Nestor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation

Background - Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Néstor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure. Results - Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope. Conclusions - Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage

Adefective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome

COMMD4 functions with the histone H2A-H2B dimer for the timely repair of DNA double-strand breaks

Genomic stability is critical for normal cellular function and its deregulation is a universal hallmark of cancer. Here we outline a previously undescribed role of COMMD4 in maintaining genomic stability, by regulation of chromatin remodelling at sites of DNA double-strand breaks. At break-sites, COMMD4 binds to and protects histone H2B from monoubiquitination by RNF20/RNF40. DNA damage-induced phosphorylation of the H2A-H2B heterodimer disrupts the dimer allowing COMMD4 to preferentially bind H2A. Displacement of COMMD4 from H2B allows RNF20/40 to monoubiquitinate H2B and for remodelling of the break-site. Consistent with this critical function, COMMD4-deficient cells show excessive elongation of remodelled chromatin and failure of both non-homologous-end-joining and homologous recombination. We present peptide-mapping and mutagenesis data for the potential molecular mechanisms governing COMMD4-mediated chromatin regulation at DNA double-strand breaks.</p

Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability

SENATAXIN AND ITS ROLE IN ATAXIA OCULOMOTOR APRAXIA TYPE 2

Neurodegenerative disorders are heterogeneous in nature and include ataxia oculomotor apraxia (AOA) syndromes that are characterised by cerebellar ataxia and a combination of different ophthalmological and neurological symptoms. AOA includes ataxia-telangiectasia (A-T), ataxia-telangiectasia like disorder (A-TLD), ataxia oculomotor apraxia type 1 (AOA1) and ataxia oculomotor apraxia type 2 (AOA2). The gene mutated in AOA2, SETX encodes a novel protein, senataxin, which shares homology to the yeast Sen1p protein, a DNA/RNA helicase. The C-terminus of senataxin contains a classical seven-motif domain found in the superfamily I of helicases. Senataxin is therefore a putative DNA/RNA helicase protein. Due to its homology with Sen1p, senataxin is hypothesised to play a role in RNA metabolism and as a nuclear helicase which plays a role in splicing. To characterise and elucidate the function of senataxin, anti-senataxin antibodies were generated against the N- and C-terminal regions of the protein. Using these antibodies the expression of senataxin in cell lines and the lack of senataxin in cells derived from AOA2 patients was shown. Furthermore, by using immunofluorescence and cellular fractionation, senataxin was demonstrated to be a nucleoplasmic protein, absent from nucleoli and with only negligible amounts of protein present in the cytoplasm. Sensitivity of AOA2 cells to DNA damaging agents was investigated. Data presented here show that similar to A-T, A-TLD and AOA1, AOA2 is also characterised by a defective response to DNA damage. AOA2 cells showed an increased sensitivity to the DNA damaging agent H2O2 and sensitivity to H2O2 was rescued by transfecting full-length SETX cDNA into these cells. However, unlike A-T cells, AOA2 cells showed a normal response to ionising radiation. A normal response to UV radiation was also observed. No defect in the repair of DNA single strand breaks was observed, however, the kinetics of re-sealing H2O2-induced DNA double strand breaks was found to be slower in AOA2 cells compared to controls. However, the rate of repair of IR-induced double strand breaks was normal, suggesting that senataxin may protect cells against oxidative DNA damage and play a role in the repair of DNA double strand breaks that arise as a result of oxidative DNA damage. In order to elucidate the biological function of senataxin, senataxin interacting proteins were identified by immunoprecipitation followed by either mass spectrometry or immunoblotting. Using this approach, senataxin was found to interact with nucleolin, heterogeneous nuclear ribonucleolar protein M, polyadenylate binding protein II, poly(A) binding protein I, spliceosomal protein 155, RNA Polymerase II and SMN, strongly suggesting a role for senataxin in all aspects of RNA metabolism including transcription and splicing. The sites of interaction between senataxin and nucleolin, spliceosomal protein 155, RNA Polymerase II and SMN were mapped and were found to be DNA or RNA independent. Chromatin immunoprecipitation assays were used to determine whether senataxin played a biological role similar to Sen1p in the transcriptional regulation of SOD1, CYC, IMPDH2, CypA and RPL36 genes. The ChIP assay showed that there was significantly less RNA Polymerase II binding to the SOD1, IMPDH2, CYC and RPL36 genomic loci in AOA2 cells compared with control cells. This differential binding of RNA Polymerase II to these genomic loci in AOA2 cells was also reflected in mRNA levels. RT-PCR demonstrated a good correlation between the reduced level of binding of RNA Polymerase II seen for SOD1, IMPDH2, CYC and RPL36 with the ChIP assay, in mRNA levels, suggesting that senataxin, similar to Sen1p, could be required for the transcriptional regulation of these genes. A double reporter assay was used to determine RNA splicing efficiency in cells where the expression of SETX was knocked-down by RNAi. This assay showed that there was a reduced splicing efficiency in cells where SETX was knocked-down compared to control cells, indicating that senataxin may be an essential factor required for splicing. Furthermore, senataxin was also shown to play a role in alternative splicing of the minigenes Tra2β1 and SRp20 and the splicing construct HC5 (derived from the human tropomycin gene). RT-PCR demonstrated that in the absence of senataxin, the splicing pattern of the minigenes Tra2β1 and SRp20 and the HC5 construct was altered, suggesting that senataxin played a role in alternative splice-site selection. This was also observed for both the endogenous human Transformer-2-beta gene (Tra2β1) and the human serine-arginine rich protein 20 (SRp20) genes, which is in agreement with a role for senataxin in alternative splice-site selection. Together, these data show that like other AOA syndromes, AOA2 is also characterised by a defect in the repair of DNA damage and that similar to Sen1p, senataxin also plays a role in RNA metabolism. In conclusion it can be hypothesised that the progressive neurological defect seen in AOA2 patients may result due to mutations in SETX which result in the misregulation of transcription, splicing and alternative splice-site selection. Some of these misregulated genes may include those involved in oxidative stress and DNA damage, since data presented in this thesis has shown that AOA2 cells are in a state of oxidative stress and have a defect in the repair of DNA double strand breaks that arise as a result of oxidative DNA damage in cells

