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1.
Molecules ; 27(21)2022 Oct 26.
Article in English | MEDLINE | ID: mdl-36364114

ABSTRACT

The use of cisplatin as a chemotherapeutic drug is impeded by the development of drug resistance. Combination therapies of a chemosensitizer for cisplatin have been studied, but with little success, and the search for an effective combination therapy is continuing. Our earlier reports have shown that Zanthoxylum armatum DC. extract enhances the apoptotic effect of cisplatin in cancer cell lines. In this study, we purified and identified the bioactive phytocompound through bio-assay-guided purification, using column chromatography and HPLC. Chemical characterization using NMR and mass spectrometry revealed the compound as planispine A, with molecular structure C25H30O6 and molecular weight, 426.16 g/mol. Planispine A was found to inhibit cancer cell proliferation in a dose-dependent manner and to sensitize the cancer cells to cisplatin-augmented apoptotic cell death, in a caspase-dependent manner. A combination of planispine A and cisplatin induced S-phase cell cycle arrest, and reduced the expression of survival proteins such as cyclin D1. Interestingly, planispine A inhibits the Fanconi anemia pathway, as shown by reduced FANCD2 foci formation and FANCD2 monoubiquitination, which revealed the molecular mechanism of chemo-sensitization of cancer cells to cisplatin. Evaluation of this combination therapy in cisplatin-resistant tumors may lead to more efficient cisplatin treatment.


Subject(s)
Fanconi Anemia , Neoplasms , Humans , Cisplatin/pharmacology , Neoplasms/drug therapy , Neoplasms/metabolism , Apoptosis , Cell Proliferation , Cell Line, Tumor
2.
BMC Cancer ; 22(1): 143, 2022 Feb 05.
Article in English | MEDLINE | ID: mdl-35123428

ABSTRACT

BACKGROUND: We have previously reported that a new intronless gene for casein kinase 2α (CK2α), CSNK2A3, is expressed in human cells. The promoter of the well-known CK2α, CSNK2A1, displays characteristics of a housekeeping gene, whereas CSNK2A3 has a characteristic of a regulated promoter with two TATA boxes and a CAAT box. GPR68, a family of the G protein-coupled receptors, is also known as ovarian cancer G protein-coupled receptor 1 (OGR1). In the current study, we analyzed the roles of CK2α genes and neutral endopeptidase (NEP), a key enzyme that influences a variety of malignancies, in the OGR1-induced inhibition of A549 cell migration. METHODS: We analyzed the transcript expressions of both the CK2α genes (CSNK2A1 and CSNK2A3) and NEP upon OGR1 overexpression. Protein expression of CK2α and NEP were also analyzed. We further elucidated the functional roles of both CK2α and NEP in the OGR1-induced inhibition of A549 cell migration in vitro using a wound-healing assay. We also analyzed the molecular mechanisms involved in the OGR1-induced inhibition of lung cancer cell migration. RESULTS: The findings of this study showed that OGR1 upregulated the expression of CSNK2A3 but not CSNK2A1 in the A549 cells. The findings further suggested OGR1 also upregulates the expression of NEP. The OGR1-induced inhibition of A549 cell migration was abrogated completely by inhibition of CK2α activity, whereas partial abrogation (~ 30%) was observed in the presence of NEP inhibition. The results also revealed that OGR1 regulates CSNK2A3 via activation of Rac1/cdc42 and MAPKs pathways. CK2 is ubiquitously expressed, and in contrast, is believed to be a constitutively active enzyme, and its regulation appears to be independent of known second messengers. CONCLUSION: In the current study, we report for the first time the OGR1-induced regulation of CSNK2A3, CK2αP, and NEP in A549 cancer cells. Our study also decoded the downstream cellular proteins of OGR1 as well as the molecular mechanism involved in OGR1-induced inhibition of A549 cell migration. The findings of this research suggest the potential therapeutic targets to inhibit lung cancer progression.


