[Method of Inducible Knockdown of Essential Genes in OSC Cell Culture of Drosophila melanogaster].

An RNA interference-based method was proposed to achieve an inducible knockdown of genes essential for cell viability. In the method, a genetic cassette in which a copper ion-dependent inducible metallothionein promoter controls expression of a siRNA precursor is inserted into a genomic pre-integrated transgene by CRIPSR/Cas9 technology. The endogenous siRNA source allows the gene knockdown in cell cultures that are refractory to conventional transfection with exogenous siRNA. The efficiency of the method was demonstrated in Drosophila ovarian somatic cell culture (OSC) for two genes that are essential for oogenesis: Cul3, encoding a component of the multiprotein ubiquitin-ligase complex with versatile functions in proteostasis, and cut, encoding a transcription factor regulating differentiation of ovarian follicular cells.

Knockdown of cullin 3 inhibits progressive phenotypes and increases chemosensitivity in cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is an extremely aggressive malignancy arising from the epithelial cells lining the bile ducts. It presents a substantial global health issue, with the highest incidence rates, ranging from 40­100 cases/100,000 individuals, found in Southeast Asia, where liver fluke infection is endemic. In Europe and America, incidence rates range from 0.4­2 cases/100,000 individuals. Globally, mortality rates range from 0.2­2 deaths/100,000 person­years and are increasing in most countries. Chemotherapy is the primary treatment for advanced CCA due to limited options from late­stage diagnosis, but its efficacy is hindered by drug­resistant phenotypes. In a previous study, proteomics analysis of drug­resistant CCA cell lines (KKU­213A­FR and KKU­213A­GR) and the parental KKU­213A line identified cullin 3 (Cul3) as markedly overexpressed in drug­resistant cells. Cul3, a scaffold protein within CUL3­RING ubiquitin ligase complexes, is crucial for ubiquitination and proteasome degradation, yet its role in drug­resistant CCA remains to be elucidated. The present study aimed to elucidate the role of Cul3 in drug­resistant CCA cell lines. Reverse transcription­quantitative PCR and western blot analyses confirmed significantly elevated Cul3 mRNA and protein levels in drug­resistant cell lines compared with the parental control. Short interfering RNA­mediated Cul3 knockdown sensitized cells to 5­fluorouracil and gemcitabine and inhibited cell proliferation, colony formation, migration and invasion. In addition, Cul3 knockdown induced G0/G1 cell cycle arrest and suppressed key cell cycle regulatory proteins, cyclin D, cyclin­dependent kinase (CDK)4 and CDK6. Bioinformatics analysis of CCA patient samples using The Cancer Genome Atlas data revealed Cul3 upregulation in CCA tissues compared with normal bile duct tissues. STRING analysis of upregulated proteins in drug­resistant CCA cell lines identified a highly interactive Cul3 network, including COMM Domain Containing 3, Ariadne RBR E3 ubiquitin protein ligase 1, Egl nine homolog 1, Proteasome 26S Subunit Non­ATPase 13, DExH­box helicase 9 and small nuclear ribonucleoprotein polypeptide G, which showed a positive correlation with Cul3 in CCA tissues. Knocking down Cul3 significantly suppressed the mRNA expression of these genes, suggesting that Cul3 may act as an upstream regulator of them. Gene Ontology analysis revealed that the majority of these genes were categorized under binding function, metabolic process, cellular anatomical entity, protein­containing complex and protein­modifying enzyme. Taken together, these findings highlighted the biological and clinical significance of Cul3 in drug resistance and progression of CCA.

The Proteasome and Cul3-Dependent Protein Ubiquitination Is Required for Gli Protein-Mediated Activation of Gene Expression in the Hedgehog Pathway.

