Biochemistry (Scholarly Articles)

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SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation
(Wiley Open Access and Federation of European Biochemical Societies, 2022-01-24) Onwubiko, Nichodemus O.; Scheffel, Felicia; Tessmer, Ingrid; Nasheuer, Heinz-Peter
The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase ¿-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that while Tag(131-627) (V350E/P417D) and Tag(131-627) (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag(1-708) and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.
Population‐based identity‐by‐descent mapping combined with exome sequencing to detect rare risk variants for schizophrenia
(Wiley, 2019-02-23) Harold, Denise; Connolly, Siobhan; Riley, Brien P.; Kendler, Kenneth S.; McCarthy, Shane E.; McCombie, William R.; Richards, Alex; Owen, Michael J.; O'Donovan, Michael C.; Walters, James; Wellcome Trust Case Control Consortium 2; Schizophrenia Working Group of the Psychiatric Genomics Consortium; Donohoe, Gary; Gill, Michael; Corvin, Aiden; Morris, Derek W.; National Institutes of Health; Science Foundation Ireland; Wellcome Trust
Genome‐wide association studies (GWASs) are highly effective at identifying common risk variants for schizophrenia. Rare risk variants are also important contributors to schizophrenia etiology but, with the exception of large copy number variants, are difficult to detect with GWAS. Exome and genome sequencing, which have accelerated the study of rare variants, are expensive so alternative methods are needed to aid detection of rare variants. Here we re‐analyze an Irish schizophrenia GWAS dataset (n = 3,473) by performing identity‐by‐descent (IBD) mapping followed by exome sequencing of individuals identified as sharing risk haplotypes to search for rare risk variants in coding regions. We identified 45 rare haplotypes (>1 cM) that were significantly more common in cases than controls. By exome sequencing 105 haplotype carriers, we investigated these haplotypes for functional coding variants that could be tested for association in independent GWAS samples. We identified one rare missense variant in PCNT but did not find statistical support for an association with schizophrenia in a replication analysis. However, IBD mapping can prioritize both individual samples and genomic regions for follow‐up analysis but genome rather than exome sequencing may be more effective at detecting risk variants on rare haplotypes.
The awarding of the first honorary Doctor of Science by the Queen's University in Ireland to William King – a journey of scientific curiosity
(Royal Irish Academy, 2020) Murray, John; Dingsdale, Ann; Nasheuer, Heinz-Peter; Harper, David A.T.
William King (1809 86) was the founding Professor of Mineralogy and Geology at Queen s College Galway (QCG), one of three regional colleges opened in 1849 to provide secular university-level education in Ireland. King came from a modest background and despite lacking third-level qualifications, began publishing on palaeontological and geological matters in the 1840s. These early contributions aided his application for the professorship in Galway, particularly his seminal 1850 monograph on the Permian fossils of England, which was in preparation at the time. During his first two decades at QCG, King maintained an up-to-date teaching programme in geology and palaeontology, played a key role in establishing the natural history museum and further developed his research portfolio. He investigated several topics of international interest, including the supposed earliest fossils of living organisms and the emerging evidence for fossil humans. King s achievements were impressive, particularly as he was essentially self-taught and also considering the isolated and poor economic standing of Galway at the time. The Queen s University in Ireland (QUI) bestowed its first ever honorary Doctor of Science on William King in 1870 in recognition of his distinguished geological research, and also to mark a refocussing of the university curriculum to better reflect the importance of science. King s award came at a time when the education system was coming under increasing scrutiny in Ireland, and as part of these reforms QUI was dissolved in 1882 and replaced by the Royal University of Ireland. One of the final acts of QUI was to award a large number of former graduates with master s degrees. A select few were conferred with honorary doctorates, including King s eldest son, William Jr., who had been amongst the first students to enter QCG in 1849 and, after graduating, enjoyed a distinguished career with the Geological Survey of India. Father and son thus achieved the unique honour of being the first and last recipients of a Doctor of Science (honoris causa) from QUI for their geological endeavours.
