DNA Damage-binding Protein Research Articles

Functional characterization of importin α8 as a classical nuclear localization signal receptor

Importin α8 has recently been identified as an importin α family member based on its primary structure and binding ability to importin β1 and to several karyophilic proteins. However, there has been no experimental evidence that importin α8 actually functions in the nuclear transport of classical nuclear localization signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin α8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin β1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin α8-binding proteins, only 62 of which overlap with those of importin α1, the closest importin α family member. Among the importin α8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin α8/β1 pathway. Furthermore, we found that the other subtypes of importin α, which were also identified as importin α8-binding candidates, indeed form heterodimers with importin α8. Notably, we found that these importin α8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin α1. From these findings, we propose that importin α8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin α8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin β binding domain.

DDB2 modulates TGF-β signal transduction in human ovarian cancer cells by downregulating NEDD4L.

The expression of DNA damage-binding protein 2 (DDB2) has been linked to the prognosis of ovarian cancer and its underlying transcription regulatory function was proposed to contribute to the favorable treatment outcome. By applying gene microarray analysis, we discovered neural precursor cell expressed, developmentally downregulated 4-Like (NEDD4L) as a previously unidentified downstream gene regulated by DDB2. Mechanistic investigation demonstrated that DDB2 can bind to the promoter region of NEDD4L and recruit enhancer of zeste homolog 2 histone methyltransferase to repress NEDD4L transcription by enhancing histone H3 lysine 27 trimethylation (H3K27me3) at the NEDD4L promoter. Given that NEDD4L plays an important role in constraining transforming growth factor β signaling by targeting activated Smad2/Smad3 for degradation, we investigated the role of DDB2 in the regulation of TGF-β signaling in ovarian cancer cells. Our data indicate that DDB2 enhances TGF-β signal transduction and increases the responsiveness of ovarian cancer cells to TGF-β-induced growth inhibition. The study has uncovered an unappreciated regulatory mode that hinges on the interaction between DDB2 and NEDD4L in human ovarian cancer cells. The novel mechanism proposes the DDB2-mediated fine-tuning of TGF-β signaling and its downstream effects that impinge upon tumor growth in ovarian cancers.

FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex.

The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.

Evaluation of gene expression changes in human primary lung epithelial cells following 24-hr exposures to inorganic arsenic and its methylated metabolites and to arsenic trioxide.

The concentration response for altered gene expression in primary lung epithelial cells was determined following two treatments with arsenicals: (1) a mixture of trivalent arsenic compounds representative of urinary arsenic concentrations in exposed human populations, and (2) arsenite (As2 O3 ) a common form of inhaled arsenic dust that is frequently used in both in vivo and in vitro experimental exposures. Biochemical assays did not detect any evidence of cytotoxicity at the concentrations used, apart from a concentration-related increase in cellular heme oxygenase that was also indicated by the genomic analysis. Cell signal pathway enrichment analysis indicated similar responses to both treatments, with concentration-related responses in pathways related to cell adhesion, cytoskeleton remodeling, development (morphogenesis), cell cycle control, and to a lesser extent inflammatory responses. These cellular responses to arsenic were consistent with those observed in a previous study with primary uroepithelial cells. Benchmark dose analysis also demonstrated similar potency of the two treatments as well as comparable sensitivity of the two cell types. A number of genes showing similar concentration-dependent expression across individuals in both bladder and lung cells were identified, including heme oxygenase 1, thioredoxin reductase, DNA damage binding protein 2, and thrombomodulin. The data on human primary lung cells from this study, together with the data from human primary uroepithelial cells, support a conclusion that biological responses to arsenic by human cells under study conditions are unlikely to occur at concentrations below 0.1 µM. Environ. Mol. Mutagen. 56:477-490, 2015. © 2015 Wiley Periodicals, Inc.

