The effects of altered membrane fatty acid composition on the toxic interactions of heavy metals with Saccharomyces Cerevisiae

The effects of altered membrane fatty acid composition on the toxic interactions of heavy metals with Saccharomyces cerevisiae were examined. Saccharomyces cerevisiae was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous PUF As resulted in them comprising greater than 65% and 40% of the total fatty acids in whole-cell and plasma membrane lipids, and nuclear membrane lipids, respectively. Incorporation of the exogenous PUF As had no discernible adverse effects on cell division. However, inhibition of cell division in the presence of Cd(N03)2 was accentuated by growth in the presence of the di-unsaturated fatty acid linoleate. Furthermore, susceptibility to both Cd2+ - and Cu2+ -induced plasma membrane permeabilisation and whole cell toxicity was markedly accentuated in PUF A-enriched cells, and increased with the degree of fatty acid unsaturation. The increased sensitivity ofPUFA-enriched cells to membrane permeabilisation and whole-cell toxicity was correlated with increased levels of lipid peroxidation in these cells. Cu2+ - and Cd2+_ induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements. Levels of the lipid peroxidation products thiobarbituric acid-reactive substances (TBARS) and conjugated dienes were markedly higher in PUF A-enriched cells, compared with unsupplemented cells, following exposure to cadmium or copper. Thus, lipid peroxidation was demonstrated as a major means of heavy metal toxicity in a microorganism for the first time. In addition, the effects ofPUFA-enrichment on the interactions of heavy metals with cellular nucleic acids were examined. Exposure ofPUFA-enriched cells to the redox-active metals chromium and copper resulted in the uncoupling of DNA synthesis from cell division, leading to sequential S phases. For example, DNA levels of up to 8C were evident in 18:3-enriched cells after only 4.5 h exposure to 100 JJ.M Cu(N03h. Using flow cytometry, the heterogeneity in susceptibility to copper toxicity of exponential phase S. cerevisiae was also examined. Susceptibility towards copper toxicity was demonstrated to be cell cycle stage-dependent, whereby G2/M phase cells were found to be the most susceptible towards copper toxicity. Staining with the oxidantsensitive probe 2',7' -dichlorodihydrofluorescein diacetate (H2DCFDA) revealed that the greater copper sensitivity of G2/M phase cells correlated with elevated endogenous levels of reactive oxygen species in these cells

Application of a Biphasic Mathematical Model of Cancer Cell Drug Response for Formulating Potent and Synergistic Targeted Drug Combinations to Triple Negative Breast Cancer Cells

Triple negative breast cancer is a collection of heterogeneous breast cancers that are immunohistochemically negative for estrogen receptor, progesterone receptor, and ErbB2 (due to deletion or lack of amplification). No dominant proliferative driver has been identified for this type of cancer, and effective targeted therapy is lacking. In this study, we hypothesized that triple negative breast cancer cells are multi-driver cancer cells, and evaluated a biphasic mathematical model for identifying potent and synergistic drug combinations for multi-driver cancer cells. The responses of two triple negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, to a panel of targeted therapy drugs were determined over a broad range of concentrations. The analyses of the drug responses by the biphasic mathematical model revealed that both cell lines were indeed dependent on multiple drivers, and inhibitors of individual drivers caused a biphasic response: a target-specific partial inhibition at low nM concentrations, and an off-target toxicity at μM concentrations. We further demonstrated that combinations of drugs, targeting each driver, cause potent, synergistic, and cell-specific cell killing. Immunoblotting analysis of the effects of the individual drugs and drug combinations on the signaling pathways supports the above conclusion. These results support a multi-driver proliferation hypothesis for these triple negative breast cancer cells, and demonstrate the applicability of the biphasic mathematical model for identifying effective and synergistic targeted drug combinations for triple negative breast cancer cells

A distinct role for recombination repair factors in an early cellular response to transcription-replication conflicts

Transcription–replication (T–R) conflicts are profound threats to genome integrity. However, whilst much is known about the existence of T–R conflicts, our understanding of the genetic and temporal nature of how cells respond to them is poorly established. Here, we address this by characterizing the early cellular response to transient T–R conflicts (TRe). This response specifically requires the DNA recombination repair proteins BLM and BRCA2 as well as a non-canonical monoubiquitylation-independent function of FANCD2. A hallmark of the TRe response is the rapid co-localization of these three DNA repair factors at sites of T–R collisions. We find that the TRe response relies on basal activity of the ATR kinase, yet it does not lead to hyperactivation of this key checkpoint protein. Furthermore, specific abrogation of the TRe response leads to DNA damage in mitosis, and promotes chromosome instability and cell death. Collectively our findings identify a new role for these well-established tumor suppressor proteins at an early stage of the cellular response to conflicts between DNA transcription and replication