Inhibition of human BK polyomavirus replication by small noncoding RNAs
(American Society for Microbiology Journals, 2011) Tikhanovich, Irina; Liang, Bo; Seoighe, Cathal; Folk, William R.; Nasheuer, Heinz-Peter
Small noncoding RNAs regulate a variety of cellular processes, including genomic imprinting, chromatin remodeling, replication, transcription, and translation. Here, we report small replication-regulating RNAs (srRNAs) that specifically inhibit DNA replication of the human BK polyomavirus (BKV) in vitro and in vivo. srRNAs from FM3A murine mammary tumor cells were enriched by DNA replication assay-guided fractionation and hybridization to the BKV noncoding control region (NCCR) and synthesized as cDNAs. Selective mutagenesis of the cDNA sequences and their putative targets suggests that the inhibition of BKV DNA replication is mediated by srRNAs binding to the viral NCCR, hindering early steps in the initiation of DNA replication. Ectopic expression of srRNAs in human cells inhibited BKV DNA replication in vivo. Additional srRNAs were designed and synthesized that specifically inhibit simian virus 40 (SV40) DNA replication in vitro. These observations point to novel mechanisms for regulating DNA replication and suggest the design of synthetic agents for inhibiting replication of polyomaviruses and possibly other viruses.
Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy
(Public Library of Science, 2012-04-19) Broderick, Ronan; Ramadurai, Sivaramakrishnan; Tóth, Katalin; Togashi, Denisio M.; Ryder, Alan G.; Langowski, Jörg; Nasheuer, Heinz-Peter
Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.
Cell cycle-dependent formation of Cdc45-Claspin complexes in human cells is compromized by UV-mediated DNA damage
(Wiley, 2013-08-02) Broderick, Ronan; Rainey, Michael D.; Santocanale, Corrado; Nasheuer, Heinz-Peter
The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays an important role in the intra-S-phase checkpoint. Its interactions with other replication proteins during the cell cycle and after intra-S-phase checkpoint activation are only partially characterized. In the present study, we show that the C terminal part of Cdc45 may mediate its interactions with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A and DNA polymerase are maximal during the S phase. Following UVC-induced DNA damage, Cdc45-Claspin complex formation is reduced, whereas the binding of Cdc45 to replication protein A is not affected. We also show that treatment of cells with UCN-01 and phosphatidylinositol 3-kinase-like kinase inhibitors does not rescue the UV-induced destabilization of Cdc45-Claspin interactions, suggesting that the loss of the interaction between Cdc45 and Claspin occurs upstream of ataxia telangiectasia and Rad 3-related activation in the intra-S-phase checkpoint.Structured digital abstract Clapsin physically interacts with Cdc45 by anti bait coimmunoprecipitation (View interaction) Cdc45 physically interacts with RPA32, Clapsin and p125 Pol delta by pull down (View interaction) Cdc45 physically interacts with RPA32 by pull down (View interaction) Cdc45 physically interacts with Clapsin by pull down (View interaction) Cdc45 physically interacts with Clapsin and RPA32 by pull down (View interaction) RPA32 physically interacts with Cdc45 by anti bait coimmunoprecipitation (View interaction)
Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences
(Wiley, 2018-04-16) Ali, Rizwan; Ramadurai, Sivaramakrishnan; Barry, Frank; Nasheuer, Heinz-Peter; Science Foundation Ireland; Else Kröner‐Fresenius‐Stiftung
The modulation of expression levels of fluorescent fusion proteins (FFPs) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule-based techniques are very sensitive to high expression levels of FFPs. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase (TK) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway (R) system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and FFPs containing these FPs. Two promoter constructs, TK2ST and TKTSC, were established, which have optimal low expression levels suitable for FCS studies in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5'-UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5'-UTR.
