DNA Damage-binding Protein Research Articles

DCAF13 promotes breast cancer cell proliferation by ubiquitin inhibiting PERP expression.

Evolutionarily conserved DDB1‐and CUL4‐associated factor 13 (DCAF13) is a recently discovered substrate receptor for the cullin RING‐finger ubiquitin ligase 4 (CRL4) E3 ubiquitin ligase that regulates cell cycle progression. DCAF13 is overexpressed in many cancers, although its role in breast cancer is currently elusive. In this study we demonstrate that DCAF13 is overexpressed in human breast cancer and that its overexpression closely correlates with poor prognosis, suggesting that DCAF13 may serve as a diagnostic marker and therapeutic target. We knocked down DCAF13 in breast cancer cell lines using CRISPR/Cas9 and found that DCAF13 deletion markedly reduced breast cancer cell proliferation, clone formation, and migration both in vitro and in vivo. In addition, DCAF13 deletion promoted breast cancer cell apoptosis and senescence, and induced cell cycle arrest in the G1/S phase. Genome‐wide RNAseq analysis and western blotting revealed that loss of DCAF13 resulted in both mRNA and protein accumulation of p53 apoptosis effector related to PMP22 (PERP). Knockdown of PERP partially reversed the hampered cell proliferation induced by DCAF13 knockdown. Co‐immunoprecipitation assays revealed that DCAF13 and DNA damage‐binding protein 1 (DDB1) directly interact with PERP. Overexpression of DDB1 significantly increased PERP polyubiquitination, suggesting that CRL4DCAF13 E3 ligase targets PERP for ubiquitination and proteasomal degradation. In conclusion, DCAF13 and the downstream effector PERP occupy key roles in breast cancer proliferation and potentially serve as prognostics and therapeutic targets.

Inter-individual Differences in Radiosensitivity based on CDKN1A, GADD45A, DDB2, BCL2 and TRX Gene Expression in Human Lymphocytes

Transcriptomic signatures of radioresponsive genes are recently explored as a powerful tool for biodosimetry. As cytogenetics remains the gold standard and a robust approach for wide-scale testing during radiologic emergencies, the utility of molecular signatures remains to be adequately validated. The present study analyzed the expression profiles of five highly responsive genes to radiation: B-cell lymphoma 2 (BCL2), DNA damage-binding protein 2 (DDB2), cyclin-dependent kinase inhibitor 1A (CDKN1A), growth arrest and DNA-damage-inducible alpha (GADD45A) and thioredoxin (TRX). Ex vivo exposure of peripheral blood lymphocytes from 17 healthy subjects, aged 21 to 53 years, at 2 Gy radiation appeared to mobilize the index genes involved in cell cycle arrest (GADD45A and CDKN1A) and the binding to DNA lesion to facilitate excision repair (DDB2). However, the transcription of pro-survival protein BCL2 and redox repair antioxidant protein TRX were not as reliable as molecular biodosimeters considering the variability among individual responses. Nevertheless, the significant correlations observed among the genes emphasize their synchronized roles during DNA damage and redox response. Gender-based differences in gene expression were not detected. These findings indicated the diverse transcriptional regulation of p53-dependent pathways in radiation-exposed lymphocytes. Further validation in more patients during healthy and diseased states could contribute to the clinical application of gene-based radiation biodosimetry.

<i>BrDDB1A</i> maintains regular growth of Chinese cabbage via UV tolerance

Normal plant growth and development ensures the timely maturity and expected yield of crops. The repair mechanism of genome damage under adverse circumstance is essential for maintaining regular plant growth. Herein, two allelic growth retardation mutants of Chinese cabbage, <italic>grm1</italic> and <italic>grm2</italic>, were obtained from EMS-mutagenized populations of wild-type ‘FT’ isolated-cultured microspores and seeds, respectively. Both mutants stably inherited and exhibited stunted growth with smaller leafy-heads. Genetic analysis and allelism test manifested that the mutated trait was triggered by a same single recessive nuclear gene. Via BSR-Seq, <italic>Brgrm1</italic> was mapped to a target region including 20 genes on chromosome A09. Whole-genome re-sequencing revealed that <italic>BraA09g024830.3C</italic> in <italic>grm1</italic> had a single base (A) deletion in the 17th exon, leading to a termination codon (TAG). Genotyping showed that the mutated phenotype co-segregated with the InDel in recombinants of the closest linkage markers. In addition, cloning of <italic>BraA09g024830.3C</italic> in <italic>grm2</italic> found that a base substitution (G-A) occurred in the last base of the 1st intron causing an additional 263-bp retention in coding sequences, which in turn led to a termination codon (TAA). <italic>BraA09g024830.3C</italic> (<italic>BrDDB1A</italic>) is a homolog of <italic>Arabidopsis thaliana</italic> <italic>Damaged DNA Binding Protein 1A</italic> (<italic>DDB1A</italic>), a key gene of UV tolerance involved in DNA damage repair. The two mutants exhibited normal plant growth identical with wild-type under an extremely low UV radiation. Our results demonstrated that <italic>BrDDB1A</italic> contributes to maintaining regular plant growth in Chinese cabbage, which provide insights into elucidating the molecular mechanism underlying Chinese cabbage growth and development.