Senataxin and Its Role in Ataxia Oculomotor Apraxia Type 2

Neurodegenerative disorders are heterogeneous in nature and include ataxia oculomotor apraxia (AOA) syndromes that are characterised by cerebellar ataxia and a combination of different ophthalmological and neurological symptoms. AOA includes ataxia-telangiectasia (A-T), ataxia-telangiectasia like disorder (A-TLD), ataxia oculomotor apraxia type 1 (AOA1) and ataxia oculomotor apraxia type 2 (AOA2). The gene mutated in AOA2, SETX encodes a novel protein, senataxin, which shares homology to the yeast Sen1p protein, a DNA/RNA helicase. The C-terminus of senataxin contains a classical seven-motif domain found in the superfamily I of helicases. Senataxin is therefore a putative DNA/RNA helicase protein. Due to its homology with Sen1p, senataxin is hypothesised to play a role in RNA metabolism and as a nuclear helicase which plays a role in splicing. To characterise and elucidate the function of senataxin, anti-senataxin antibodies were generated against the N- and C-terminal regions of the protein. Using these antibodies the expression of senataxin in cell lines and the lack of senataxin in cells derived from AOA2 patients was shown. Furthermore, by using immunofluorescence and cellular fractionation, senataxin was demonstrated to be a nucleoplasmic protein, absent from nucleoli and with only negligible amounts of protein present in the cytoplasm. Sensitivity of AOA2 cells to DNA damaging agents was investigated. Data presented here show that similar to A-T, A-TLD and AOA1, AOA2 is also characterised by a defective response to DNA damage. AOA2 cells showed an increased sensitivity to the DNA damaging agent H2O2 and sensitivity to H2O2 was rescued by transfecting full-length SETX cDNA into these cells. However, unlike A-T cells, AOA2 cells showed a normal response to ionising radiation. A normal response to UV radiation was also observed. No defect in the repair of DNA single strand breaks was observed, however, the kinetics of re-sealing H2O2-induced DNA double strand breaks was found to be slower in AOA2 cells compared to controls. However, the rate of repair of IR-induced double strand breaks was normal, suggesting that senataxin may protect cells against oxidative DNA damage and play a role in the repair of DNA double strand breaks that arise as a result of oxidative DNA damage. In order to elucidate the biological function of senataxin, senataxin interacting proteins were identified by immunoprecipitation followed by either mass spectrometry or immunoblotting. Using this approach, senataxin was found to interact with nucleolin, heterogeneous nuclear ribonucleolar protein M, polyadenylate binding protein II, poly(A) binding protein I, spliceosomal protein 155, RNA Polymerase II and SMN, strongly suggesting a role for senataxin in all aspects of RNA metabolism including transcription and splicing. The sites of interaction between senataxin and nucleolin, spliceosomal protein 155, RNA Polymerase II and SMN were mapped and were found to be DNA or RNA independent. Chromatin immunoprecipitation assays were used to determine whether senataxin played a biological role similar to Sen1p in the transcriptional regulation of SOD1, CYC, IMPDH2, CypA and RPL36 genes. The ChIP assay showed that there was significantly less RNA Polymerase II binding to the SOD1, IMPDH2, CYC and RPL36 genomic loci in AOA2 cells compared with control cells. This differential binding of RNA Polymerase II to these genomic loci