The Simple Chordate \u3cem\u3eCiona intestinalis\u3c/em\u3e Has a Reduced Complement of Genes Associated with Fanconi Anemia

Fanconi anemia (FA) is a human genetic disease characterized by congenital defects, bone marrow failure, and increased cancer risk. FA is associated with mutation in one of 24 genes. The protein products of these genes function cooperatively in the FA pathway to orchestrate the repair of DNA interstrand cross-links. Few model organisms exist for the study of FA. Seeking a model organism with a simpler version of the FA pathway, we searched the genome of the simple chordate Ciona intestinalis for homologs of the human FA-associated proteins. BLAST searches, sequence alignments, hydropathy comparisons, maximum likelihood phylogenetic analysis, and structural modeling were used to infer the likelihood of homology between C. intestinalis and human FA proteins. Our analysis indicates that C. intestinalis indeed has a simpler and potentially functional FA pathway. The C. intestinalis genome was searched for candidates for homology to 24 human FA and FA-associated proteins. Support was found for the existence of homologs for 13 of these 24 human genes in C. intestinalis. Members of each of the three commonly recognized FA gene functional groups were found. In group I, we identified homologs of FANCE, FANCL, FANCM, and UBE2T/FANCT. Both members of group II, FANCD2 and FANCI, have homologs in C. intestinalis. In group III, we found evidence for homologs of FANCJ, FANCO, FANCQ/ERCC4, FANCR/RAD51, and FANCS/BRCA1, as well as the FA-associated proteins ERCC1 and FAN1. Evidence was very weak for the existence of homologs in C. intestinalis for any other recognized FA genes. This work supports the notion that C. intestinalis, as a close relative of vertebrates, but having a much reduced complement of FA genes, offers a means of studying the function of certain FA proteins in a simpler pathway than that of vertebrate cells

Modulation of the Fanconi anemia pathway \u3ci\u3evia\u3c/i\u3e chemically induced changes in chromatin structure

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci. The regulation and function of FANCD2 and FANCI, however, is poorly understood. In addition, how chromatin state impacts pathway activation is also unknown. In this study, we have examined the influence of chromatin state on the activation of the FA pathway. We describe potent activation of FANCD2 and FANCI monoubiquitination and nuclear foci formation following treatment of cells with the histone methyltransferase inhibitor BRD4770. BRD4770-induced activation of the pathway does not occur via the direct induction of DNA damage or via the inhibition of the G9a histone methyltransferase, a mechanism previously proposed for this molecule. Instead, we show that BRD4770-inducible FANCD2 and FANCI monoubiquitination and nuclear foci formation may be a consequence of inhibition of the PRC2/EZH2 chromatin-modifying complex. In addition, we show that inhibition of the class I and II histone deacetylases leads to attenuated FANCD2 and FANCI monoubiquitination and nuclear foci formation. Our studies establish that chromatin state is a major determinant of the activation of the FA pathway and suggest an important role for the PRC2/EZH2 complex in the regulation of this critical tumor suppressor pathway

The PTEN Phosphatase Functions Cooperatively with the Fanconi Anemia Proteins in DNA Crosslink Repair

Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and increased cancer risk. The FA proteins function primarily in DNA interstrand crosslink (ICL) repair. Here, we have examined the role of the PTEN phosphatase in this process. We have established that PTEN-deficient cells, like FA cells, exhibit increased cytotoxicity, chromosome structural aberrations, and error-prone mutagenic DNA repair following exposure to ICL-inducing agents. The increased ICL sensitivity of PTEN-deficient cells is caused, in part, by elevated PLK1 kinase-mediated phosphorylation of FANCM, constitutive FANCM polyubiquitination and degradation, and the consequent inefficient assembly of the FA core complex, FANCD2, and FANCI into DNA repair foci. We also establish that PTEN function in ICL repair is dependent on its protein phosphatase activity and ability to be SUMOylated, yet is independent of its lipid phosphatase activity. Finally, via epistasis analysis, we demonstrate that PTEN and FANCD2 function cooperatively in ICL repair

Proteomic responses to elevated ocean temperature in ovaries of the ascidian \u3cem\u3eCiona intestinalis\u3c/em\u3e

