, thereby promoting the transcriptional activation of CDKN1A. Further investigation revealed a positive coexpression of WDR20 and c-Myc in a tissue microarray with 88 HCC clinical samples. By employing three patient-derived organoids from individuals with HCC, we have validated the decrease in c-Myc expression and the significant induction of senescence and growth inhibition following silencing of WDR20. This study not only uncovers the biological function of WDR20 and elucidates the molecular mechanism underlying its negative regulation of HCC cellular senescence but also highlight the potential of WDR20 as a promising target for HCC therapy.

Proteomic analysis of giant panda testicular tissue of different age groups

Background The reproductive ability of male giant pandas has been a major complicating factor in the ex-situ conservation of the species. While it is well known that the testis produces sperm and secretes androgens, a process that requires precise regulation of various proteins, at present, there has been no systematic study on the composition of proteins in the testis of the giant pandas. Therefore, this study aims to apply proteomics to explore the regulation of proteins in the testes of giant pandas. Methods Samples from the testes of three giant pandas (22 years, 18 years, 8 days) were studied to assess the protein’s function. A label-free quantitative method was used to isolate testicular proteins from each male, 139,039 peptides and 11,435 proteins were obtained. Results Gene Ontology (GO) annotates most of the proteins involved in the processes of protein phosphorylation, oxidation-reduction, proteolysis, and signal transduction. KEGG pathway indicated that most of the proteins were involved in the pathway of signal transduction, transport, and catabolism. The protein kinase and WD40 repeats were involved in protein-protein interaction, which in turn regulates gene expression in the testicular tissue of giant pandas. Conclusions This study is the first to conduct an in-depth proteomic analysis of testicular tissue in giant pandas. The results revealed the important role of proteins in testicular tissue on spermatogenesis, testosterone production, and testicular microenvironment, providing clues for further research on male giant panda reproduction.

Structure of a step II catalytically activated spliceosome from Chlamydomonas reinhardtii.

Pre-mRNA splicing, a fundamental step in eukaryotic gene expression, is executed by the spliceosomes. While there is extensive knowledge of the composition and structure of spliceosomes in yeasts and humans, the structural diversity of spliceosomes in non-canonical organisms remains unclear. Here, we present a cryo-EM structure of a step II catalytically activated spliceosome (C* complex) derived from the unicellular green alga Chlamydomonas reinhardtii at 2.6 Å resolution. This Chlamydomonas C* complex comprises 29 proteins and four RNA elements, creating a dynamic assembly that shares a similar overall architecture with yeast and human counterparts but also has unique features of its own. Distinctive structural characteristics include variations in protein compositions as well as some noteworthy RNA features. The splicing factor Prp17, with four fragments and a WD40 domain, is engaged in intricate interactions with multiple protein and RNA components. The structural elucidation of Chlamydomonas C* complex provides insights into the molecular mechanism of RNA splicing in plants and understanding splicing evolution in eukaryotes.

FBW7-Mediated Degradation of CHD3 Suppresses Hepatocellular Carcinoma Metastasis and Stemness to Enhance Oxaliplatin Sensitivity.

Ubiquitination plays a key role in various cancers, and F-box and WD repeat domain containing 7 (FBW7) is a tumor suppressor that targets several cancer-causing proteins for ubiquitination. This paper set out to pinpoint the role of FBW7 in hepatocellular carcinoma (HCC). The target proteins of FBW7 and the expression of hromodomain helicase DNA binding protein 3 (CHD3) were analyzed in liver HCC (LIHC) samples using the BioSignal Data website. The effects of CHD3 and FBW7 on HCC cell viability, migration, invasion and stemness were investigated through cell counting kit (CCK)-8, wound healing, transwell and sphere formation assays. Detection on CHD3 and FBW7 expressions as well as their relationship was performed employing quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunoprecipitation, ubiquitination and western blot analyses. The prediction of Ubibrowser revealed CHD3 as a target protein of FBW7. The data of starBase exhibited a higher expression level of CHD3 in LIHC samples relative to normal samples. CHD3 was upregulated in HCC cells. CHD3 knockdown inhibited HCC cell proliferation, migration, invasion, stemness and oxaliplatin sensitivity. FBW7 targeted CHD3 for ubiquitination. FBW7 overexpression restrained HCC cell migration, invasion and stemness, and attenuated the effects of overexpressed CHD3 on promoting migration, invasion, stemness and oxaliplatin resistance in HCC cells. FBW7 overexpression suppresses HCC cell metastasis, stemness and oxaliplatin resistance via targeting CHD3 for ubiquitylation and degradation.