The tomato DDI2, a PCNA ortholog, associating with DDB1-CUL4 complex is required for UV-damaged DNA repair and plant tolerance to UV stress

CULLIN 4 (CUL4)-DAMAGED DNA binding protein 1 (DDB1)-based ubiquitin E3 ligase modulates diverse cellular processes including repair of damaged genomic DNA. In this study, an uncharacterized gene termed as DDB1-Interacting protein 2 (DDI2) was identified in yeast two-hybrid screening with bait gene DDB1. The co-immunoprecipitation (co-IP) assays further demonstrated that DDI2 is associated with tomato DDB1-CUL4 complex in vivo. It appears that DDI2 encodes an ortholog of proliferating cell nuclear antigen (PCNA). Confocal microscope observation indicated that DDI2-GFP fusion protein was localized in nuclei. The expression of DDI2 gene is constitutive but substantially enhanced by UV-C irradiation. The transgenic tomato plants with overexpression or knockdown of DDI2 gene displayed the increased or decreased tolerance, respectively, to UV-C stress and chemical mutagen cisplatin. The quantitative analysis of UV-induced DNA lesions indicated that the dark repair of DNA damage was accelerated in DDI2 overexpression lines but delayed in knockdown lines. Conclusively, tomato DDI2 gene is required for UV-induced DNA damage repair and plant tolerance to UV stress. In addition, fruits of DDI2 transgenic plants are indistinguishable from that of wild type, regarding fresh weight and nutrient quality. Therefore, overexpression of DDI2 offers a suitable strategy for genetic manipulation of enhancing plant tolerance to UV stress.

Genome-wide identification, sequence characterization, and protein-protein interaction properties of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat family members in Solanum lycopersicum.

One hundred DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family genes were identified in the S. lycopersicum genome. The DWD genes encode proteins presumably functioning as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. These findings provide candidate genes and a research platform for further gene functionality and molecular breeding study. A subclass of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family proteins has been demonstrated to function as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. However, little information is available about the cognate subfamily genes in tomato (S. lycopersicum). In this study, based on the recently released tomato genome sequences, 100 tomato genes encoding DWD proteins that potentially interact with DDB1 were identified and characterized, including analyses of the detailed annotations, chromosome locations and compositions of conserved amino acid domains. In addition, a phylogenetic tree, which comprises of three main groups, of the subfamily genes was constructed. The physical interaction between tomato DDB1 and 14 representative DWD proteins was determined by yeast two-hybrid and co-immunoprecipitationassays. The subcellular localization of these 14 representative DWD proteins was determined. Six of them were localized in both nucleus and cytoplasm, seven proteins exclusively in cytoplasm, and one protein either in nucleus and cytoplasm, or exclusively in cytoplasm. Comparative genomic analysis demonstrated that the expansion of these subfamily members in tomato predominantly resulted from two whole-genome triplication events in the evolution history.

Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2

Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

The thalidomide-binding domain of cereblon defines the CULT domain family and is a new member of the β-tent fold.

Despite having caused one of the greatest medical catastrophies of the last century through its teratogenic side-effects, thalidomide continues to be an important agent in the treatment of leprosy and cancer. The protein cereblon, which forms an E3 ubiquitin ligase compex together with damaged DNA-binding protein 1 (DDB1) and cullin 4A, has been recently indentified as a primary target of thalidomide and its C-terminal part as responsible for binding thalidomide within a domain carrying several invariant cysteine and tryptophan residues. This domain, which we name CULT (cereblon domain of unknown activity, binding cellular ligands and thalidomide), is also found in a family of secreted proteins from animals and in a family of bacterial proteins occurring primarily in δ-proteobacteria. Its nearest relatives are yippee, a highly conserved eukaryotic protein of unknown function, and Mis18, a protein involved in the priming of centromeres for recruitment of CENP-A. Searches for distant homologs point to an evolutionary relationship of CULT, yippee, and Mis18 to proteins sharing a common fold, which consists of two four-stranded β-meanders packing at a roughly right angle and coordinating a zinc ion at their apex. A β-hairpin inserted into the first β-meander extends across the bottom of the structure towards the C-terminal edge of the second β-meander, with which it forms a cradle-shaped binding site that is topologically conserved in all members of this fold. We name this the β-tent fold for the striking arrangement of its constituent β-sheets. The fold has internal pseudosymmetry, raising the possibility that it arose by duplication of a subdomain-sized fragment.