Ciona intestinalis, a common sea squirt, exhibits lower reproductive success at the upper extreme of the water temperatures it experiences in coastal New England. In order to understand the changes in protein expression associated with elevated temperatures, and possible response to global temperature change, we reared C. intestinalis from embryos to adults at 18°C (a temperature at which they reproduce normally at our collection site in Rhode Island) and 22°C (the upper end of the local temperature range). We then dissected ovaries from animals at each temperature, extracted protein, and measured proteomic levels using shotgun mass spectrometry (LC-MS/MS). 1532 proteins were detected at a 1% false discovery rate present in both temperature groups by our LC-MS/MS method. 62 of those proteins are considered up- or down-regulated according to our statistical criteria. Principal component analysis shows a clear distinction in protein expression pattern between the control (18°C) group and high temperature (22°C) group. Similar to previous studies, cytoskeletal and chaperone proteins are upregulated in the high temperature group. Unexpectedly, we find evidence that proteolysis is downregulated at the higher temperature. We propose a working model for the high temperature response in C. intestinalis ovaries whereby increased temperature induces upregulation of signal transduction pathways involving PTPN11 and CrkL, and activating coordinated changes in the proteome especially in large lipid transport proteins, cellular stress responses, cytoskeleton, and downregulation of energy metabolism

Insight into the Regulation of the Fanconi Anemia D2 protein, a Major DNA Repair Protein

Fanconi anemia (FA) is a rare genetic disease characterized by birth defects, bone marrow failure and increased cancer risk. FA is caused by mutation of 22 genes. The proteins coded by these genes function in the FA pathway, which repairs DNA damage, and maintains chromosome stability. Following exposure to DNA damaging agents, the pathway is activated, which leads to the transfer of a small signaling protein, ubiquitin, onto FANCD2. How this important step is regulated remains poorly understood. Using in silico molecular modeling, we recently identified four putative phosphorylation sites proximal to the site of FANCD2 monoubiquitination, lysine 561 (K561). Phosphorylation is another form of protein modification, and involves the replacement of a hydroxyl group in the side chain of an amino acid (serine (S), threonine (T), or tyrosine (Y)) with a phosphate group. Phosphates are added to proteins by enzymes called kinases, and removed by phosphatases. The newly identified phosphorylation sites are candidate cyclin-dependent kinase (CDK) sites, kinases that play a major role in regulating cell cycle progression. We hypothesize that FANCD2 is phosphorylated by CDKs, thereby regulating its activity during the cell cycle. The goal of my honors project was to determine if FANCD2 is phosphorylated by CDKs. We planned to test this hypothesis by purifying a fragment of FANCD2 containing the four-phosphorylation sites and performing an in vitro CDK kinase assay. First, I used molecular visualization software to design a fragment of FANCD2 containing the four-phosphorylation sites, without disrupting protein secondary structure. I then designed PCR primers to amplify this region and to clone this fragment into the pET-28a plasmid using restriction enzyme cloning. Two plasmids were generated: pET-28a-FANCD2-WT, containing the wild-type FANCD2 sequence, and pET-28a-FANCD2-TA, containing the FANCD2 sequence mutated at the putative phosphorylation sites. Immobilized metal affinity chromatography will be used to purify the FANCD2-WT and FANCD2-TA proteins. I will then incubate these purified proteins with several different cyclin-CDK pairs to determine if FANCD2 is phosphorylated by CDK and if mutation of the putative phosphorylation sites blocks phosphorylation. We anticipate that these experiments will greatly improve our understanding of the regulation of the FANCD2 protein

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions

Understanding the histone DNA repair code: H4K20me2 makes its mark

Chromatin is a highly compact structure that must be rapidly rearranged in order for DNA repair proteins to access sites of damage and facilitate timely and efficient repair. Chromatin plasticity is achieved through multiple processes, including the posttranslational modification of histone tails. In recent years, the impact of histone posttranslational modification on the DNA damage response has become increasingly well recognized, and chromatin plasticity has been firmly linked to efficient DNA repair. One particularly important histone posttranslational modification process is methylation. Here, we focus on the regulation and function of H4K20 methylation (H4K20me) in the DNA damage response and describe the writers, erasers, and readers of this important chromatin mark as well as the combinatorial histone posttranslational modifications that modulate H4K20me recognition. Finally, we discuss the central role of H4K20me in determining if DNA double-strand breaks (DSB) are repaired by the error-prone, nonhomologous DNA end joining pathway or the error-free, homologous recombination pathway. This review article discusses the regulation and function of H4K20me2 in DNA DSB repair and outlines the components and modifications that modulate this important chromatin mark and its fundamental impact on DSB repair pathway choice