ERα determines the chemo-resistant function of mutant p53 involving the switch between lincRNA-p21 and DDB2 expressions

Mutant p53 (mutp53) commonly loses its DNA binding affinity to p53 response elements (p53REs) and fails to induce apoptosis fully. However, the p53 mutation does not predict chemoresistance in all subtypes of breast cancers, and the critical determinants remain to be identified. In this study, mutp53 was found to mediate chemotherapy-induced long intergenic noncoding RNA-p21 (lincRNA-p21) expression by targeting the G-quadruplex structure rather than the p53RE on its promoter to promote chemosensitivity. However, estrogen receptor alpha (ERα) suppressed mutp53-mediated lincRNA-p21 expression by hijacking mutp53 to upregulate damaged DNA binding protein 2 (DDB2) transcription for subsequent DNA repair and chemoresistance. Levels of lincRNA-p21 positively correlated with the clinical responses of breast cancer patients to neoadjuvant chemotherapy and had an inverse correlation with the ER status and DDB2 level. In contrast, the carboplatin-induced DDB2 expression was higher in ER-positive breast tumor tissues. These results demonstrated that ER status determines the oncogenic function of mutp53 in chemoresistance by switching its target gene preference from lincRNA-p21 to DDB2 and suggest that induction of lincRNA-p21 and targeting DDB2 would be effective strategies to increase the chemosensitivity of mutp53 breast cancer patients.

Effect of high-intensity light on the photosynthetic activity, pigment content and expression of light-dependent genes of photomorphogenetic Solanum lycopersicum hp mutants

The relationship between photosynthesis, pigment accumulation, and the expression of key light-regulated genes in Solanum lycopersicum hp-1, hp-2 and hp-1.2 photomorphogenetic mutants under conditions of high-intensity light (2000 μm (photons) m−2s−1) was studied.The hp-2 mutant (LA3006) and the hp-1 mutants (LA4012 and LA3538) are deficient in DET1 (De-etiolated 1 and DDB1 (DNA DAMAGE-BINDING PROTEIN 1), respectively, which are components of the CDD complex (COP10, DDB1, DET1). HP mutants are superproducers of various pigments and are sensitive to light. We have shown that HIL (high-intensity light) causes a decrease in PSII activity after 24 and 72 h of irradiation, which was partially restored after 72 h in the WT. The photosynthetic rate noticeably decreased only in LA4012 and LA3538 after 24 h of irradiation. After 72 h, the photosynthetic rate decreased in all mutants, with the exception of hp-1.2 LA0279, but the decrease was most noticeable in LA4012, yet significant changes in the respiration rate were absent. The LA0279 mutant was more capable of accumulating anthocyanin in the cells of the subepidermal parenchyma and chlorenchyma, as well as in the cells at the base of large multicellular glandular trichomes and in the mesophyll. Another important difference was the accumulation of increased amounts of antheraxanthin and phenolic compounds in the leaves of LA0279 after 72 h of HIL irradiation. Unlike LA4012, LA3006, LA0279, and LA3538 sowed a significantincrease in the expression levels of CHS, HY5, and FLS genes after 24 h, which may be one of the reasons for the higher adaptive potential of those three mutants. In addition to that in LA3538, strong light-induced stress led to an increased level of flavonol synthase (FLS) expression in the LA3006, LA0279, and LA4012 mutants.We hypothesize that the photosynthetic apparatus (PA) of the LA0279 mutant, which is deficient in the DET1 and DDB1 genes, is most adapted to prolonged HIL. Most likely, the resistance of PA mutants to HIL is due to a variety of factors, which, in addition to the redistribution of carotenoids, may include morphological features associated with the accumulation of anthocyanin in the epidermis, subepidermal layer, mesophyll and trichomes of leaves and with an increase in leaf thickness.