Nucleotide Excision Repair (NER) Is Frequently Impaired and Affects Outcome in Multiple Myeloma (MM)

IntroductionMultiple Myeloma (MM) is a heterogeneous disease characterized by genomic instability and eventual poor outcome. Aberrations in DNA repair-related pathways have been considered to explain the instability. Nucleotide excision repair (NER) is an important pathway involved in the removal of bulky adducts and DNA crosslinks induced by various genotoxins. Little is known about the relationship between NER in MM biology and patient outcomes. Here we assess the role of NER in MM.MethodsWe evaluated NER efficiency in a panel of MM cell lines (n=18), with a functional assay based on the purified DNA-Damage Binding protein 2 (DDB2) complex (DDB2 proteo-probe, Dreze et al. 2014). NER proficiency was correlated with cytogenetic characteristics, p53 status, sequencing data, gene expression profile, and with melphalan (MLP) sensitivity evaluated by CellTiterGlo (CTG). We then evaluated NER efficiency in patient samples and interrogated the role of NER in MM patients by correlating expression of NER genes with survival (OS) in a cohort of 170 patients (IFM 2005-01) homogeneously treated with alkylating agents.ResultsNER, measured as the amount of (6-4) photoproducts remaining 2 hours after UV irradiation, showed variability between MM cell lines. Out of 18 cell lines, 7 exhibited various levels of NER deficiencies, defined as less than 90% repair at 2 hours (4 cell lines 90-70% and 3 cell lines <60%). The other 11 cell lines presented more than 90% of repair.P53 loss of function did not associate with NER deficiency. Notably, all t(4;14) cell lines tested (n=5) showed a NER repair rate > 90%. NER deficient cell lines (NER <90%) were sensitive to melphalan. However all melphalan sensitive cells did not exhibit NER deficiency, This suggests that other DNA repair pathways are involved in the repair of melphalan-induced lesions. Furthermore, we performed the assay in patient samples showing variable levels of NER, which may reflect different disease status and prognosis. Whole genome sequencing data from 6 NER deficient cell lines revealed missense mutations in critical NER genes in 2 of these cell lines. MM1S and MM1R cells showed mutations in the Xeroderma Pigmentosum Complementation Group A (XPA) gene (mutation D70H), and MM1R was also mutated in the Cockayne syndrome, ERCC6 gene (mutation L682I). Gene expression profile comparison in 12 of these showed a positive correlation between expression of NER genes and NER efficiency.We next studied expression of 20 NER genes in 170 patients treated with high dose melphalan (IFM 2005-01). The analysis revealed a significant negative correlation between 5 overexpressed NER genes (ERCC3, ERCC4, ERCC6, MMS19 and NTHL1) and overall survival (OS).ConclusionNER efficiency is heterogeneous in MM, in part due to acquired mutations. Impairment of NER is associated with outcome as well as may contribute to genomic instability. Ability to proficiently measure NER in patient samples provides us an opportunity to now evaluate NER as a prognostic marker in myeloma. DisclosuresNo relevant conflicts of interest to declare.