Alkoxyalkyl esters of nucleotide analogs inhibit polyomavirus DNA replication and large T antigen activities
(American Society for Microbiology Journals, 2020-12-07) Onwubiko, Nichodemus O.; Diaz, Suraya; Krecmerova, Marcela; Nasheuer, Heinz-Peter
Polyomavirus-related infections are ubiqutious in immunocompromised individuals and in some cases are intractable and fatal. Due to lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections has been repurposed as anti-polyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as anti-polyomavirus agents are still under research. Here, polyomavirus large tumor antigens (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl-ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system only utilizing purified proteins. We present evidence that DNA helicase, and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl-ester derivatives of cidofovir suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting ssDNA and dsDNA binding activities of Tags. These findings show that the alkoxyalkyl-ester derivatives of cidofovir are effective in vitro without undergoing further conversions and conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.
SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication
(Oxford University Press (OUP), 2020-03-04) Onwubiko, Nichodemus O.; Borst, Angela; Diaz, Suraya A.; Passkowski, Katharina; Scheffel, Felicia; Tessmer, Ingrid; Nasheuer, Heinz-Peter
DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (K-D values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.
Cognitive analysis of schizophrenia risk genes that function as epigenetic regulators of gene expression
(Wiley, 2016-10-20) Whitton, Laura; Cosgrove, Donna; Clarkson, Christopher; Harold, Denise; Kendall, Kimberley; Richards, Alex; Mantripragada, Kiran; Owen, Michael J.; O'Donovan, Michael C.; Walters, James; Hartmann, Annette; Konte, Betina; Rujescu, Dan; Gill, Michael; Corvin, Aiden; Rea, Stephen; Donohoe, Gary; Morris, Derek W.
Epigenetic mechanisms are an important heritable and dynamic means of regulating various genomic functions, including gene expression, to orchestrate brain development, adult neurogenesis, and synaptic plasticity. These processes when perturbed are thought to contribute to schizophrenia pathophysiology. A core feature of schizophrenia is cognitive dysfunction. For genetic disorders where cognitive impairment is more severe such as intellectual disability, there are a disproportionally high number of genes involved in the epigenetic regulation of gene transcription. Evidence now supports some shared genetic aetiology between schizophrenia and intellectual disability. GWAS have identified 108 chromosomal regions associated with schizophrenia risk that span 350 genes. This study identified genes mapping to those loci that have epigenetic functions, and tested the risk alleles defining those loci for association with cognitive deficits. We developed a list of 350 genes with epigenetic functions and cross-referenced this with the GWAS loci. This identified eight candidate genes: BCL11B, CHD7, EP300, EPC2, GATAD2A, KDM3B, RERE, SATB2. Using a dataset of Irish psychosis cases and controls (n = 1235), the schizophrenia risk SNPs at these loci were tested for effects on IQ, working memory, episodic memory, and attention. Strongest associations were for rs6984242 with both measures of IQ (P = 0.001) and episodic memory (P = 0.007). We link rs6984242 to CHD7 via a long range eQTL. These associations were not replicated in independent samples. Our study highlights that a number of genes mapping to risk loci for schizophrenia may function as epigenetic regulators of gene expression but further studies are required to establish a role for these genes in cognition. (C) 2016 Wiley Periodicals, Inc.
Genes influenced by MEF2C contribute to neurodevelopmental disease via gene expression changes that affect multiple types of cortical excitatory neurons
(Oxford University Press (OUP), 2020-09-25) Cosgrove, Donna; Whitton, Laura; Fahey, Laura; Ó Broin, Pilib; Donohoe, Gary; Morris, Derek W.; Brain and Behavior Research Foundation; Irish Research Council
Myocyte enhancer factor 2 C (MEF2C) is an important transcription factor during neurodevelopment. Mutation or deletion of MEF2C causes intellectual disability (ID) and common variants within MEF2C are associated with cognitive function and schizophrenia risk. We investigated if genes influenced by MEF2C during neurodevelopment are enriched for genes associated with neurodevelopmental phenotypes, and if this can be leveraged to identify biological mechanisms and individual brain cell types affected. We used a set of 1055 genes that were differentially expressed in the adult mouse brain following early embryonic deletion of Mef2c in excitatory cortical neurons. Using GWAS data, we found these differentially expressed genes (DEGs) to be enriched for genes associated with schizophrenia, intelligence and educational attainment but not autism spectrum disorder (ASD). For this gene-set, genes that overlap with target genes of the Fragile X mental retardation protein (FMRP) are a major driver of these enrichments. Using trios data, we found these DEGs to be enriched for genes containing de novo mutations reported in ASD and ID, but not schizophrenia. Using single cell RNA-seq data, we identified that a number of different excitatory glutamatergic neurons in the cortex were enriched for these DEGs including deep layer pyramidal cells and cells in the retrosplenial cortex, entorhinal cortex and subiculum, and these cell types are also enriched for FMRP target genes. The involvement of MEF2C and FMRP in synapse elimination suggests that disruption of this process in these cell types during neurodevelopment contributes to cognitive function and risk of neurodevelopmental disorders.