CRL4B complex promotes the development of pancreatic cancer by inhibiting secreted frizzled related protein 1

Objective: To investigate the role of CUL4B-RING E3 ubiquitin ligase (CRL4B) complex in pancreatic tumorigenesis and the molecular mechanism. Methods: Pancreatic cells were divided into control group (transfected with negative control lentivirus), shCUL4B group (transfected with CUL4B lentivirus), shDDB1 group [transfected with DNA damage binding protein 1 (DDB1) lentivirus], and shCUL4B+ siSFRP1 group (transfected with CUL4B lentivirus and SFRP1-siRNA). RNA-seq was performed in pancreatic cancer cell lines with CUL4B and DDB1 knocked down respectively, to identify the target genes regulated by CRL4B complex. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of target genes. Chromatin immunoprecipitation (ChIP) assay was used to identify the target genes directly regulated by CUL4B and DDB1. Western blot was used to detect the protein expression levels of the epithelial-mesenchymal transition (EMT) markers. The EdU cell proliferation test was used to detect cell proliferation ability. The scratch repair test and Transwell cell invasion test were used to detect cell migration and invasion ability. Finally, the sequencing data of pancreatic cancer-related tumor samples and normal samples in GEO, TCGA and GTEx databases were used to analyze the expression correlations of CUL4B, DDB1 and their downstream target genes. Results: RNA-seq results showed that target genes regulated by CRL4B complex involved in a number of malignant tumor-related signaling pathways. qRT-PCR results verified that the mRNA expression levels of the target genes of CUL4B or DDB1 knockdown groups were higher than those of the control group, and the difference was statistically significant (P<0.05). ChIP-PCR results showed that CRL4B complex directly bound to the promoter regions of the target genes, NME1 and SFRP1, and the enrichment of monoubiquitination of lysine at 119 of histone H2A (H2AK119ub1) in the promoter region of target gene was reduced after CUL4B knockdown. The proliferation rate in PANC-1 cell line of the control group was (32.10±3.58)%, higher than (13.95±1.66)% in the shCUL4B group and (22.38±0.77)% in the shCUL4B+ siSFRP1 group (P<0.05). The proliferation rate in AsPC-1 cell line of the control group was (35.47±7.80)%, higher than (19.60±3.58)% in the shCUL4B group and (30.09±0.81)% in the shCUL4B+ siSFRP1 group (P<0.05). The scratch repair experiment showed that the migration rate of PANC-1 cell line control group was (53.18±3.70)%, higher than that (17.46±2.62)% in the shCUL4B group and (44.99±9.18)% in the shCUL4B + siSFRP1 group (P<0.05). Western blot showed the expression levels of epithelial markers including α-catenin and γ-catenin in the control group were 1.00±0.03 and 1.01±0.11, respectively, lower than 1.44±0.01 and 1.21±0.06 in the shCUL4B group (P<0.05). The expression levels of mesenchymal markers including fibronectin and vimentin in the control group were 1.01±0.14 and 1.02±0.18, respectively, higher than 1.53±0.13 and 1.22±0.07 in the shCUL4B+ siSFRP1 group (P<0.05). The cell metastasis rate of the control group was (100.00±3.96)%, higher than the (35.49±0.34)% in the shCUL4B group and (107.06±2.77)% in the shCUL4B+ siSFRP1 group, the difference was statistically significant (P<0.05). The expressions of CUL4B and DDB1 were significantly upregulated in the pancreatic cancer tissues, and were negatively correlated with the expression of SFRP1 (r=-0.342 and r=-0.264, respectively). Conclusions: CRL4B complex inhibits the transcription of target gene SFRP1 and promotes the development of pancreatic cancer. Moreover, CRL4B complex is upregulated in pancreatic cancer, which provide a potential of therapeutic target for pancreatic cancer.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

Effect of the regulator of G-protein signaling 2 on the proliferation and invasion of oral squamous cell carcinoma cells and its molecular mechanism.