Mechanism of Lenalidomide Activity in Multiple Myeloma

Lenalidomide is a derivative of thalidomide, a drug developed in the 1950s as a sedative and treatment for morning sickness that became infamous for causing limb deformations (phocomelia) and other birth defects when used by pregnant women. Following the discovery in the 1990s that thalidomide inhibits the release of tumor necrosis factor (TNF) and blocks angiogenesis, researchers began studying thalidomide in other diseases, including cancer. Thalidomide demonstrated high in vitro activity in multiple myeloma and high response rates in clinical trials, leading to its accelerated approval by the FDA in 2006. In addition to the direct antiproliferative effects on multiple myeloma cells, thalidomide and its more potent derivatives, lenalidomide and pomalidomide, have pleiotropic effects on immune cells and are therefore called immunomodulatory drugs (IMiDs). IMiDs enhance the release of interleukin-2 (IL-2) and interferon-γ (IFN-γ) from activated T cells, inhibit the immunosuppressive activity of regulatory T cells, and increase natural killer (NK) cell-mediated cytotoxicity. In peripheral blood monocytes (PBMCs) IMiDs inhibit the release of TNF and other cytokines including interleukin-6 (IL-6), a critical growth factor for multiple myeloma cells. Recently, cereblon (CRBN) was identified as the common primary target for all IMiDs. CRBN forms an E3 ubiquitin ligase together with DNA damage-binding protein 1 (DDB1), cullin 4A (CUL4A), and regulators of cullins (ROC1) CRBN-CRL4. Interaction with this enzymatic complex has been shown to be essential for most properties of IMiDs including teratogenicity, antiproliferative effects in multiple myeloma and some of the immunomodulatory properties. More recently, it was demonstrated that lenalidomide and its analogues activate the CRBN-CRL4 E3 ligase to ubiquitinate and degrade two members of the Ikaros family of zinc finger transcription factors: Ikaros (IKZF1) and Aiolos (IKZF3). IKZF1 and IKZF3 are key regulators in lymphopoiesis and essential for lymphoid progenitor differentiation into effector cells. While IKZF1 and IKZF3 deletions and loss of function mutations are frequent in acute lymphoblastic leukemia, mature B-cell lymphomas like multiple myeloma and chronic lymphocytic leukemia have high IKZF1 and IKZF3 expression. Inactivation of IKZF1 and IKZF3 results in growth inhibition in multiple myeloma. Conversely, over-expression of IKZF1 or IKZF3 confers lenalidomide resistance, demonstrating that degradation of IKZF1 and IKZF3 is responsible for the direct cytotoxic effects of lenalidomide in multiple myeloma. One of the transcriptional targets of IKZF1 and IKZF3 is interferon regulatory factor 4 (IRF4), a transcription factor that is essential for proliferation and survival of multiple myeloma cells that is down-regulated after lenalidomide-induced degradation of IKZF1 and IKZF3. At the IL-2 locus, IKZF3 is a transcriptional repressor that is de-repressed after lenalidomide-induced degradation of IKZF3; explaining one of the immunomodulatory properties of lenalidomide. While IKZF1 and IKZF3 degradation is likely involved in the other effects of lenalidomide on T cell subsets and NK cells, it is unlikely that this accounts for all of its properties. Limb deformations, for instance, do not occur in mice with germline genetic inactivation of IKZF1 or IKZF3. Similarly, it is unlikely that degradation of the lymphoid transcription factors IKZF1 and IKZF3 accounts for the specific activity of lenalidomide in myelodysplastic syndrome with chromosome 5q deletion. Since most of these effects have been shown to depend on CRBN, it is conceivable that they result from lenalidomide-induced alteration of other substrates of the CRBN-CRL4 E3 ubiquitin ligase. Future studies aiming to identify the substrates responsible for each of the biological effects of IMiDs could enable the development of more specific drugs that modify ubiquitination of different sets of proteins with fewer side effects. DisclosuresNo relevant conflicts of interest to declare.

Light and COP1 regulate level of overexpressed DET1 protein

de-etiolated 1 (det1) and constitutive photomorphogenic 1 (cop1) were initially identified as constitutively photomorphogenic Arabidopsis mutants, exhibiting light-grown phenotypes in the dark. Subsequently, both were shown to be components of Damaged DNA Binding protein 1 (DDB1)/CULLIN4-type complexes. Arabidopsis has two DDB1 homologues, DDB1A and DDB1B, and DDB1A mutants enhance det1 phenotypes. Here we examine ddb1a cop1 double mutants and find that ddb1a weakly enhances some cop1 phenotypes but not others, suggesting developmental regulation of COP1–DDB1A interaction. DET1 loss of function strongly enhances cop1 phenotypes. Here we show that MycDET1 overexpression also enhances cop1 phenotypes, thus MycDET1 overexpression in cop1 mutants also generates loss of function effects. Finally, the effect of the cop1 mutant background on the biochemical properties of MycDET1 was examined. MycDET1 levels were found to be lower in the dark than in the light and this difference required COP1. In summary, both DDB1A loss of function and MycDET1 overexpression enhance cop1 phenotypes, while cop1 mutants fail to exhibit light regulation of MycDET1 levels.