Many cellular processes are regulated by proteasome-mediated protein degradation, including regulation of signaling pathways and gene expression. Among the pathways regulated by the ubiquitin-proteasome system is the Hedgehog pathway and its downstream effectors, the Gli transcription factors. Here we provide evidence that proteasomal activity is necessary for maintaining the activation of the Hedgehog pathway, and this crucial event takes place at the level of Gli proteins. We undertook extensive work to demonstrate the specificity of the observed phenomenon by ruling out the involvement of primary cilium, impaired nuclear import, failed dissociation from Sufu, microtubule stabilization, and stabilization of Gli repressor forms. Moreover, we showed that proteasomal-inhibition-mediated Hedgehog pathway downregulation is not restricted to the NIH-3T3 cell line. We demonstrated, using CRISPR/Ca9 mutagenesis, that neither Gli1, Gli2, nor Gli3 are solely responsible for the Hedgehog pathway downregulation upon proteasome inhibitor treatment, and that Cul3 KO renders the same phenotype. Finally, we report two novel E3 ubiquitin ligases, Btbd9 and Kctd3, known Cul3 interactors, as positive Hedgehog pathway regulators. Our data pave the way for a better understanding of the regulation of gene expression and the Hedgehog signaling pathway.

Photoreceptor-targeted extracellular vesicles-mediated delivery of Cul7 siRNA for retinal degeneration therapy.

Rationale: Photoreceptor loss is a primary pathological feature of retinal degeneration (RD) with limited treatment strategies. RNA interference (RNAi) has emerged as a promising method of gene therapy in regenerative medicine. However, the transfer of RNAi therapeutics to photoreceptors and the deficiency of effective therapeutic targets are still major challenges in the treatment of RD. Methods: In this study, photoreceptor-derived extracellular vesicles (PEVs) conjugated with photoreceptor-binding peptide MH42 (PEVsMH42) were prepared using the anchoring peptide CP05. Transcriptome sequencing was applied to investigate the potential therapeutic target of RD. We then engineered PEVsMH42 with specific small-interfering RNAs (siRNAs) through electroporation and evaluated their therapeutic efficacy in N-methyl-N-nitrosourea (MNU)-induced RD mice and Pde6ßrd1/rd1 mutant mice. Results: PEVsMH42 were selectively accumulated in photoreceptors after intravitreal injection. Cullin-7 (Cul7) was identified as a novel therapeutic target of RD. Taking advantage of the established PEVsMH42, siRNAs targeting Cul7 (siCul7) were efficiently delivered to photoreceptors and consequently blocked the expression of Cul7. Moreover, suppression of Cul7 effectively protected photoreceptors to alleviate RD both in MNU-induced mouse model and Pde6ßrd1/rd1 mutant mouse model. Mechanistically, PEVsMH42 loaded with siCul7 (PEVsMH42-siCul7)-induced Cul7 downregulation was responsible for preventing Cul7-mediated glutathione peroxidase 4 (Gpx4) ubiquitination and degradation, resulting in the inhibition of photoreceptor ferroptosis. Conclusions: In summary, PEVsMH42-siCul7 attenuate photoreceptor ferroptosis to treat RD by inhibiting Cul7-induced ubiquitination of Gpx4. Our study develops a PEVs-based platform for photoreceptor-targeted delivery and highlights the potential of PEVsMH42-siCul7 as effective therapeutics for RD.

Identification and Characterization of Novel Small-Molecule Enhancers of the CUL3LZTR1 E3 Ligase KRAS Complex.

The RAS family of GTPases is among the most frequently mutated proteins in human cancer, creating a high clinical demand for therapies that counteract their signaling activity. An important layer of regulation that could be therapeutically exploited is the proteostatic regulation of the main RAS GTPases KRAS, NRAS, and HRAS, as well as the closely related members, MRAS and RIT1, by the leucine zipper-like transcriptional regulator 1 cullin 3 RING E3 ubiquitin ligase complex (CUL3LZTR1). Genetic inactivation of LZTR1, as observed in different cancer entities and Noonan syndrome leads to enhanced RAS GTPase abundance and altered MAPK pathway activation state. Novel therapeutic approaches to interfere with hyperactive RAS signaling, thereby complementing existing treatments, are highly sought after. Motivated by the growing arsenal of molecular glue degraders, we report the identification of novel chemical fragments that enhance the protein-protein interaction (PPI) of the KRAS-LZTR1 complex. We established a split-luciferase-based reporter assay that monitors the RAS GTPase-LZTR1 interaction in a scalable format, capable of capturing chemical, as well as mutational perturbations. Using this screening system, in combination with a small fragment library, we identified two fragments, C53 and Z86, that enhance the interaction of the KRAS-LZTR1 complex in a dose-dependent manner. Further orthogonal validation experiments using proximity biotinylation (BioID), thermal shift assays, and NMR spectroscopy demonstrated fragment-dependent enhanced recruitment of endogenous LZTR1 and physical engagement of KRAS. The two fragments, which potentiate the KRAS-LZTR1 interaction, serve as starting points for fragment-based drug discovery. Additionally, the assay we introduced is amenable to high-throughput screening to further explore the pharmacological modulation of the CUL3LZTR1-RAS GTPase complex.