This study aims to investigate the effect of the regulator of G-protein signaling 2 (RGS2) on the proliferation and invasion of oral squamous cell carcinoma (OSCC) cells and its potential molecular mechanism. Metho⁃ds The expression status and clinical significance of RGS2 in head and neck squamous cell carcinomas and matched adjacent normal tissues were evaluated using TCGA database. Three OSCC cell lines (i.e., SCC-9, Cal27, and Fadu) were overexpressed with RGS2, and the effect of RGS2 on cell proliferation and invasion was determined using the Transwell, clone formation, and cell counting kit (CCK)-8 assays. Moreover, the yeast two-hybrid scree-ning and co-immunoprecipitation (Co-IP) assays were conducted to detect the correlation of RGS2, four and a half LIM domains protein 1 (FHL1), and damage DNA-binding protein 1 (DDB1). The expression level of RGS2 in OSCC was significantly lower than that in matched adjacent normal tissues (P=0.023). The high RGS2 expression level was negatively correlated with lymphovascular invasion (P<0.001). After transfection with lentiv-RGS2, the expression of RGS2 was increased, and the invasion and proliferation abilities of OSCC cell lines were evidently inhibited. FHL1 could competitively bind with RGS2, which decreased the integration of DDB1 and RGS2, inhibited the ubiquitination process of RGS2, and maintained the stability of the RGS2 protein. RGS2 plays an important role in the inhibition of OSCC proliferation and invasion. The structure stability of RGS2 is competitively regulated by FHL1 and DDB1.

A DNA repair disorder caused by de novo monoallelic DDB1 variants is associated with a neurodevelopmental syndrome

The DNA damage-binding protein 1 (DDB1) is part of the CUL4-DDB1 ubiquitin E3 ligase complex (CRL4), which is essential for DNA repair, chromatin remodeling, DNA replication, and signal transduction. Loss-of-function variants in genes encoding the complex components CUL4 and PHIP have been reported to cause syndromic intellectual disability with hypotonia and obesity, but no phenotype has been reported in association with DDB1 variants. Here, we report eight unrelated individuals, identified through Matchmaker Exchange, with de novo monoallelic variants in DDB1, including one recurrent variant in four individuals. The affected individuals have a consistent phenotype of hypotonia, mild to moderate intellectual disability, and similar facies, including horizontal or slightly bowed eyebrows, deep-set eyes, full cheeks, a short nose, and large, fleshy and forward-facing earlobes, demonstrated in the composite face generated from the cohort. Digital anomalies, including brachydactyly and syndactyly, were common. Three older individuals have obesity. We show that cells derived from affected individuals have altered DDB1 function resulting in abnormal DNA damage signatures and histone methylation following UV-induced DNA damage. Overall, our study adds to the growing family of neurodevelopmental phenotypes mediated by disruption of the CRL4 ubiquitin ligase pathway and begins to delineate the phenotypic and molecular effects of DDB1 misregulation.

Application of fluorescent-based technology detecting protein-protein interactions to monitor the binding of hepatitis B virus X protein to DNA-damage-binding protein 1.

The hepatitis B virus X protein (HBx) and the V protein of paramyxovirus simian virus 5 (SV5-V) interact with DNA damage-binding protein 1 (DDB1), a cellular enzyme involved in DNA repair and cell cycle regulation, to stimulate viral activity. DDB1 has several cellular substrates, and the amino acid sequences of the binding sites in the viral proteins and their substrates are notably dissimilar. To determine whether HBx binds preferentially to DDB1, despite differences in the amino acid sequences, we developed a system to monitor DDB1 binding in living cells through a protein-protein visuali­zation system, designated fluorescent-based technology detecting protein-protein interactions (Fluoppi). HBx in association with DDB1 formed clear fluorescent puncta. The number of these fluorescent puncta increased with an increase in the amount of HBx. The binding of HBx to DDB1 inhibited the cellular substrate DDB1-CUL4A-associated factor 9 (DCAF9) from binding to DDB1. The inhibitor nitazoxanide prevented the viral proteins HBx and SV5-V from binding to DDB1 but did not inhibit the binding of DCAF9 or HBx(ΔNC), which constitutes the binding site of HBx. Our results demonstrate that the Fluoppi system is useful for monitoring the binding of HBx to DDB1 as well as for examining the effect of drugs on DDB1-Hbx binding.