CRL4DCAF1 is required in activated oocytes for follicle maintenance and ovulation.

In mammals, oocytes within the primordial follicles require a number of essential factors to maintain their survival. However, the survival factors for activated oocytes have been poorly characterized. Recently we reported that damaged DNA binding protein-1 (DDB1), the linker subunit of the cullin ring-finger ubiquitin E3 ligase-4 (CRL4) complex, and its substrate adaptor, DDB1-CUL4 associated factor-1 (DCAF1), were essential for primordial follicle maintenance. In this study we specifically deleted these in the oocytes of growing follicles, to investigate if DDB1 and DCAF1 were also survival factors for activated oocytes. In the ovaries of Ddb1(fl/fl);Zp3-Cre mice, the primordial follicle pool was intact, but awakened oocytes and growing follicles beyond the primary stage were rapidly depleted. In the ovaries of Dcaf1(fl/fl);Pten(fl/fl);Gdf9-Cre and Ddb1(fl/fl);Pten(fl/fl);Gdf9-Cre mice, global primordial follicle activation was stimulated by enhanced PI3K signaling, but the awakened oocytes were rapidly lost due to no CRL4(DCAF1) activity. These mouse models provided original evidence that CRL4(DCAF1) was essential for maintaining oocyte survival, not only those in dormancy at the primordial follicle stage, but also naturally awakened oocytes and those awakened by hyper-activation of PI3K signaling. Interestingly, the oocyte-specific Ddb1 or Dcaf1 knockout mice had ovulation defects even before oocyte exhaustion. CRL4(DCAF1) within oocytes was required for cumulus expansion and ovulation-related somatic gene expression in a cell non-autonomous manner. Granulosa cells that surrounded these Ddb1 or Dcaf1-deleted oocytes exhibited increased rates of apoptosis and showed poor responses to ovulation signals. These results suggested that CRL4 in oocytes also regulated granulosa cell functions in a cell non-autonomous manner.

Abstract 3874: Loss of DDB2 enhances the tumorigenicity of ovarian cancer cells through expanding cancer stem-like cell population

Abstract Ovarian cancer is an extremely aggressive disease associated with a high percentage of recurrence and chemotherapy resistance. DNA damage binding protein 2 (DDB2) is a 48-kDa protein originally identified as a component of the damage-specific DNA-binding heterodimeric complex DDB. Besides the known ability of DDB2 protein to bind ultraviolet (UV)-damaged DNA and serving as the initial damage recognition factor during nucleotide excision repair (NER), DDB2 was also reported to be involved in the promotion of apoptosis to reduce cisplatin resistance of ovarian cancer cells, control of cell migration, invasiveness and breast tumor progression, as well as suppression of metastasis of colon cancer through a transcriptional regulatory mechanism. Here we show that DDB2 is downregulated in human ovarian serous adenocarcinomas. DDB2 overexpression in human ovarian cancer cell line CP70 suppressed its capability to recapitulate tumors in athymic nude mice, as well as the growth of these cells cultured in semisolid media. Flow cytometry analyses indicate that DDB2 overexpression in CP70 cells reduced the abundance of ALDH+ cells, which are believed to be cancer stem-like cells (CSCs) in ovarian cancer. In contrast, downregulation of DDB2 in ovarian cancer cell line A2780 increased the percentage of ALDH+ cells. In addition, we analyzed the expression level of DDB2 in the putative CSCs isolated from various ovarian cancer cell lines, and demonstrated that DDB2 expression is downregulated in all tested CSCs in comparison with their parental bulk tumor cells. Overexpression of DDB2 in the ovarian CSCs reduced the expression of CSC marker Nanog, as well as inhibited the self-renewal of these CSCs. In addition, overexpression of DDB2 in CSCs isolated from ovarian cancer cell line SKOV-3 also decreased the tumorigenicity of these cells in NOD/SCID mice. Further analysis of publicly available gene expression array datasets revealed that low DDB2 expression correlates with poor outcomes among ovarian cancer patients, suggesting that loss of DDB2 may promote ovarian tumor relapse through expanding CSC population. Given that the DDB2 protein level is downregulated in ovarian tumor cells, targeting DDB2 expression could be a promising strategy to eradicate CSCs, and helps to halt ovarian cancer relapse. (Supported by NIH R01 grant CA151248 to QEW) Citation Format: Chunhua Han, Ran Zhao, Xingluo Liu, Amit Kumar Srivastava, Li Gong, Hsiaoyin Mao, Meihua Qu, Weiqiang Zhao, Jianhua Yu, Qi-En Wang. Loss of DDB2 enhances the tumorigenicity of ovarian cancer cells through expanding cancer stem-like cell population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3874. doi:10.1158/1538-7445.AM2014-3874