Ribosomal Protein S4 X-Linked as a Novel Modulator of MDM2 Stability by Suppressing MDM2 Auto-Ubiquitination and SCF Complex-Mediated Ubiquitination.

Mouse double minute 2 (MDM2) is an oncoprotein that is frequently overexpressed in tumors and enhances cellular transformation. Owing to the important role of MDM2 in modulating p53 function, it is crucial to understand the mechanism underlying the regulation of MDM2 levels. We identified ribosomal protein S4X-linked (RPS4X) as a novel binding partner of MDM2 and showed that RPS4X promotes MDM2 stability. RPS4X suppressed polyubiquitination of MDM2 by suppressing homodimer formation and preventing auto-ubiquitination. Moreover, RPS4X inhibited the interaction between MDM2 and Cullin1, a scaffold protein of the Skp1-Cullin1-F-box protein (SCF) complex and an E3 ubiquitin ligase for MDM2. RPS4X expression in cells enhanced the steady-state level of MDM2 protein. RPS4X was associated not only with MDM2 but also with Cullin1 and then blocked the MDM2/Cullin1 interaction. This is the first report of an interaction between ribosomal proteins (RPs) and Cullin1. Our results contribute to the elucidation of the MDM2 stabilization mechanism in cancer cells, expanding our understanding of the new functions of RPs.

The small CRL4CSA ubiquitin ligase component DDA1 regulates transcription-coupled repair dynamics.

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.

The cullin Rtt101 promotes ubiquitin-dependent DNA-protein crosslink repair across the cell cycle.

DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1. Using an in vivo inducible Top1-mimicking DPC system, we reveal the significant impact of Rtt101 ubiquitination on DPC removal across different cell cycle phases. High-throughput methods coupled with next-generation sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a main contributor to DPC repair throughout the yeast cell cycle.

Functional characterization of Cullin-1-RING ubiquitin ligase (CRL1) complex in Leishmania infantum.

Cullin-1-RING ubiquitin ligases (CRL1) or SCF1 (SKP1-CUL1-RBX1) E3 ubiquitin ligases are the largest and most extensively investigated class of E3 ligases in mammals that regulate fundamental processes, such as the cell cycle and proliferation. These enzymes are multiprotein complexes comprising SKP1, CUL1, RBX1, and an F-box protein that acts as a specificity factor by interacting with SKP1 through its F-box domain and recruiting substrates via other domains. E3 ligases are important players in the ubiquitination process, recognizing and transferring ubiquitin to substrates destined for degradation by proteasomes or processing by deubiquitinating enzymes. The ubiquitin-proteasome system (UPS) is the main regulator of intracellular proteolysis in eukaryotes and is required for parasites to alternate hosts in their life cycles, resulting in successful parasitism. Leishmania UPS is poorly investigated, and CRL1 in L. infantum, the causative agent of visceral leishmaniasis in Latin America, is yet to be described. Here, we show that the L. infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 -putative) form a LinfCRL1 complex structurally similar to the H. sapiens CRL1. Mass spectrometry analysis of the LinfSkp1 and LinfCul1 interactomes revealed proteins involved in several intracellular processes, including six F-box proteins known as F-box-like proteins (Flp) (data are available via ProteomeXchange with identifier PXD051961). The interaction of LinfFlp 1-6 with LinfSkp1 was confirmed, and using in vitro ubiquitination assays, we demonstrated the function of the LinfCRL1(Flp1) complex to transfer ubiquitin. We also found that LinfSKP1 and LinfRBX1 knockouts resulted in nonviable L. infantum lineages, whereas LinfCUL1 was involved in parasite growth and rosette formation. Finally, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L. infantum. Thus, a new class of E3 ubiquitin ligases has been described in L. infantum with functions related to various parasitic processes that may serve as prospective targets for leishmaniasis treatment.