Subject(s)
Cell Movement/genetics , Neprilysin/metabolism , Ovarian Neoplasms/genetics , Receptors, G-Protein-Coupled/metabolism , A549 Cells , Casein Kinase II/metabolism , Cell Line, Tumor , Female , Humans , Lung Neoplasms/genetics , Up-Regulation/genetics
3.
Biochem Biophys Res Commun ; 511(3): 498-503, 2019 04 09.
Article in English | MEDLINE | ID: mdl-30803761

ABSTRACT

Indepth studies of protein-protein interactions are essential for discovering the molecular mechanisms and the biological context of protein functions. Even though previous study on the purification of SPIN1 interacting protein complex has shown Spindlin docking protein (SPIN.DOC) as the most abundant interacting protein partner; the study on the molecular function of SPIN.DOC is limited. Since the role of SPIN1 has been previously documented as a histone code reader and transcriptional coactivator of Wnt signaling, SPIN.DOC may probably involve in epigenetic regulation and Wnt signaling. This study aims to purify SPIN.DOC interacting protein complex and characterize the molecular function of SPIN.DOC. The finding of this study revealed that the suppression of SPIN.DOC expression in HEK293 cells by shRNA, slightly destabilized SPIN1 without any change in its chromatin localization. However, knockdown of SPIN1 decreased the expression and chromatin localization of SPIN.DOC. Nevertheless, overexpression of SPIN.DOC increased the expression and chromatin localization of SPIN1 but no change in the SPIN.DOC protein expression and chromatin localization when SPIN1 is overexpressed. TOPflash reporter assays revealed that SPIN.DOC regulates gene expression in Wnt signaling pathway and act as transcriptional repressor. Further, we show that C-terminal deleted mutant of SPIN.DOC is unable to interact with SPIN1. Unlike the wild type SPIN.DOC which acts as transcriptional repressor, overexpression of C-terminal deletion mutant activates Wnt signaling suggesting that SPIN.DOC-SPIN1 complex may act as transcriptional repressor. Overall, our data revealed new molecular functions of SPIN.DOC.


Subject(s)
Cell Cycle Proteins/metabolism , Co-Repressor Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Phosphoproteins/metabolism , Wnt Signaling Pathway , HEK293 Cells , HeLa Cells , Histone Code , Humans , Protein Interaction Maps
4.
Sci Rep ; 8(1): 15183, 2018 10 12.
Article in English | MEDLINE | ID: mdl-30315192

ABSTRACT

Increasing incidence of drug resistance is ascertained to be the main obstacles in limiting the virus among the human immunodeficiency virus (HIV) infected individuals. This study investigates the drug resistance mutations (DRMs), genetic variants and origin of transmitted drug resistance of HIV-1 among the HIV-1 infected wives of intravenous drug users (IDUs) in Manipur. 44 HIV pol gene sequences were generated from 56 blood samples by viral gene amplification and sequencing. Sequences were then analysed for drug resistance, genetic variants and origin. The result revealed that among the treatment naive cases, 35.7% had Transmitted Drug Resistance Mutations (TDRMs) while among treatment experienced cases, 50% had Acquired Drug Resistant Mutations (ADRMs). These TDRMs and ADRMs conferred resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and/or protease inhibitors (PIs). Majority of the isolated HIV-1 sequences (77.3%) were subtype C while 9.1% was discordant subtype, 6.8% was subtype B, 4.5% was CRF_01AE and 2.3% was URF_BC. TDRM strains were found to be introduced from Myanmar, Vietnam and mainland India. This study also reveals the appearance of CRF_01AE for the first time in Manipur. The finding of this study indicates high prevalence of drug resistant mutations and complex molecular epidemiology in Manipur.


Subject(s)
Anti-Retroviral Agents/therapeutic use , Drug Resistance, Viral/drug effects , Drug Users , HIV Infections/drug therapy , HIV-1/drug effects , HIV-1/genetics , Spouses , Adult , Anti-Retroviral Agents/pharmacology , Antiretroviral Therapy, Highly Active , Drug Resistance, Viral/genetics , Female , Genetic Variation , Genotype , Geography , HIV Infections/virology , Humans , India , Mutation/genetics , Phylogeny
5.
Sci Rep ; 8(1): 4090, 2018 03 06.
Article in English | MEDLINE | ID: mdl-29511228