Genes regulated by BCL11B during T‐cell development are enriched for de novo mutations found in schizophrenia patients
(Wiley, 2020-07-29) Fahey, Laura; Donohoe, Gary; Ó Broin, Pilib; Morris, Derek W.
While abnormal neurodevelopment contributes to schizophrenia (SCZ) risk, there is also evidence to support a role for immune dysfunction in SCZ.BCL11B, associated with SCZ in genome-wide association study (GWAS), is a transcription factor that regulates the differentiation and development of cells in the central nervous and immune systems. Here, we use functional genomics data from studies ofBCL11Bto investigate the contribution of neuronal and immune processes to SCZ pathophysiology. We identified the gene targets of BCL11B in brain striatal cells (n = 223 genes), double negative 4 (DN4) developing T cells (n = 114 genes) and double positive (DP) developing T cells (n = 518 genes) using an integrated analysis of RNA-seq and ChIP-seq data. No gene-set was enriched for genes containing common variants associated with SCZ but the DP gene-set was enriched for genes containing missense de novo mutations (DNMs;p= .001) using data from 3,447 SCZ trios. Post hoc analysis revealed the enrichment to be stronger for DP genes negatively regulated by BCL11B. Biological processes enriched for genes negatively regulated by BCL11B in DP gene-set included immune system development and cytokine signaling. These analyses, leveraging a GWAS-identified SCZ risk gene and data on gene expression and transcription factor binding, indicate that DNMs in immune pathways contribute to SCZ risk.
Altered gene regulation as a candidate mechanism by which ciliopathy gene SDCCAG8 contributes to schizophrenia and cognitive function
(Oxford University Press (OUP), 2019-12-23) Flynn, Mairéad; Whitton, Laura; Donohoe, Gary; Morrison, Ciaran G.; Morris, Derek W.; College of Science, National University of Ireland, Galway; Irish Research Council; Thomas Crawford Hayes Trust Research Fellowship
Mutations in genes that encode centrosomal/ciliary proteins cause severe cognitive deficits, while common single-nucleotide polymorphisms in these genes are associated with schizophrenia (SZ) and cognition in genome-wide association studies. The role of these genes in neuropsychiatric disorders is unknown. The ciliopathy gene SDCCAG8 is associated with SZ and educational attainment (EA). Genome editing of SDCCAG8 caused defects in primary ciliogenesis and cilium-dependent cell signalling. Transcriptomic analysis of SDCCAG8-deficient cells identified differentially expressed genes that are enriched in neurodevelopmental processes such as generation of neurons and synapse organization. These processes are enriched for genes associated with SZ, human intelligence (IQ) and EA. Phenotypic analysis of SDCCAG8-deficent neuronal cells revealed impaired migration and neuronal differentiation. These data implicate ciliary signalling in the aetiology of SZ and cognitive dysfunction. We found that centrosomal/ciliary genes are enriched for association with IQ, suggesting altered gene regulation as a general model for neurodevelopmental impacts of centrosomal/ciliary genes.