Argyrophilic nucleolar organizing regions in patients with Xeroderma Pigmentosum Group E

Xeroderma pigmentosum E (XP-E) is a rare autosomal recessive inherited genodermatosis associated with less severe sun burning with minimal sun exposure. The affected individuals acquire abnormal pigmentation and are at high risk for skin cancer [1] . Notably, the DNA damage-binding protein 2 (DDB2) gene is responsible for XP- E disease [2] .

Phase separation of Arabidopsis EMB1579 controls transcription, mRNA splicing, and development.

Tight regulation of gene transcription and mRNA splicing is essential for plant growth and development. Here we demonstrate that a plant-specific protein, EMBRYO DEFECTIVE 1579 (EMB1579), controls multiple growth and developmental processes in Arabidopsis. We demonstrate that EMB1579 forms liquid-like condensates both in vitro and in vivo, and the formation of normal-sized EMB1579 condensates is crucial for its cellular functions. We found that some chromosomal and RNA-related proteins interact with EMB1579 compartments, and loss of function of EMB1579 affects global gene transcription and mRNA splicing. Using floral transition as a physiological process, we demonstrate that EMB1579 is involved in FLOWERING LOCUS C (FLC)-mediated repression of flowering. Interestingly, we found that EMB1579 physically interacts with a homologue of Drosophila nucleosome remodeling factor 55-kDa (p55) called MULTIPLE SUPPRESSOR OF IRA 4 (MSI4), which has been implicated in repressing the expression of FLC by forming a complex with DNA Damage Binding Protein 1 (DDB1) and Cullin 4 (CUL4). This complex, named CUL4-DDB1MSI4, physically associates with a CURLY LEAF (CLF)-containing Polycomb Repressive Complex 2 (CLF-PRC2). We further demonstrate that EMB1579 interacts with CUL4 and DDB1, and EMB1579 condensates can recruit and condense MSI4 and DDB1. Furthermore, emb1579 phenocopies msi4 in terms of the level of H3K27 trimethylation on FLC. This allows us to propose that EMB1579 condensates recruit and condense CUL4-DDB1MSI4 complex, which facilitates the interaction of CUL4-DDB1MSI4 with CLF-PRC2 and promotes the role of CLF-PRC2 in establishing and/or maintaining the level of H3K27 trimethylation on FLC. Thus, we report a new mechanism for regulating plant gene transcription, mRNA splicing, and growth and development.

DDB1 promotes the proliferation and hypertrophy of chondrocytes during mouse skeleton development

The proliferation and hypertrophy of chondrocytes play important roles in endochondral ossification, which is tightly regulated during skeleton development. However, the regulation mechanism remains largely unknown. Here we show that DDB1 (Damaged DNA Binding Protein 1) has a critical function in the development of growth plates. Using chondrocyte-specific DDB1 knockout mice, we found that DDB1 deletion in chondrocytes results in dwarfism due to the aberrant skeleton development. The structure of growth plate in tibia becomes disordered at P21, not in femur. But at P70, the changes are severer in femur than tibia. Chondrocyte proliferation and differentiation are attenuated and asynchronous in both tibia and femur at P7 and P21. Furthermore, DDB1 deficiency induces p27 upregulation and subsequent cell cycle arrest in primary chondrocytes. Therefore, our data reveal that DDB1 is essential for the skeleton development by controlling chondrocyte proliferation and differentiation.

YTHDF2 promotes mitotic entry and is regulated by cell cycle mediators.

The N6-methyladenosine (m6A) modification regulates mRNA stability and translation. Here, we show that transcriptomic m6A modification can be dynamic and the m6A reader protein YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) promotes mRNA decay during cell cycle. Depletion of YTHDF2 in HeLa cells leads to the delay of mitotic entry due to overaccumulation of negative regulators of cell cycle such as Wee1-like protein kinase (WEE1). We demonstrate that WEE1 transcripts contain m6A modification, which promotes their decay through YTHDF2. Moreover, we found that YTHDF2 protein stability is dependent on cyclin-dependent kinase 1 (CDK1) activity. Thus, CDK1, YTHDF2, and WEE1 form a feedforward regulatory loop to promote mitotic entry. We further identified Cullin 1 (CUL1), Cullin 4A (CUL4A), damaged DNA-binding protein 1 (DDB1), and S-phase kinase-associated protein 2 (SKP2) as components of E3 ubiquitin ligase complexes that mediate YTHDF2 proteolysis. Our study provides insights into how cell cycle mediators modulate transcriptomic m6A modification, which in turn regulates the cell cycle.