ABSTRACT

Zerumbone is a known anti-cancer herbal compound. However, the actual protein target is not fully understood or known. This investigation focus on the association of zerumbone in HCT116 colon cancer cell proliferation and its link with TNF-alpha. The study shows that with the increasing concentration of zerumbone, there was a reduction of HCT116 cells proliferation based on the cell line study and hence higher TNF-alpha inhibition based on the TNF-alpha assay. The study also emphasizes on the computational aspect by investigating the molecular docking analysis of zerumbone against TNF-alpha. The docked complex was further validated using molecular dynamics simulation studies. The docking analysis observed that alpha-beta unsaturated carbonyl scaffold is an important moiety for the anti-cancer activity of zerumbone. Furthermore, the DFT analysis also confirms the reactivity nature of zerumbone based on the frontier molecular orbital analysis.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , HCT116 Cells/drug effects , Immunologic Factors/pharmacology , Sesquiterpenes/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Antineoplastic Agents/chemistry , Colon , HCT116 Cells/physiology , Humans , Immunologic Factors/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Sesquiterpenes/chemistry , Tumor Necrosis Factor-alpha/chemistry
6.
J Med Virol ; 89(6): 989-999, 2017 06.
Article in English | MEDLINE | ID: mdl-27869320

ABSTRACT

According to the Joint National Programme on HIV/AIDS (UNAIDS), the northeastern region of India has the highest HIV prevalence in the country. This study was conducted to determine the current HIV-1 molecular epidemiology of Manipur, a state in northeast India. Blood samples from HIV-1 seropositive subjects were collected between June 2011 and February 2014. The partial regions of HIV-1 genes; pol and tat-vpu-env were independently amplified, sequenced, analyzed, and genotyped. Based on all sequences generated from 110 samples using pol and/or tat-vpu-env gene, the overall HIV-1 genotypes distribution of Manipur was as follows: 65.45% (72/110) subtype C, 32.73% (36/110) unique recombinant forms (URFs), and 1.82% (2/110) subtype B. The distribution of HIV-1 genotypes among the risk groups was: heterosexual: 58.33% (35/60) subtype C, 38.33% (23/60) URFs, and 3.34% (2/60) subtype B; intravenous drug users (IDUs): 85.36% (35/41) subtype C, 9.76% (4/41) URFs, and 4.88% (2/41) subtype B; mother to child (MTC): 50% (3/6) URFs and 50% (3/6) subtype C and blood transfusion: 100% (3/3) subtype C. The findings for the first time revealed the emergence of URFs of HIV-1 in Manipur which is predominant among the sexual and MTC risk groups as compared to IDUs. Taking together, this study illustrated that Manipur is the "recombinant hotspot of HIV" of India. The results will provide the clinical importance for continuous monitoring of HIV-infections in order to design appropriate prevention measures to limit the spread of new HIV infections.


Subject(s)
Genotype , HIV Infections/epidemiology , HIV Infections/virology , HIV-1/classification , HIV-1/genetics , Adult , Female , HIV-1/isolation & purification , Human Immunodeficiency Virus Proteins/genetics , Humans , India/epidemiology , Male , Middle Aged , Molecular Epidemiology , Polymerase Chain Reaction , Prevalence , RNA, Viral/genetics , Recombination, Genetic , Sequence Analysis, DNA , Young Adult
7.
Biol Res ; 48: 46, 2015 Aug 20.
Article in English | MEDLINE | ID: mdl-26290043