MiR-182-5p promotes the proliferation and invasion of hilar cholangiocarcinoma cells by inhibiting FBXW7

BackgroundHilar cholangiocarcinoma (HCCA) is a common type of cholangiocarcinoma (CHOL) that originates from the right and/or left hepatic duct near the biliary tract confluence. The objective of this study is to investigate the impact of miR-182-5p on the proliferation and invasion of HCCA cells and identify a potential target for HCCA treatment.MethodsHCCA tissues were collected and HCCA cells were cultured. miR-182-5p and F-box and WD repeat domain containing 7 (FBXW7) were detected. After transfection of miR-182-5p inhibitor into HCCA cells, cell proliferation and invasion were detected by cell counting 8-kit and Transwell assay. FBXW7 expression was detected by Western blot. The targeted relationship between miR-182-5p and FBXW7 3’UTR was verified by dual-luciferase report assay. si-FBXW7 and miR-182-5p inhibitor were transfected into cells for combined experiments. HCCA cells with lowly-expressed miR-182-5p were injected into nude mice to establish the xenograft tumor model, and subsequent observations were made on tumor growth and gene expression changes.ResultsmiR-182-5p exhibited high expression levels in both HCCA tissues and cell lines. Inhibiting miR-182-5p effectively suppressed the proliferation and migration of HCCA cells. miR-182-5p bounded to FBXW7 3 ‘UTR and inhibited FBWX7 expression. Suppressing FBXW7 expression partially reversed the inhibitory effect of miR-182-5p inhibitor on HCCA cell proliferation and invasion. Silencing miR-182-5p could inhibit the HCCA growth in vivo.ConclusionmiR-182-5p promoted the proliferation and invasion of HCCA cells by targeting and inhibiting FBXW7 expression.

A comprehensive study of highly repetitive WD40 proteins in cyanobacteria

The WD40 repeat-containing proteins are ancient proteins regulating various cellular and biological processes in eukaryotes. WD40 proteins are extensively studied in eukaryotes, and their genome-wide characterisation has revealed their hidden potential. On the contrary, in-depth taxonomic and functional description of WD40 proteins in prokaryotes, particularly in cyanobacteria, remains largely unexplored. In this study, we have comprehensively analysed cyanobacterial WD40 proteins and detailed comparisons among different cyanobacterial orders. About 7000 WD40 proteins were identified in all six cyanobacterial orders accounting for 22–43 % of all WD40s. While their abundance was less in Chroococcales, Pleurocapsales and, Stigonematales, the WD40s were profoundly present in Nostcales and Oscillatoriales, exhibiting multifarious functions such as cell signalling, transcription factors, catalytic enzymes and scaffold etc. Current systemic analysis showed that most WD40 proteins contain multiple WD40 domains, as indicated by their repeat numbers and average confidence scores. The observation also indicated that most WD40 proteins have complex hydrogen bond networks. Their taxonomic distribution and gene neighbourhood analysis revealed topical or newly repeated duplication events form most WD40s. Further, the studies confirmed that the recently formed WD40 proteins are highly repetitive with higher structural stability. Overall, the result of this study has described an assembly of cyanobacterial WD40 proteins and highlighting their evolution, distribution and probable functions.