A comparative analysis indicates SLC7A11 expression regulate the prognostic value of KEAP1-NFE2L2-CUL3 mutations in human uterine corpus endometrial carcinoma.

Uterine corpus endometrial cancer (UCEC) is a third most common malignancy in women with a poor prognosis in advanced stages. In this study, we performed an integrated comparative analysis of exome and transcriptome data from The Cancer Genome Atlas (TCGA) of Lung Adenocarcinoma (LUAD), and UCEC patients. Our multi-omics analysis shows that the UCEC patients carrying mutations in the KEAP1-NFE2L2-CUL3 genes were associated with better progression-free survival (PFS), whereas the KEAP1-NFE2L2-CUL3 mutation in LUAD showed poor outcomes. Functional annotations and correlative expression studies show that genes, particularly GCLC and GCLM related to glutathione synthesis are expressed at lower levels in the KEAP1-NFE2L2-CUL3 mutant UCEC compared to LUAD. This events result in glutathione deficiency and it may compromise to combat intracellular reactive oxygen species (ROS). However, the expression of genes involved in the glutathione recycling process was not affected. On the other hand, cellular import of cystine is high due to increased SLC7A11 expression in UCEC. Because glutathione synthesis is impaired, the unconverted cysteine accumulates in cells, leading to di-sulfite stress. Apart from NRF2, ARID1A is one of the positive regulators of SLC7A11. In support, UCEC patients with co-occurrence of KEAP1-NFE2L2-CUL3 and ARID1A mutation shows significantly decreased PFS with decline of SLC7A11 expression as compared to patients carrying only KEAP1-NFE2L2-CUL3 mutation. Thus, we hypothesize that the KEAP1-NFE2L2-CUL3 mutation in UCEC leads to uncontrollable ROS with di-sulfite stress, reflecting a favorable clinical outcome.

Prognostic potential of CUL3 ligase with differential roles in luminal A and basal type breast cancer tumors.

Breast cancer is a prevalent and significant cause of mortality in women, and manifests as six molecular subtypes. Its further histologic classification into non-invasive ductal or lobular carcinoma (DCIS) and invasive carcinoma (ILC or IDC) underscores its heterogeneity. The ubiquitin-proteasome system plays a crucial role in breast cancer, with inhibitors targeting the 26S proteasome showing promise in clinical treatment. The Cullin-RING ubiquitin ligases, including CUL3, have direct links to breast cancer. This study focuses on CUL3 as a potential biomarker, leveraging high-throughput sequencing, gene expression profiling, experimental and data analysis tools. Through comprehensive analysis using databases like GEPIA2 and UALCAN, as well as TCGA datasets, CUL3's expression and its association with prognostic values were assessed. Additionally, the impact of CUL3 overexpression was explored in MCF-7 and MDA-MB-231 breast cancer cell lines, revealing distinct differences in molecular and phenotypic characteristics. We further profiled its expression and localization in breast cancer tissues identifying prominent differences between luminal A and TNBC tumors. Conclusively, CUL3 was found to be associated with cell cycle progression, and DNA damage response, exhibiting diverse roles depending on the tumor's molecular type. It exhibits a tendency to act as an oncogene in triple-negative tumors and as a tumor suppressor in luminal A types, suggesting a potential significance in breast cancer progression and therapeutic directions.

CCT and Cullin1 Regulate the TORC1 Pathway to Promote Dendritic Arborization in Health and Disease.

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.

Association of CUL4B with the pathogenesis of age-related cataract.