ABSTRACT

BACKGROUND: Clinical use of chemotherapeutic drug, cisplatin is limited by its toxicity and drug resistance. Therefore, efforts continue for the discovery of novel combination therapies with cisplatin, to increase efficacy and reduce its toxicity. Here, we screened 16 medicinal plant extracts from Northeast part of India and found that leaf extract of Zanthoxylum armatum DC. (ZALE) induced cytotoxicity as well as an effect on the increasing of the efficiency of chemotherapeutic drugs (cisplatin, mitomycin C and camptothecin). This work shows detail molecular mechanism of anti-cancer activity of ZALE and its potential for combined treatment regimens to enhance the apoptotic response of chemotherapeutic drugs. RESULTS: ZALE induced cytotoxicity, nuclear blebbing and DNA fragmentation in HeLA cells suggesting apoptosis induction in human cervical cell line. However, the apoptosis induced was independent of caspase 3 activation and poly ADP ribose polymerase (PARP) cleavage. Further, ZALE activated Mitogen-activated protein kinases (MAPK) pathway as revealed by increased phosphorylation of extracellular-signal-regulated kinases (ERK), p38 and c-Jun N-terminal kinase (JNK). Inhibition of ERK activation but not p38 or JNK completely blocked the ZALE induced apoptosis suggesting an ERK dependent apoptosis. Moreover, ZALE generated DNA double strand breaks as suggested by the induction γH2AX foci formation. Interestingly, pretreatment of certain cancer cell lines with ZALE, sensitized the cancer cells to cisplatin and other chemotherapeutic drugs. Enhanced caspase activation was observed in the synergistic interaction among chemotherapeutic drugs and ZALE. CONCLUSION: Purification and identification of the bio-active molecules from the ZALE or as a complementary treatment for a sequential treatment of ZALE with chemotherapeutic drugs might be a new challenger to open a new therapeutic window for the novel anti-cancer treatment.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cisplatin/pharmacology , Plant Extracts/pharmacology , Zanthoxylum/chemistry , Apoptosis/drug effects , Enzyme Activation/drug effects , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/drug effects
8.
Asian Pac J Cancer Prev ; 16(12): 4927-35, 2015.
Article in English | MEDLINE | ID: mdl-26163617

ABSTRACT

Northeastern India is a major nasopharyngeal carcinoma (NPC) high risk-area although the rest of the country has very low incidence. A case-control study of 105 NPC cases and 115 controls was conducted to identify the potential risk factors for NPC development in this region. Information was collected by interviewer about socio-demographic characteristics, cigarette smoking, alcohol consumption, dietary history, occupational history, and a family history of cancer. Epstein-Barr viral load was assayed from the blood DNA by real time PCR. Associations between GSTs genotypes, cytochrome P450 family including CYP1A1, CYP2E1 and CYP2A6 polymorphisms and susceptibility to relationship between the diseases were studied using PCR-RFLP assay. Results indicate that Epstein-Barr virus load was significantly higher in patients compared to controls (p<0.0001). Furthermore, concentration of blood EBV-DNA was significantly higher in advanced stage disease (Stage III and IV) than in early stage disease (Stage I and II) (p<0.05). Presence of CYP2A6 variants that reduced the enzyme activity was significantly less frequent in cases than controls. Smoked meat consumption, exposure to smoke, living in poorly ventilated house and alcohol consumption were associated with NPC development among the population of Northeastern India. Thus, overall our study revealed that EBV viral load and genetic polymorphism of CYP2A6 along with living practices which include smoked meat consumption, exposure to smoke, living in poorly ventilated houses and alcohol consumption are the potential risk factors of NPC in north eastern region of India. Understanding of the risk factors and their role in the etiology of NPC are helpful forpreventive measures and screening.


Subject(s)
Genetic Predisposition to Disease/genetics , Nasopharyngeal Neoplasms/etiology , Nasopharyngeal Neoplasms/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Viral/immunology , Carcinoma , Case-Control Studies , Cells, Cultured , Cytochrome P-450 Enzyme System/genetics , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/genetics , Epstein-Barr Virus Infections/immunology , Female , Herpesvirus 4, Human/immunology , Humans , India , Male , Middle Aged , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/immunology , Nasopharyngeal Neoplasms/virology , Polymorphism, Genetic/genetics , Risk Factors , Smoking/adverse effects , Viral Load/genetics , Young Adult
9.
Biol. Res ; 48: 1-9, 2015. ilus, graf, tab
Article in English | LILACS | ID: biblio-950810