Abstract 4909: Novel radiation mitigators and cancer chemoprevention drugs

Abstract The possibility of a radiation disaster from a nuclear detonation or accident has existed for over 50 years and spawned much of the basic research in radiobiology in the 1950-60s. The recent Fukushima accident was yet another reminder that there remains a dire need to develop novel therapies against radiation-induced toxicities. Here we report on the development of two novel radiation countermeasure therapies: Yel001 and Yel002. These small, biologically active, drug-like molecules were uncovered in the DEL high throughput assay reducing radiation-induced cyto- and geno-toxicity in yeast. Radiation-modulating activity was further confirmed in yeast plate-based DEL Assay: addition of either Yel001 or Yel002 to irradiated cultures reduced cell death and genomic instability. Further, Yel compounds increases survival to 75% in vivo following an LD100/30 dose of ionizing radiation (IR) with the first therapeutic injection administered 24 hours post exposure followed by injections at 48,72,96, and 120 hours. Additionally, treatment with Yel001 and Yel002 compounds reduces radiation-induced leukemia from 90% to to 50% and 40% respectively. Of note, treatment with either Yel001 or Yel002 reduced spontaneous leukemia rate from 10% to 0%. Furthermore, Yel002 significantly prolonged the life of Atm deficient mice. Treatment with Yel002 following IR accelerates the recovery of the hematopoietic cells and protects stem cells after sub-lethal exposures. In addition, treatment with Yel002 reduces I131, EMS, MMS, UV, cigarette smoke extract as well as nitrogen mustard induced toxicity as well as genotoxicity showing a broad application spectrum. Proteomics shows that Yel002 induces DNA repair mediated by Atm signaling, homologous recombination, nonhomologous end joining, base excision repair, DNA damage binding proteins and chromosome segregation functions. Toxicity has not been observed in neither in vitro or in vivo administrations, even in a two generation teratogenicity assay. Overall, Yel compounds have much potential as stockpile therapies for radiation-induced lethality and cancer as well as spontaneous cancer: they are highly effective when administered up to 24hours post exposure, they reduce radiation-induced sequelae such as leukemia, and appear to have an acceptable toxicity profile. Citation Format: Robert H. Schiestl, Yelena Rivina, Michael Davoren. Novel radiation mitigators and cancer chemoprevention drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4909. doi:10.1158/1538-7445.AM2014-4909

Abstract 2391: USP7 deubiquitinates XPC in response to ultraviolet light irradiation

Abstract Ultraviolet light (UV)-induced Xeroderma pigmentosum complementation group C (XPC) protein ubiquitination is mediated by an E3 ubiquitin ligase complex containing UV damaged-DNA binding protein. Here, we report that ubiquitin specific protease 7 (USP7) deubiquitinates XPC during NER. We have demonstrated that transiently compromising cellular USP7, by siRNA or specific inhibitor HBX41108, leads to accumulation of ubiquitinated forms of XPC. However, complete USP7 disruption causes an ubiquitin-mediated XPC degradation upon cellular irradiation. We show that USP7 interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further showed that valosin-containing protein (VCP)/p97 is required for UV-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and co-localizes with XPC. Inhibition of VCP/p97 causes an accumulation of ubiquitinated XPC on DNA damaged chromatin. Moreover, USP7 disruption severely impairs the repair of cyclobutane pyrimidine dimers (CPD) and, to a lesser extent, affects the repair of 6-4 photoproducts (6-4PP). Taken together, our findings have uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis (This work was supported by grants from NIH). Citation Format: Jinshan He, Qianzheng Zhu, Nidhi Sharma, Gulzar Wani, Chunhua Han, Jiang Qian, Kyle Pentz, Qi-en Wang, Altaf A Wani. USP7 deubiquitinates XPC in response to ultraviolet light irradiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2391. doi:10.1158/1538-7445.AM2014-2391