Hepatitis B Virus HBx Protein Mediates the Degradation of Host Restriction Factors through the Cullin 4 DDB1 E3 Ubiquitin Ligase Complex.

The hepatitis B virus (HBV) regulatory HBx protein is required for infection, and its binding to cellular damaged DNA binding protein 1 (DDB1) is critical for this function. DDB1 is an adaptor protein for the cullin 4A Really Interesting New Gene (RING) E3 ubiquitin ligase (CRL4) complex and functions by binding cellular DDB1 cullin associated factor (DCAF) receptor proteins that recruit substrates for ubiquitination and degradation. We compared the proteins found in the CRL4 complex immunoprecipitated from uninfected versus HBV-infected hepatocytes from human liver chimeric mice for insight into mechanisms by which HBV and the cell interact within the CRL4 complex. Consistent with its role as a viral DCAF, HBx was found in the HBV CRL4 complexes. In tissue culture transfection experiments, we showed that HBx expression led to decreased levels of known restriction factor structural maintenance of chromosomes protein 6 (SMC6) and putative restriction factors stromal interaction molecule 1 (STIM1, zinc finger E-box binding homeobox 2 (ZEB2), and proteasome activator subunit 4 (PSME4). Moreover, silencing of these proteins led to increased HBV replication in the HepG2-sodium taurocholate cotransporting polypeptide (NTCP) infection model. We also identified cellular DCAF receptors in CRL4 complexes from humanized mice. Increasing amounts of HBx did not reveal competitive DCAF binding to cullin4 (CUL4)-DDB1 in plasmid-transfected cells. Our results suggest a model in which HBx benefits virus replication by directly or indirectly degrading multiple cellular restriction factors.

Small molecule NSC1892 targets the CUL4A/4B-DDB1 interactions and causes impairment of CRL4DCAF4 E3 ligases to inhibit colorectal cancer cell growth.

Cullin 4A and 4B (CUL4A and 4B) function as oncogenes in colorectal cancer (CRC) cells. Both of them conservatively associate with DNA damage-binding protein 1 (DDB1) and DDB1-CUL4-associated factor 4 (DCAF4) to form Cullin-RING E3 ligases known as CRL4DCAF4, which specifically ubiquitinate and degrade tumor suppressor ST7 (suppression of tumorigenicity 7). Knockdown either CUL4A/4B or DDB1 significantly inhibits tumor cell growth in vitro and in vivo. Thus, targeting these CRL4DCAF4 components and their interactions may be an effective strategy for the therapy of CRC. In this study, we developed an in vitro AlphaScreen assay to identify small molecules targeting the CUL4A-DDB1 interaction. We obtained a compound NSC1892, which strongly disrupted the CUL4A-DDB1 interaction (IC50 = 1.8 μM). Oncogenic phenotype analyses indicated that NSC1892 showed significant cytotoxicity to decrease cell proliferation, colony formation and invasion in CRC cells. Biochemical analyses demonstrated that NSC1892 treatment did not change CUL4A and CUL4B protein levels, but caused the degradation of DDB1, thereby leading to the impaired assembly of CRL4DCAF4 E3 ligases and resulting in the accumulation of ST7. The administration of NSC1892 in mice also significantly inhibited tumor growth through degrading DDB1 and accumulating ST7. Interestingly, NSC1892 also showed promising cytotoxicity to decrease the growth of other CUL4A- or CUL4B-overexpressing tumor cells such as SKOV3 ovarian cells and Saos2 osteosarcoma cells. Our results provide a new avenue for the development of a therapeutic compound targeting tumors through disrupting the CUL4-DDB1 interaction.

The small molecule PSSM0332 disassociates the CRL4ADCAF8 E3 ligase complex to decrease the ubiquitination of NcoR1 and inhibit the inflammatory response in a mouse sepsis-induced myocardial dysfunction model.