Genome-wide identification of the TRAF gene family in humpback grouper (Cromileptes altivelis) and analysis of their expression in response to Vibrio harveyi challenge

TRAF (Tumor necrosis factor receptor-associated factor) proteins are key mediators of signal transduction in cell signaling and immune regulation within the toll-like receptor (TLR) and tumor necrosis factor (TNFR) superfamily. Despite the importance of TRAF genes in teleost innate immunity, study on their functions in C. altivelis is limited. This study utilized bioinformatics methods to identify and named eight TRAF genes (CaTRAF2a, CaTRAF2a-like, CaTRAF2b, CaTRAF3, CaTRAF4a, CaTRAF5, CaTRAF6 and CaTRAF7) in C. altivelis. Phylogenetic, syntenic and molecular evolution revealed that all CaTRAF members were evolutionarily conserved in teleost. Domain analysis indicated the presence of a conserved N-terminal RING finger domain in all CaTRAF proteins. Most CaTRAF proteins also featured a MATH domain at the C-terminal, with the exception of CaTRAF7 which contained seven repeat WD40 domains. In addition, qRT-PCR was used to detect the expression patterns of nine different tissues and eight different embryonic development stages of healthy fish, and it was found that there were spatial and tissue specificities among the members. HE staining revealed evident pathological lesions in the tissues post V. harveyi infection. Atrophy and significant bending of the gill lamellae were observed in the gills, while irregular cell shapes, increased fat vacuoles, and enlarged cell volume were noted in the liver. Intestinal tissues displayed thickening of the muscle layer, elongation of intestinal villi, and increased folds. Moreover, the expression of TRAF gene changed significantly after V. harveyi infection. These results would help to clarify the molecular role of CaTRAF gene in the regulation of immune and inflammatory responses in C. altivelis.

Evidence for a hydrogen sulfide-sensing E3 ligase in yeast.

In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor that activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the trans-sulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30's WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30 dynamically senses the flow of sulfur metabolites through the trans-sulfuration pathway to regulate the synthesis of these special amino acids.

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.

Transcriptome-wide Identification of Nine Tandem Repeat Protein Families in Roselle (Hibiscus sabdariffa L.).

Plants are rich in tandem repeats-containing proteins. It is postulated that the occurrence of tandem repeat gene families facilitates the adaptation and survival of plants in adverse environmental conditions. This study intended to identify the tandem repeats in the transcriptome of a high potential tropical horticultural plant, roselle (Hibiscus sabdariffa L.). A total of 92,974 annotated de novo assembled transcripts were analysed using in silico approach, and 6,541 transcripts that encoded proteins containing tandem repeats with length of 20-60 amino acid residues were identified. Domain analysis revealed a total of nine tandem repeat protein families in the transcriptome of roselle, which are the Ankyrin repeats (ANK), Armadillo repeats (ARM), elongation factor-hand domain repeats (EF-hand), Huntingtin, elongation factor 3, protein phosphatase 2A, yeast kinase TOR1 repeats (HEAT), Kelch repeats (Kelch), leucine rich repeats (LRR), pentatricopeptide repeats (PPR), tetratricopeptide repeats (TPR) and WD40 repeats (WD40). Functional annotation analysis further matched 6,236 transcripts to 1,045 known proteins that contained tandem repeats including proteins implicated in plant development, protein-protein interaction, immunity and abiotic stress responses. The findings provide new insights into the occurrence of tandem repeats in the transcriptome and lay the foundation to elucidate the functional associations between tandem peptide repeats (TRs) and proteins in roselle and facilitate the identification of novel biotic and abiotic response related tandem repeats genes that may be useful in breeding improved varieties.

Mesenchymal Stem Cells-Derived Exosomal miR-223-3p Alleviates Ocular Surface Damage and Inflammation by Downregulating Fbxw7 in Dry Eye Models.