in AOA2 cells was also reflected in mRNA levels. RT-PCR demonstrated a good correlation between the reduced level of binding of RNA Polymerase II seen for SOD1, IMPDH2, CYC and RPL36 with the ChIP assay, in mRNA levels, suggesting that senataxin, similar to Sen1p, could be required for the transcriptional regulation of these genes. A double reporter assay was used to determine RNA splicing efficiency in cells where the expression of SETX was knocked-down by RNAi. This assay showed that there was a reduced splicing efficiency in cells where SETX was knocked-down compared to control cells, indicating that senataxin may be an essential factor required for splicing. Furthermore, senataxin was also shown to play a role in alternative splicing of the minigenes Tra2β1 and SRp20 and the splicing construct HC5 (derived from the human tropomycin gene). RT-PCR demonstrated that in the absence of senataxin, the splicing pattern of the minigenes Tra2β1 and SRp20 and the HC5 construct was altered, suggesting that senataxin played a role in alternative splice-site selection. This was also observed for both the endogenous human Transformer-2-beta gene (Tra2β1) and the human serine-arginine rich protein 20 (SRp20) genes, which is in agreement with a role for senataxin in alternative splice-site selection. Together, these data show that like other AOA syndromes, AOA2 is also characterised by a defect in the repair of DNA damage and that similar to Sen1p, senataxin also plays a role in RNA metabolism. In conclusion it can be hypothesised that the progressive neurological defect seen in AOA2 patients may result due to mutations in SETX which result in the misregulation of transcription, splicing and alternative splice-site selection. Some of these misregulated genes may include those involved in oxidative stress and DNA damage, since data presented in this thesis has shown that AOA2 cells are in a state of oxidative stress and have a defect in the repair of DNA double strand breaks that arise as a result of oxidative DNA damage in cells

Combination Therapy With Histone Deacetylase Inhibitors (HDACi) for the Treatment of Cancer: Achieving the Full Therapeutic Potential of HDACi

Genetic and epigenetic changes in DNA are involved in cancer development and tumor progression. Histone deacetylases (HDACs) are key regulators of gene expression that act as transcriptional repressors by removing acetyl groups from histones. HDACs are dysregulated in many cancers, making them a therapeutic target for the treatment of cancer. Histone deacetylase inhibitors (HDACi), a novel class of small-molecular therapeutics, are now approved by the Food and Drug Administration as anticancer agents. While they have shown great promise, resistance to HDACi is often observed and furthermore, HDACi have shown limited success in treating solid tumors. The combination of HDACi with standard chemotherapeutic drugs has demonstrated promising anticancer effects in both preclinical and clinical studies. In this review, we summarize the research thus far on HDACi in combination therapy, with other anticancer agents and their translation into preclinical and clinical studies. We additionally highlight the side effects associated with HDACi in cancer therapy and discuss potential biomarkers to either select or predict a patient’s response to these agents, in order to limit the off-target toxicity associated with HDACi

Failure of amino acid homeostasis causes cell death following proteasome inhibition