PURPOSE: Age-related cataract (ARC) is the most common cause of visual impairment and blindness in older adults. However, the role of CUL4B in the ARC remains unclear. Therefore, we investigated CUL4B expression and its effects on apoptosis. MATERIALS AND METHODS: CUL4B expression levels were detected by a quantitative real-time polymerase chain reaction from the anterior lens capsules of patients with ARC and HLE-B3 cells treated with different concentrations of H2O2. CUL4B expression was silenced by siRNA transfection to evaluate apoptosis. CUL4B and apoptotic proteins B cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), caspase-3, cleaved caspase-3, Bax, Bak, and Bid were assessed using western blot analysis. Apoptosis was monitored using the TUNEL assay. RESULTS: CUL4B expression was downregulated in the anterior lens capsules (P < 0.0001) and H2O2-treated HLE-B3 cells (P = 0.0405). CUL4B protein levels were significantly lower in 100 µmol/L (P = 0.0012) and 200 µmol/L (P = 0.0041) H2O2-treated HLE-B3 cells than in the untreated cells. CUL4B expression was significantly knocked down at the mRNA (P = 0.0043) and protein levels (P = 0.0002) in HLE-B3 cells. Bcl-2 (P = 0.0199), Mcl-1 (P = 0.0042), and caspase-3 (P = 0.0142) were significantly downregulated, whereas cleaved caspase-3 (P = 0.0089) and Bak (P = 0.009) were significantly upregulated in the knockdown group. The TUNEL assay showed a greater induction of apoptosis. CONCLUSIONS: CUL4B downregulation promotes the apoptosis of lens epithelial cells. Our study may help in understanding the role of CUL4B in ARC pathogenesis.

A Cullin 5-based complex serves as an essential modulator of ORF9b stability in SARS-CoV-2 replication.

The ORF9b protein, derived from the nucleocapsid's open-reading frame in both SARS-CoV and SARS-CoV-2, serves as an accessory protein crucial for viral immune evasion by inhibiting the innate immune response. Despite its significance, the precise regulatory mechanisms underlying its function remain elusive. In the present study, we unveil that the ORF9b protein of SARS-CoV-2, including emerging mutant strains like Delta and Omicron, can undergo ubiquitination at the K67 site and subsequent degradation via the proteasome pathway, despite certain mutations present among these strains. Moreover, our investigation further uncovers the pivotal role of the translocase of the outer mitochondrial membrane 70 (TOM70) as a substrate receptor, bridging ORF9b with heat shock protein 90 alpha (HSP90α) and Cullin 5 (CUL5) to form a complex. Within this complex, CUL5 triggers the ubiquitination and degradation of ORF9b, acting as a host antiviral factor, while HSP90α functions to stabilize it. Notably, treatment with HSP90 inhibitors such as GA or 17-AAG accelerates the degradation of ORF9b, leading to a pronounced inhibition of SARS-CoV-2 replication. Single-cell sequencing data revealed an up-regulation of HSP90α in lung epithelial cells from COVID-19 patients, suggesting a potential mechanism by which SARS-CoV-2 may exploit HSP90α to evade the host immunity. Our study identifies the CUL5-TOM70-HSP90α complex as a critical regulator of ORF9b protein stability, shedding light on the intricate host-virus immune response dynamics and offering promising avenues for drug development against SARS-CoV-2 in clinical settings.

Long Noncoding RNA MSL3P1 Regulates CUL3 mRNA Cytoplasmic Transport and Stability and Promotes Lung Adenocarcinoma Metastasis.

Lung adenocarcinoma (LUAD) is the most prevalent histological type of lung cancer. Previous studies have reported that specific long noncoding RNAs (lncRNA) are involved in cancer development and progression. The phenotype and mechanism of ENST00000440028, named MSL3P1, an lncRNA referred to as a cancer-testis gene with potential roles in tumorigenesis and progression, have not been reported. MSL3P1 is overexpressed in LUAD tumor tissues, which is significantly associated with clinical characteristics, metastasis, and poor clinical prognosis. MSL3P1 promotes the metastasis of LUAD in vitro and in vivo. The enhancer reprogramming in LUAD tumor tissue is the major driver of the aberrant expression of MSL3P1. Mechanistically, owing to the competitive binding to CUL3 mRNA with ZFC3H1 protein (a protein involved in targeting polyadenylated RNA to exosomes and promoting the degradation of target mRNA), MSL3P1 can prevent the ZFC3H1-mediated RNA degradation of CUL3 mRNA and transport it to the cytoplasm. This activates the downstream epithelial-to-mesenchymal transition signaling pathway and promotes tumor invasion and metastasis. Implications: This study indicates that lncRNA MSL3P1 regulates CUL3 mRNA stability and promotes metastasis and holds potential as a prognostic biomarker and therapeutic target in LUAD.

A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22.

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.

Enhanced ApcMin/+ adenoma formation after epithelial CUL4B deletion by recruitment of myeloid-derived suppressor cells.