ABSTRACT

BACKGROUND: Clinical use of chemotherapeutic drug, cisplatin is limited by its toxicity and drug resistance. Therefore, efforts continue for the discovery of novel combination therapies with cisplatin, to increase efficacy and reduce its toxicity. Here, we screened 16 medicinal plant extracts from Northeast part of India and found that leaf extract of Zanthoxylum armatum DC. (ZALE) induced cytotoxicity as well as an effect on the increasing of the efficiency of chemotherapeutic drugs (cisplatin, mitomycin C and camptothecin). This work shows detail molecular mechanism of anti-cancer activity of ZALE and its potential for combined treatment regimens to enhance the apoptotic response of chemotherapeutic drugs. RESULTS: ZALE induced cytotoxicity, nuclear blebbing and DNA fragmentation in HeLA cells suggesting apoptosis induction in human cervical cell line. However, the apoptosis induced was independent of caspase 3 activation and poly ADP ribose polymerase (PARP) cleavage. Further, ZALE activated Mitogen-activated protein kinases (MAPK) pathway as revealed by increased phosphorylation of extracellular-signal-regulated kinases (ERK), p38 and c-Jun N-ter-minal kinase (JNK). Inhibition of ERK activation but not p38 or JNK completely blocked the ZALE induced apoptosis suggesting an ERK dependent apoptosis. Moreover, ZALE generated DNA double strand breaks as suggested by the induction γH2AX foci formation. Interestingly, pretreatment of certain cancer cell lines with ZALE, sensitized the cancer cells to cisplatin and other chemotherapeutic drugs. Enhanced caspase activation was observed in the synergistic interaction among chemotherapeutic drugs and ZALE. CONCLUSION: Purification and identification of the bio-active molecules from the ZALE or as a complementary treatment for a sequential treatment of ZALE with chemotherapeutic drugs might be a new challenger to open a new therapeutic window for the novel anti-cancer treatment.


Subject(s)
Humans , Plant Extracts/pharmacology , Cisplatin/pharmacology , Zanthoxylum/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , HeLa Cells , Apoptosis/drug effects , Mitogen-Activated Protein Kinases/drug effects , JNK Mitogen-Activated Protein Kinases/drug effects , Enzyme Activation/drug effects
10.
Nat Commun ; 5: 2987, 2014.
Article in English | MEDLINE | ID: mdl-24390579

ABSTRACT

The conserved MHF1-MHF2 (MHF) complex functions in the activation of the Fanconi anaemia pathway of the DNA damage response, in regulating homologous recombination, and in DNA replication fork maintenance. MHF facilitates the processing of multiple types of branched DNAs by the DNA translocase FANCM. Here we report the crystal structure of a human MHF-DNA complex that reveals the DNA-binding mode of MHF. The structure suggests that MHF prefers branched DNA over double-stranded DNA because it engages two duplex arms. Biochemical analyses verify that MHF preferentially engages DNA forks or various four-way junctions independent of the junction-site structure. Furthermore, genetic experiments provide evidence that the observed DNA-binding interface of MHF is important for cellular resistance to DNA damage. These results offer insights into how the MHF complex recognizes branched DNA and stimulates FANCM activity at such a structure to promote genome maintenance.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , DNA Damage/genetics , DNA Helicases/metabolism , DNA Repair/genetics , DNA-Binding Proteins/metabolism , DNA/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Crystallography, X-Ray , DNA Helicases/genetics , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Humans , Models, Molecular , Protein Structure, Tertiary
11.
J Biol Chem ; 288(47): 33500-33508, 2013 Nov 22.
Article in English | MEDLINE | ID: mdl-24108125

ABSTRACT

Genomic instability and a predisposition to cancer are hallmarks of Bloom syndrome, an autosomal recessive disease arising from mutations in the BLM gene. BLM is a RecQ helicase component of the BLM-Topo III α-RMI1-RMI2 (BTR) complex, which maintains chromosome stability at the spindle assembly checkpoint (SAC). Other members of the BTR complex include Topo IIIa, RMI1, and RMI2. All members of the BTR complex are essential for maintaining the stable genome. Interestingly, the BTR complex is posttranslationally modified upon SAC activation during mitosis, but its significance remains unknown. In this study, we show that two proteins that interact with BLM, RMI1 and RMI2, are phosphorylated upon SAC activation, and, like BLM, RMI1, and RMI2, are phosphorylated in an MPS1-dependent manner. An S112A mutant of RMI2 localized normally in cells and was found in SAC-induced coimmunoprecipitations of the BTR complex. However, in RMI2-depleted cells, an S112A mutant disrupted the mitotic arrest upon SAC activation. The failure of cells to maintain mitotic arrest, due to lack of phosphorylation at Ser-112, results in high genomic instability characterized by micronuclei, multiple nuclei, and a wide distribution of aberrantly segregating chromosomes. We found that the S112A mutant of RMI2 showed defects in redistribution between the nucleoplasm and nuclear matrix. The phosphorylation at Ser-112 of RMI2 is independent of BLM and is not required for the stability of the BTR complex, BLM focus formation, and chromatin targeting in response to replication stress. Overall, this study suggests that the phosphorylation of the BTR complex is essential to maintain a stable genome.


Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Instability/physiology , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/metabolism , M Phase Cell Cycle Checkpoints/physiology , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , RecQ Helicases/metabolism , Amino Acid Substitution , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , DNA Topoisomerases, Type I/genetics , DNA-Binding Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Multiprotein Complexes/genetics , Mutation, Missense , Nuclear Proteins/genetics , Phosphorylation/physiology , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , RecQ Helicases/genetics , Serine/genetics , Serine/metabolism , Spindle Apparatus/genetics , Spindle Apparatus/metabolism
12.
Cancer Res ; 73(14): 4300-10, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23698467

ABSTRACT

Fanconi anemia (FA) is a genome instability syndrome that has been associated with both cancer predisposition and bone marrow failure. FA proteins are involved in cellular response to replication stress in which they coordinate DNA repair with DNA replication and cell-cycle progression. One regulator of the replication stress response is the ATP-dependent DNA translocase FANCM, which we have shown to be hyperphosphorylated in response to various genotoxic agents. However, the significance of this phosphorylation remained unclear. Here, we show that genotoxic stress-induced FANCM phosphorylation is ATR-dependent and that this modification is highly significant for the cellular response to replication stress. We identified serine (S1045) residue of FANCM that is phosphorylated in response to genotoxic stress and this effect is ATR-dependent. We show that S1045 is required for FANCM functions including its role in FA pathway integrity, recruiting FANCM to the site of interstrand cross links, preventing the cells from entering mitosis prematurely, and efficient activation of the CHK1 and G2-M checkpoints. Overall, our data suggest that an ATR-FANCM feedback loop is present in the FA and replication stress response pathways and that it is required for both efficient ATR/CHK1 checkpoint activation and FANCM function.


Subject(s)
DNA Helicases/genetics , DNA Helicases/metabolism , Serine/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Division/physiology , Cell Line , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Replication , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , G2 Phase/physiology , HEK293 Cells , HeLa Cells , Humans , Mitosis/genetics , Mutation , Phosphorylation , Protein Kinases/genetics , Protein Kinases/metabolism , Serine/genetics , Signal Transduction
13.
Structure ; 18(9): 1159-70, 2010 Sep 08.
Article in English | MEDLINE | ID: mdl-20826342

ABSTRACT

Mutations in BLM, a RecQ-like helicase, are linked to the autosomal recessive cancer-prone disorder Bloom's syndrome. BLM associates with topoisomerase (Topo) IIIα, RMI1, and RMI2 to form the BLM complex that is essential for genome stability. The RMI1-RMI2 heterodimer stimulates the dissolution of double Holliday junction into non-crossover recombinants mediated by BLM-Topo IIIα and is essential for stabilizing the BLM complex. However, the molecular basis of these functions of RMI1 and RMI2 remains unclear. Here we report the crystal structures of multiple domains of RMI1-RMI2, providing direct confirmation of the existence of three oligonucleotide/oligosaccharide binding (OB)-folds in RMI1-RMI2. Our structural and biochemical analyses revealed an unexpected insertion motif in RMI1N-OB, which is important for stimulating the dHJ dissolution. We also revealed the structural basis of the interaction between RMI1C-OB and RMI2-OB and demonstrated the functional importance of the RMI1-RMI2 interaction in genome stability maintenance.