Sepsis-induced myocardial dysfunction (SIMD) is a life-threatening complication caused by inflammation, but how it is initiated is still unclear. Several studies have shown that extracellular high mobility group box 1 (HMGB1), an important cytokine triggering inflammation, is overexpressed during the pathogenesis of SIMD, but the underlying mechanism regarding its overexpression is still unknown. Herein, we discovered that CUL4A (cullin 4A) assembled an E3 ligase complex with RBX1 (ring-box 1), DDB1 (DNA damage-binding protein 1), and DCAF8 (DDB1 and CUL4 associated factor 8), termed CRL4ADCAF8, which ubiquitinated and degraded NcoR1 (nuclear receptor corepressor 1) in an LPS-induced SIMD mouse model. The degradation of NcoR1 failed to form a complex with the SP1 transcription factor, leading to the upregulation of HMGB1. Mature HMGB1 functioned as an effector to induce the expression of proinflammatory cytokines, causing inflammation and resulting in SIMD pathology. Using an in vitro AlphaScreen technology, we identified three small molecules that could inhibit the CUL4A-RBX1 interaction. Of them, PSSM0332 showed the strongest ability to inhibit the ubiquitination of NcoR1, and its administration in SIMD mice exhibited promising effects on decreasing the inflammatory response. Collectively, our results reveal that the CRL4ADCAF8 E3 ligase is critical for the initiation of SIMD by regulating the expression of HMGB1 and proinflammatory cytokines. Our results suggest that PSSM0332 is a promising candidate to inhibit the inflammatory response in the pathogenesis of SIMD, which will provide a new option for the therapy of SIMD.

Knockdown of Ubiquitin-Specific Protease 53 Enhances the Radiosensitivity of Human Cervical Squamous Cell Carcinoma by Regulating DNA Damage-Binding Protein 2.

Background:Cervical cancer ranks fourth in incidence and mortality among women. Ubiquitin-specific protein 53 binds to damage-specific DNA binding protein 2 and affects the biological properties of colon cancer. Damage-specific DNA binding protein is involved in nucleotide excision repair, which can repair DNA damage. However, the mechanism by which ubiquitin-specific protein 53 regulates the radiosensitivity of cervical cancer through damage-specific DNA binding protein remains unclear.Methods:Tissue samples from 40 patients with cervical squamous cell carcinoma who received radiotherapy were examined by immunohistochemistry to detect the expression of ubiquitin-specific protein 53, and clinical data were collected for statistical analysis. The cell cycle was detected by flow cytometry in Siha cells transfected with Si-USP53 and exposed to 8 Gy irradiation. Cell viability was determined by the CCK8 method in cells transfected with Si-USP53 and exposed to 0, 2, 4, 6, 8, or 10 Gy. The expression of damage-specific DNA binding protein, cyclin-dependent kinase 1, and cell cycle checkpoint kinase 2 was detected in cells transfected with Si-USP53.Results:The expression of ubiquitin-specific protein 53 in the tissues of patients with cervical squamous cell carcinoma was correlated with the sensitivity to radiotherapy. Knockdown of ubiquitin-specific protein 53 in Siha cells downregulated damage-specific DNA binding protein and caused G2/M cell cycle arrest and decreased the survival rate of cells in response to radiation.Conclusion:Ubiquitin-specific protein 53–induced cell cycle arrest and affected the radiotherapy sensitivity of tumors through damage-specific DNA binding protein.

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System.

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Role of Damage DNA-Binding Protein 1 in Pancreatic Cancer Progression and Chemoresistance.

Damaged DNA-binding protein 1 (DDB1) recruits nucleotide excision pathway proteins to form the UV-damaged DNA-binding protein complex and is required for DNA repair. DDB1 was reported to participate in apoptosis and chemoresistance regulation in several cancers. However, little is known about the function of DDB1 in pancreatic adenocarcinoma (PDAC). In this study, we reported that DDB1 functions as a tumor-promoting factor in PDAC by regulating cancer cell proliferation, epithelial-mesenchymal transition (EMT) and chemoresistance. Compared to normal pancreatic tissues, PDAC tissues had high expression levels of DDB1, and this high expression was positively correlated with poor prognosis. Furthermore, reductions in cell proliferation and EMT were observed in DDB1-deficient PDAC cell lines. Intriguingly, we also found that abrogation of DDB1 expression increased PDAC cell sensitivity to gemcitabine (GEM). Mechanistically, DDB1 knockdown was associated with an increase in deoxycytidine kinase expression in vivo and in vitro. In summary, our work demonstrated that DDB1 promotes PDAC progression and chemoresistance and may serve as a potential predictive marker and therapeutic target for PDAC treatment.