Our previous study indicated that exosomes derived from mouse adipose-derived mesenchymal stem cells (mADSC-Exos) alleviated the benzalkonium chloride (BAC)-induced mouse dry eye model. However, the specific active molecules in mADSC-Exos that contribute to anti-dry eye therapy remain unidentified. In this study, we aimed to investigate the efficacy and mechanisms of miR-223-3p derived from mADSC-Exos in dry eye models. Enzyme-linked immunosorbent assay (ELISA) experiments were conducted to determine miR-223-3p derived from mADSC-Exos that exerted anti-inflammatory effects on hyperosmolarity-induced mouse corneal epithelial cells (MCECs). The therapeutic efficacy of miR-223-3p was evaluated in mice with dry eye induced by either BAC or scopolamine (Scop). Mice were randomly assigned to 5 groups: sham, model, miR-223-3p overexpression, miR-223-3p knockdown, and 0.1% pranoprofen (positive group). Post-treatment, the severity of dry eye symptoms, and the pro-inflammatory cytokine levels were assessed. The effect of miR-223-3p on silencing the target gene was verified using ELISA and dual luciferase reporter assays. The mADSC-Exos that knocked out miR-223-3p did not reduce interleukin (IL)-6 content. Supplementing with miR-223-3p could restore the reduction of IL-6. The miR-223-3p effectively ameliorated ocular surface damage and decreased pro-inflammatory cytokines or chemokines in both BAC- and Scop-induced mouse dry eye models. Furthermore, miR-223-3p inhibited cell apoptosis. F-box and WD repeat domain-containing 7 (Fbxw7) was the potential direct target of miR-223-3p. The miR-223-3p suppressed the 3'-untranslated region of Fbxw7. The Fbxw7 knockdown suppressed hyperosmolarity-induced inflammation in MCECs. The mADSC-derived exosomal miR-223-3p mitigates ocular surface damage and inflammation, indicating its potential as a promising treatment option for dry eye.

Identification of dystrophin Dp71dΔ71-associated proteins in PC12 cells by quantitative proteomics

Dystrophin Dp71 is essential for the development of the nervous system. Its alteration is associated with intellectual disability. Different Dp71 isoforms are generated by alternative splicing; however, their functions have not been fully described. Here, we identified Dp71dΔ71-associated proteins to understand the complex functions. PC12 cells, stably transfected with pTRE2pur-Myc/Dp71dΔ71 or pTRE2pur-Myc empty vector (EV), were analyzed by immunoprecipitation followed with quantitative proteomics with data-independent acquisition and ion mobility separation. We used the Top3 method to quantify absolutely every protein detected. A total of 106 proteins were quantified with Progenesis QI software and the database UP000002494. Seven new proteins associated with Dp71dΔ71 were selected with at least 2-fold quantity between immunoprecipitated proteins of PC12-Myc/Dp71dΔ71 versus PC12-EV cells. These results revealed new proteins that interact with Dp71dΔ71, including β-Tubulin, S-adenosylmethionine synthase isoform type-2, adapter molecule crk, helicase with zinc finger 2, WD repeat domain 93, cyclin-L2 and myosin-10, which are related to cell migration and/or cell growth. The results lay the foundation for future research on the relationship between these proteins and Dp71 isoforms.

MiR‑25‑3p serves as an oncogenic in colorectal cancer cells by regulating the ubiquitin ligase FBXW7 function.