Differential expression patterns and a novel interaction factor of Damaged DNA Binding Protein 1A (DDB1A) and DDB1B in Arabidopsis thaliana

Damaged DNA Binding protein 1 (DDB1) is a bridging protein of CULLIN4 (CUL4)-RING E3 ligase complex, which is involved in multiple biological processes including DNA repair, parental imprinting, epigenetic regulation and flowering time. In Arabidopsis (Arabidopsis thaliana), there are two paralogous genes of DDB1, i.e. DDB1A and DDB1B. In this study, we fused the promoter sequences of DDB1A (−1 bp to 1697 bp 5’ to ATG) and DDB1B (−1 bp to 1655 bp 5’ to ATG) to the GUS gene, respectively, and we then analyzed differential expression patterns of DDB1A and DDB1B using the transgenic PDDB1A::GUS and PDDB1B::GUS plants. The results showed that DDB1B was specifically expressed in the style, and abscisic acid (ABA) might increase the expression level of DDB1A in the meristematic tissues in the light. Using yeast two-hybrid, we found that both DDB1A and DDB1B interacted with CDT2 (also referred to as DDB1-CUL4 ASSOCIATED FACTOR2, DCAF2). Transcript analysis showed that a transcript retains one intron (85 bp in length), suggesting that CDT2 may be involved in the regulation of alternative splicing (AS). This work might provide a clue for further investigating the roles of DDB1A and DDB1B in multiple physiological processes and lay a solid foundation for in depth studying the functions of CDT2 in Arabidopsis.

Statin-induced inhibition of breast cancer proliferation and invasion involves attenuation of iron transport: intermediacy of nitric oxide and antioxidant defence mechanisms.

Accumulating evidence from invitro, invivo, clinical and epidemiological studies shows promising results for the use of statins against many cancers including breast carcinoma. However, the molecular mechanisms responsible for the anti-proliferative and anti-invasive properties of statins still remain elusive. In this study, we investigated the involvement of nitric oxide, iron homeostasis and antioxidant defence mechanisms in mediating the anti-proliferative and anti-invasive properties of hydrophobic statins in MDA-MB-231, MDA-MB-453 and BT-549 metastatic triple negative breast cancer cells. Fluvastatin and simvastatin significantly increased cytotoxicity which was reversed with mevalonate. Interestingly, fluvastatin downregulated transferrin receptor (TfR1), with a concomitant depletion of intracellular iron levels in these cells. Statin-induced effects were mimicked by geranylgeranyl transferase inhibitor (GGTI-298) but not farnesyl transferase inhibitor (FTI-277). Further, it was observed that TfR1 downregulation is mediated by increased nitric oxide levels via inducible nitric oxide synthase (iNOS) expression. NOS inhibitors (asymmetric dimethylarginine and 1400W) counteracted and sepiapterin, a precursor of tetrahydrobiopterin, exacerbated statin-induced depletion of intracellular iron levels. Notably, fluvastatin increased manganese superoxide dismutase (by repressing the transcription factor DNA damage-binding protein 2), catalase and glutathione which, in turn, diminished H2 O2 levels. Fluvastatin-induced downregulation of TfR1, matrix metalloproteinase-2, -9 and inhibition of invasion were reversed in the presence of aminotriazole, a specific inhibitor of catalase. Finally, we conclude that fluvastatin, by altering iron homeostasis, nitric oxide generation and antioxidant defence mechanisms, induces triple negative breast cancer cell death.