Colorectal cancer (CRC) stands as a prevalent malignancy globally. A pivotal event in CRC pathogenesis involves the loss-of-function mutation in the APC gene, leading to the formation of benign polyps. Despite the well-established role of APC, the contribution of CUL4B to CRC initiation in the pre-tumorous stage remains poorly understood. In this investigation, we generated a murine model by crossing ApcMin/+ mice with Cul4bΔIEC mice to achieve specific deletion of Cul4b in the gut epithelium against an ApcMin/+ background. By employing histological methods, RNA-sequencing (RNA-seq), and flow cytometry, we assessed alterations and characterized the immune microenvironment. Our results unveiled that CUL4B deficiency in gut epithelium expedited ApcMin/+ adenoma formation. Notably, CUL4B in adenomas restrained the accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). In vivo inhibition of MDSCs significantly delayed the growth of CUL4B deleted ApcMin/+ adenomas. Furthermore, the addition of MDSCs to in vitro cultured ApcMin/+; Cul4bΔIEC adenoma organoids mitigated their alterations. Mechanistically, CUL4B directly interacted with the promoter of Csf3, the gene encoding granulocyte-colony stimulating factor (G-CSF) by coordinating with PRC2. Inhibiting CUL4B epigenetically activated the expression of G-CSF, promoting the recruitment of MDSCs. These findings offer novel insights into the tumor suppressor-like roles of CUL4B in regulating ApcMin/+ adenomas, suggesting a potential therapeutic strategy for CRC initiation and progression in the context of activated Wnt signaling.

Comprehensive prognostic and immunological analysis of Cullin2 in pan-cancer and its identification in hepatocellular carcinoma.

BACKGROUND: As a member of the Cullin family, Cullin2 (CUL2) is involved in the development and spread of different types of cancers. However, the precise role of CUL2 in human cancer remains largely elusive. METHODS: In this study, various databases were applied to observe the CUL2 expression. Kaplan-Meier and Spearman correlation analyses were employed to investigate the potential links between CUL2 level, patient prognosis, and the infiltration of immune cells. In addition, the association between CUL2 and the efficacy of immunotherapy in an immunotherapy cohort was investigated. Moreover, the expression and distribution of CUL2 in cells were observed using the Human Protein Atlas (THPA) database. Finally, clinical tissue specimens and in vitro function assays were conducted to validate the expressions and effects of CUL2 on the biological functions in hepatocellular carcinoma (HCC) cells. RESULTS: While there are variations in CUL2 expression across different organs and cell types, it is notably upregulated in a majority of tumor tissues. In addition, CUL2 gene mutations are common in multiple cancers with low mutation rates and CUL2 is closely related to the prognosis of some cancer's patients, some immune regulatory factors, TMB, MSI, MMR genes, and DNA methylation. Further, our results found that downregulating CUL2 inhibits the proliferation, and migration abilities. CONCLUSIONS: The expression of CUL2 has an impact on the prognosis of various tumors, and this correlation is particularly noteworthy due to its significant association with the infiltration of immune cells within tumors. CUL2 was an oncogene contributing to the progression of HCC.

Dynamic role of CUL4B in radiation-induced intestinal injury-regeneration.

CUL4B, a crucial scaffolding protein in the largest E3 ubiquitin ligase complex CRL4B, is involved in a broad range of physiological and pathological processes. While previous research has shown that CUL4B participates in maintaining intestinal homeostasis and function, its involvement in facilitating intestinal recovery following ionizing radiation (IR) damage has not been fully elucidated. Here, we utilized in vivo and in vitro models to decipher the role of CUL4B in intestinal repair after IR-injury. Our findings demonstrated that prior to radiation exposure, CUL4B inhibited the ubiquitination modification of PSME3, which led to the accumulation of PSME3 and subsequent negative regulation of p53-mediated apoptosis. In contrast, after radiation, CUL4B dissociated from PSME3 and translocated into the nucleus at phosphorylated histones H2A (γH2AX) foci, thereby impeding DNA damage repair and augmenting p53-mediated apoptosis through inhibition of BRCA1 phosphorylation and RAD51. Our study elucidated the dynamic role of CUL4B in the repair of radiation-induced intestinal damage and uncovered novel molecular mechanisms underlying the repair process, suggesting a potential therapeutic strategy of intestinal damage after radiation therapy for cancers.