Accumulating evidence indicates that the dysregulation of microRNAs (miRNAs or miRs), is associated with human malignancies and suggests a casual role of miRNAs in tumor initiation and progression. Even though it has been discovered that a number of miRNAs play significant parts in the development of colorectal cancer (CRC), it is crucial to comprehend the regulatory functions that other miRNAs play in CRC. Based on GSE183437 and GSE156719 microarray data that were obtained from Gene Expression Omnibus database, candidate miRNAs were researched. The oncogenic effects of miR‑25‑3p in different malignancies have led to its selection for additional investigation in the present study. The expression of miR‑25‑3p was verified by reverse transcription‑quantitative PCR, and its correlation with clinicopathological characteristics in patients with CRC was then investigated. In vitro assays were conducted to investigate the influence of miR‑25‑3p on the proliferative and apoptotic behaviors of HCT116 and Caco‑2 cells. The present data revealed that miR‑25‑3p exhibited one of the most significant upregulations in CRC tissues and cell lines. The expression levels of miR‑25‑3p were found to be intimately correlated with tumor size, distant metastasis, tumor‑node‑metastasis stage, and shorter overall survival rate. In terms of functionality, the downregulation of miR‑25‑3p led to the inhibition of cellular proliferation and the enhancement of apoptosis in both HCT116 and Caco‑2 cell lines. The critical tumor suppressor F‑box and WD repeat containing domain 7 (FBXW7) was identified as a direct molecular target for miR‑25‑3p, with an inverse relationship observed between the two in neoplastic tissues. Subsequent studies demonstrated that the tumor suppressive effects of miR‑25‑3p inhibitor were effectively negated by the silencing of FBXW7. Moreover, the ability of FBXW7 to inhibit the expression of several oncogenes was deemed essential for countering the anticancer effects mediated by miR‑25‑3p downregulation. These findings posit miR‑25‑3p as a promising therapeutic target and prognostic indicator for CRC.

The underlying molecular mechanisms of hormonal regulation of fruit color in fruit-bearing plants.

Fruit color is a key feature of fruit quality, primarily influenced by anthocyanin or carotenoid accumulation or chlorophyll degradation. Adapting the pigment content is crucial to improve the fruit's nutritional and commercial value. Genetic factors along with other environmental components (i.e., light, temperature, nutrition, etc.) regulate fruit coloration. The fruit coloration process is influenced by plant hormones, which alsoplay a vital role in various physiological and biochemical metabolic processes. Additionally, phytohormones play a role in the regulation of a highly conserved transcription factor complex, called MBW (MYB-bHLH-WD40). The MBW complex, which consists of myeloblastosis (MYB), basic helix-loop-helix (bHLH), and WD40 repeat (WDR) proteins, coordinates the expression of downstream structural genes associated with anthocyanin formation. In fruit production, the application of plant hormones may be important for promoting coloration. However, concerns such as improper concentration or application time must be addressed. This article explores the molecular processes underlying pigment formation and how they are influenced by various plant hormones. The ABA, jasmonate, and brassinosteroid increase anthocyanin and carotenoid formation, but ethylene, auxin, cytokinin, and gibberellin have positive as well as negative effects on anthocyanin formation. This article establishes the necessary groundwork for future studies into the molecular mechanisms of plant hormones regulating fruit color, ultimately aiding in their effective and scientific application towards fruit coloration.

WDR64, a testis-specific protein, is involved in the manchette and flagellum formation by interacting with ODF1

The WD40 repeat (WDR) domain is present in a wide range of proteins, providing sites for protein‒protein interactions. Recent studies have shown that WDR proteins play indispensable roles in spermatogenesis, such as in spermatocyte division, sperm head formation and flagellar assembly. In this study, we identified a novel testis-specific gene, WDR64, which has the typical characteristics of WD40 proteins with two β-propellers, and is highly conserved in Mammalia. RT-PCR and Western blot results revealed that WDR64 was highly expressed in testis. WDR64 protein was weakly expressed at postnatal Day 7, increased substantially at postnatal Day 28 and maintained at high levels thereafter. Further immunofluorescence demonstrated that WDR64 was localized posterior to the nucleus in steps 8–14 spermatids in line with the dynamic localization of manchette, moved to the flagella in steps 15–16 spermatids, and localized at the midpiece of the flagellum in mature spermatozoa. To explore the function of WDR64, we performed immunoprecipitation‒mass spectrometry (IP‒MS) to screen its interacting proteins and found that WDR64 interacted with ODF1 to form a complex. The WDR64/ODF1 complex is located at the manchette during nucleus shaping and finally at the midpiece of the mature spermatozoa tail, suggesting that it may be involved in the assembly of the manchette and flagella during spermiogenesis. Our findings provide the first understanding of the expression pattern of WDR64 and its potential molecular mechanism in spermiogenesis.