The ubiquitin-proteasome system targets many cellular proteins for degradation and thereby controls most cellular processes. Although it is well established that proteasome inhibition is lethal, the underlying mechanism is unknown. Here, we show that proteasome inhibition results in a lethal amino acid shortage. In yeast, mammalian cells, and flies, the deleterious consequences of proteasome inhibition are rescued by amino acid supplementation. In all three systems, this rescuing effect occurs without noticeable changes in the levels of proteasome substrates. In mammalian cells, the amino acid scarcity resulting from proteasome inhibition is the signal that causes induction of both the integrated stress response and autophagy, in an unsuccessful attempt to replenish the pool of intracellular amino acids. These results reveal that cells can tolerate protein waste, but not the amino acid scarcity resulting from proteasome inhibition

Abstract PO-031: Aldolase A (ALDOA) is required for efficient DNA double-strand break (DSB) repair

Introduction: Metabolic reprogramming, known as the Warburg effect, is one of the universal differences between cancer cells and non-cancerous cells. Glucose metabolism and DNA repair are frequently dysregulated in cancer. Metabolic pathways provide cells with nucleic acids and energy required to repair DNA. However, the underlying mechanisms that promote crosstalk between these processes are unknown. ALDOA is a glycolytic enzyme that catalyses the conversion of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. ALDOA is overexpressed in several types of cancer. In this study, we demonstrate a novel mechanism through which ALDOA directly regulates DSB repair. Methods: ALDOA was depleted from cells using siRNA and single-shot quantitative proteomics were performed. Immunofluorescence was utilized to determine the localization of ALDOA. DSB repair reporter assays were used to measure DSB break repair. Gene expression was quantified by western blot and qPCR. Immunoprecipitations were used to detect protein:protein interactions. Statistical analysis: The experiments were at least n=3, and data are presented as the means ± SEM. Statistical significance was evaluated using Student’s t-test or one-way ANOVA. Results: In order to identify ALDOA-dependent pathways, we performed quantitative mass spectrometry on ALDOA depleted cells. In addition to the expected decrease in glycolysis pathways, we also observed a significant downregulation of DNA repair proteins in ALDOA depleted cells. Further analysis showed that the ALDOA protein responds to DNA damage (IR) and migrates from the cytosol to the nucleus, suggesting that it could be directly involved in DNA damage repair. Slower clearance of γ-H2AX foci (a DSB marker), and decreased clonogenicity following irradiation (IR) treatment were also observed, indicating dysfunctional DNA repair processes. Repair of DSBs is primarily though the NHEJ (non-homologous end-joining) or HR (homologous recombination) -mediated DNA repair pathways. Silencing ALDOA led to a decrease in both NHEJ- and HR-mediated DSB repair efficiency. This disruption was likely due to the significant reduction of both the mRNA and protein of the DNA repair effector kinases, DNA-Dependent Kinase (DNA-PK) and Ataxia and Telangiectasia Mutated (ATM) in ALDOA-depleted cells. In addition to regulating the expression of DNAPK and ATM we also found that ALDOA directly interacted with both kinases, suggesting that it may have a direct role in regulating their function. Here, we define a role for ALDOA in the repair of DNA DSBs, through the regulation of DNA repair effector kinase expression and function. Conclusion: These results identify crosstalk between metabolic and DNA repair pathways and have implications for cancer treatment and tumorigenesis. The role of ALDOA in DNA repair could promote therapeutic resistance in tumors and may be a future therapeutic target to sensitize tumors to DNA-damaging agents such as radiation

Combination Therapy With Histone Deacetylase Inhibitors (HDACi) for the Treatment of Cancer: Achieving the Full Therapeutic Potential of HDACi

Genetic and epigenetic changes in DNA are involved in cancer development and tumor progression. Histone deacetylases (HDACs) are key regulators of gene expression that act as transcriptional repressors by removing acetyl groups from histones. HDACs are dysregulated in many cancers, making them a therapeutic target for the treatment of cancer. Histone deacetylase inhibitors (HDACi), a novel class of small-molecular therapeutics, are now approved by the Food and Drug Administration as anticancer agents. While they have shown great promise, resistance to HDACi is often observed and furthermore, HDACi have shown limited success in treating solid tumors. The combination of HDACi with standard chemotherapeutic drugs has demonstrated promising anticancer effects in both preclinical and clinical studies. In this review, we summarize the research thus far on HDACi in combination therapy, with other anticancer agents and their translation into preclinical and clinical studies. We additionally highlight the side effects associated with HDACi in cancer therapy and discuss potential biomarkers to either select or predict a patient’s response to these agents, in order to limit the off-target toxicity associated with HDACi