Subject(s)
Carrier Proteins/chemistry , DNA-Binding Proteins/chemistry , Nuclear Proteins/chemistry , Bloom Syndrome/metabolism , Carrier Proteins/metabolism , Crystallography, X-Ray , DNA Topoisomerases, Type I/chemistry , DNA, Cruciform/chemistry , DNA, Cruciform/metabolism , DNA-Binding Proteins/metabolism , Genomic Instability , Humans , Nuclear Proteins/metabolism , Protein Folding , Protein Subunits
14.
Mol Cell ; 37(6): 879-86, 2010 Mar 26.
Article in English | MEDLINE | ID: mdl-20347429

ABSTRACT

FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in (1) destabilization of FANCM and MHF2, (2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, (3) defective chromatin localization of FA nuclear core complex proteins, (4) elevated MMC-induced chromosome aberrations, and (5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.


Subject(s)
DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Fanconi Anemia/metabolism , Histones/metabolism , Protein Folding , Protein Multimerization , Cell Line, Tumor , DNA/metabolism , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Fanconi Anemia/genetics , Humans , Protein Binding
15.
Hum Mutat ; 30(7): E761-70, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19405097

ABSTRACT

Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA crosslinking agents. Eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and FANCM) and three non-FA proteins (FAAP100, FAAP24 and HES1) form an FA nuclear core complex, which is required for monoubiquitination of the FANCD2-FANCI dimer upon DNA damage. FANCL possesses a PHD/RING-finger domain and is a putative E3 ubiquitin ligase subunit of the core complex. In this study, we report an FA patient with an unusual presentation belonging to the FA-L complementation group. The patient lacks an obvious FA phenotype except for the presence of a café-au-lait spot, mild hypocellularity and a family history of leukemia. The molecular diagnosis and identification of the FA subgroup was achieved by FA complementation assay. We identified bi-allelic novel mutations in the FANCL gene and functionally characterized them. To the best of our knowledge, this is the second reported case belonging to the FA-L complementation group.


Subject(s)
Fanconi Anemia Complementation Group L Protein/genetics , Fanconi Anemia/genetics , Mutation , Alleles , Cafe-au-Lait Spots , Family Health , Genetic Complementation Test , Humans , Infant , Leukemia , Male
16.
Blood ; 114(1): 174-80, 2009 Jul 02.
Article in English | MEDLINE | ID: mdl-19423727

ABSTRACT

FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM(-/-) cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM(-/-) cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM(-/-) cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex-dependent and -independent manner.


Subject(s)
DNA Helicases/genetics , DNA Helicases/metabolism , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Camptothecin/pharmacology , Cell Line, Tumor , Cross-Linking Reagents/pharmacology , DNA Helicases/deficiency , Drug Resistance/genetics , Drug Resistance/physiology , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia Complementation Group A Protein/metabolism , Gene Expression , Humans , Mutation , Radiation Tolerance/genetics , Radiation Tolerance/physiology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection , Ubiquitination/genetics , Ultraviolet Rays
17.
Mutat Res ; 668(1-2): 20-6, 2009 Jul 31.
Article in English | MEDLINE | ID: mdl-19379763

ABSTRACT

Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA-crosslinking agents. Eight FA proteins (FANCA, -B, -C, -E, -F, -G, -L and -M) and three non-FA proteins (FAAP100, FAAP24 and HES1) form the FA nuclear core complex that is required for monoubiquitination of the FANCD2-FANCI dimer upon DNA damage. The other three FA proteins, FANCD1/BRCA2, FANCJ/BACH1/BRIP1 and FANCN/PALB2, act in parallel or downstream of the FANCD2-FANCI dimer. Despite the isolation and characterization of several FA proteins, the mechanism by which these proteins protect cells from DNA interstrand crosslinking agents has been unclear. This is because a majority of the FA proteins lack any recognizable functional domains that can provide insight into their function. The recently discovered FANCM (Hef) and FANCJ (BRIP1/BACH1) proteins contain helicase domains, providing potential insight into the role of FA proteins in DNA repair. FANCM with its partner, FAAP24, and FANCJ bind and metabolize a variety of DNA substrates. In this review, we focus on the discovery, structure, and function of the FANCM-FAAP24 and FANCJ proteins.


Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , DNA Helicases/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Fanconi Anemia Complementation Group Proteins/metabolism , Fanconi Anemia/metabolism , Basic-Leucine Zipper Transcription Factors/chemistry , DNA/metabolism , DNA Helicases/chemistry , DNA-Binding Proteins/chemistry , Drug Discovery , Fanconi Anemia Complementation Group Proteins/chemistry , Humans , Structure-Activity Relationship
18.
Genes Dev ; 22(20): 2856-68, 2008 Oct 15.
Article in English | MEDLINE | ID: mdl-18923083

ABSTRACT

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo IIIalpha and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo IIIalpha-BLAP75/RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo IIIalpha and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.


Subject(s)
Carrier Proteins/metabolism , DNA Helicases/physiology , DNA, Cruciform/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Oligonucleotides/metabolism , Amino Acid Sequence , Animals , Bloom Syndrome/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Nucleus/metabolism , Cells, Cultured , Chickens , Chromatin/genetics , Chromatin/metabolism , Chromatography, Affinity , Chromosome Breakage , Computational Biology , DNA Helicases/chemistry , DNA Repair , DNA Replication/drug effects , DNA Topoisomerases, Type I/physiology , DNA, Cruciform/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Fibrosarcoma/genetics , Fibrosarcoma/metabolism , Fibrosarcoma/pathology , HeLa Cells , Humans , Hydroxyurea/pharmacology , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , Microscopy, Fluorescence , Mitosis , Molecular Sequence Data , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Oligonucleotides/chemistry , Oligonucleotides/genetics , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , Phosphorylation/drug effects , Protein Folding , RNA, Small Interfering/pharmacology , RecQ Helicases , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
19.
Nat Genet ; 37(9): 958-63, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16116422

ABSTRACT

Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.


Subject(s)
Archaea/chemistry , DNA Helicases/genetics , DNA Repair , Fanconi Anemia/genetics , Hemagglutinins, Viral/genetics , Ligases/genetics , Viral Fusion Proteins/genetics , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Biological Evolution , DNA/metabolism , DNA Helicases/deficiency , DNA Helicases/metabolism , Fanconi Anemia/enzymology , Fanconi Anemia Complementation Group D2 Protein , Fanconi Anemia Complementation Group L Protein , Humans , Immunoprecipitation , Ligases/deficiency , Ligases/metabolism , Molecular Sequence Data , Mutation , Nuclear Proteins/metabolism , Phosphorylation , Protein Transport , Ubiquitin/metabolism , Viral Fusion Proteins/deficiency
20.
Oncogene ; 24(29): 4609-23, 2005 Jul 07.
Article in English | MEDLINE | ID: mdl-15897906

ABSTRACT

Histone deacetylase (HDAC) inhibitors induce differentiation and/or apoptosis in a variety of cell types by activating transcription of target genes. Activation of the death receptor (DR) pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis preferentially in cancer cells. Here, we investigated the intracellular mechanisms by which HDAC inhibitors (suberoylanilide hydroxamic acid, m-carboxycinnamic acid bis-hydroxamide, MS-275 and trichostatin A) enhance the apoptosis-inducing potential of TRAIL in breast cancer cells in vitro. A synergism in apoptosis was observed in both TRAIL-sensitive and -resistant cells upon sequential treatments with HDAC inhibitors followed by TRAIL. HDAC inhibitors synergized with TRAIL by inducing DRs DR4/TRAIL-R1 and DR5/TRAIL-R2 through NFkappaB activation and some of the proapoptotic members of the Bcl-2 family, and engaging the mitochondrial pathway. The ability of HDAC inhibitors to sensitize TRAIL-resistant cells suggests that HDAC inhibitors may induce fundamental alterations in cell signaling pathways. Thus, the sequential treatments with HDAC inhibitors followed by TRAIL may be used as a new therapeutic approach for the treatment of human cancers.


Subject(s)
Apoptosis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Membrane Glycoproteins/physiology , Tumor Necrosis Factor-alpha/physiology , Apoptosis Regulatory Proteins , Humans , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured
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