Poly(ADP-ribose)-mediated interplay of XPA and PARP1 leads to reciprocal regulation of protein function.

Poly(ADP-ribose) (PAR) is a complex and reversible post-translational modification that controls protein function and localization through covalent modification of, or noncovalent binding to target proteins. Previously, we and others characterized the noncovalent, high-affinity binding of the key nucleotide excision repair (NER) protein XPA to PAR. In the present study, we address the functional relevance of this interaction. First, we confirm that pharmacological inhibition of cellular poly(ADP-ribosyl)ation (PARylation) impairs NER efficacy. Second, we demonstrate that the XPA-PAR interaction is mediated by specific basic amino acids within a highly conserved PAR-binding motif, which overlaps the DNA damage-binding protein 2 (DDB2) and transcription factor II H (TFIIH) interaction domains of XPA. Third, biochemical studies reveal a mutual regulation of PARP1 and XPA functions showing that, on the one hand, the XPA-PAR interaction lowers the DNA binding affinity of XPA, whereas, on the other hand, XPA itself strongly stimulates PARP1 enzymatic activity. Fourth, microirradiation experiments in U2OS cells demonstrate that PARP inhibition alters the recruitment properties of XPA-green fluorescent protein to sites of laser-induced DNA damage. In conclusion, our results reveal that XPA and PARP1 regulate each other in a reciprocal and PAR-dependent manner, potentially acting as a fine-tuning mechanism for the spatio-temporal regulation of the two factors during NER.

The tomato DWD motif-containing protein DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase and plays a pivotal role in abiotic stress responses

CULLIN4(CUL4)–DAMAGED DNA BINDING PROTEIN1 (DDB1)-based ubiquitin ligase plays significant roles in multiple physiological processes via ubiquitination-mediated degradation of relevant target proteins. The DDB1–CUL4-associated factor (DCAF) acts as substrate receptor in the CUL4–DDB1 ubiquitin ligase complex and determines substrate specificity. In this study, we identified a tomato (Solanum lycopersicum) DDB1-interacting (DDI1) protein as a DCAF protein involved in response to abiotic stresses, including UV radiation, high salinity and osmotic stress. Co-immunoprecipitation and bimolecular fluorescence complementation assay indicated that DDI1 associates with CUL4–DDB1 in the nucleus. Quantitative RT-PCR analysis indicated the DDI1 gene is induced by salt, mannitol and UV-C treatment. Moreover, transgenic tomato plants with overexpression or knockdown of the DDI1 gene exhibited enhanced or attenuated tolerance to salt/mannitol/UV-C, respectively. Thus, our data suggest that DDI1 functions as a substrate receptor of the CUL4–DDB1 ubiquitin ligase, positively regulating abiotic stress response in tomato.

α-N-Methylation of Damaged DNA-binding Protein 2 (DDB2) and Its Function in Nucleotide Excision Repair

DDB2 exhibits a high affinity toward UV-damaged DNA, and it is involved in the initial steps of global genome nucleotide excision repair. Mutations in the DDB2 gene cause the genetic complementation group E of xeroderma pigmentosum, an autosomal recessive disease manifested clinically by hypersensitivity to sunlight exposure and an increased predisposition to skin cancer. Here we found that, in human cells, the initiating methionine residue in DDB2 was removed and that the N-terminal alanine could be methylated on its α-amino group in human cells, with trimethylation being the major form. We also demonstrated that the α-N-methylation of DDB2 is catalyzed by the N-terminal RCC1 methyltransferase. In addition, a methylation-defective mutant of DDB2 displayed diminished nuclear localization and was recruited at a reduced efficiency to UV-induced cyclobutane pyrimidine dimer foci. Moreover, loss of this methylation conferred compromised ATM (ataxia telangiectasia mutated) activation, decreased efficiency in cyclobutane pyrimidine dimer repair, and elevated sensitivity of cells toward UV light exposure. Our study provides new knowledge about the posttranslational regulation of DDB2 and expands the biological functions of protein α-N-methylation to DNA repair.