An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis
(Oxford University Press (OUP), 2014-01-28) Morris, Derek W.; Science Foundation Ireland; Wellcome Trust; National Institute of Mental Health
Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100 kb), rare copy-number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1564 cases and 1748 controls all from Ireland, and further extended the analysis to include an additional 5196 UK controls. We found association with duplications at chr20p12.2 (P = 0.007) and evidence of replication in large independent European schizophrenia (P = 0.052) and UK bipolar disorder case-control cohorts (P = 0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11 707 cases and 10 carriers in 21 204 controls [meta-analysis Cochran-Mantel-Haenszel P-value = 2 x 10(-4); odds ratio (OR) = 11.3, 95% CI = 3.7, infinity]. Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68 Mb with similar breakpoints across samples. By haplotype analysis and sequencing, we identified a tandem ~149 kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P = 2.5 x 10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100 kb), rare copy-number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1564 cases and 1748 controls all from Ireland, and further extended the analysis to include an additional 5196 UK controls. We found association with duplications at chr20p12.2 (P = 0.007) and evidence of replication in large independent European schizophrenia (P = 0.052) and UK bipolar disorder case-control cohorts (P = 0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11 707 cases and 10 carriers in 21 204 controls [meta-analysis Cochran-Mantel-Haenszel P-value = 2 x 10(-4); odds ratio (OR) = 11.3, 95% CI = 3.7, infinity]. Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68 Mb with similar breakpoints across samples. By haplotype analysis and sequencing, we identified a tandem ~149 kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P = 2.5 x 10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.
Excess of rare novel loss-of-function variants in synaptic genes in schizophrenia and autism spectrum disorders
(Springer Nature, 2013-10-15) Kenny, Elaine M.; Cormican, Paul; Furlong, Sarah; Heron, Eleisa; Kenny, Graham; Fahey, Ciara; Kelleher, Eric; Ennis, Sean; Tropea, Daniela; Anney, Richard; Corvin, Aiden P.; Donohoe, Gary; Gallagher, Louise; Gill, Michael; Morris, Derek W.; Health Research Board; Science Foundation Ireland
Schizophrenia (SZ) and autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that may share an underlying pathology suggested by shared genetic risk variants. We sequenced the exonic regions of 215 genes in 147 ASD cases, 273 SZ cases and 287 controls, to identify rare risk mutations. Genes were primarily selected for their function in the synapse and were categorized as: (1) Neurexin and Neuroligin Interacting Proteins, (2) Post-synaptic Glutamate Receptor Complexes, (3) Neural Cell Adhesion Molecules, (4) DISC1 and Interactors and (5) Functional and Positional Candidates. Thirty-one novel loss-of-function (LoF) variants that are predicted to severely disrupt protein-coding sequence were detected among 2 861 rare variants. We found an excess of LoF variants in the combined cases compared with controls (P=0.02). This effect was stronger when analysis was limited to singleton LoF variants (P=0.0007) and the excess was present in both SZ (P=0.002) and ASD (P=0.001). As an individual gene category, Neurexin and Neuroligin Interacting Proteins carried an excess of LoF variants in cases compared with controls (P=0.05). A de novo nonsense variant in GRIN2B was identified in an ASD case adding to the growing evidence that this is an important risk gene for the disorder. These data support synapse formation and maintenance as key molecular mechanisms for SZ and ASD.Schizophrenia (SZ) and autism spectrum disorders (ASDs) are complex neurodevelopmental disorders that may share an underlying pathology suggested by shared genetic risk variants. We sequenced the exonic regions of 215 genes in 147 ASD cases, 273 SZ cases and 287 controls, to identify rare risk mutations. Genes were primarily selected for their function in the synapse and were categorized as: (1) Neurexin and Neuroligin Interacting Proteins, (2) Post-synaptic Glutamate Receptor Complexes, (3) Neural Cell Adhesion Molecules, (4) DISC1 and Interactors and (5) Functional and Positional Candidates. Thirty-one novel loss-of-function (LoF) variants that are predicted to severely disrupt protein-coding sequence were detected among 2 861 rare variants. We found an excess of LoF variants in the combined cases compared with controls (P=0.02). This effect was stronger when analysis was limited to singleton LoF variants (P=0.0007) and the excess was present in both SZ (P=0.002) and ASD (P=0.001). As an individual gene category, Neurexin and Neuroligin Interacting Proteins carried an excess of LoF variants in cases compared with controls (P=0.05). A de novo nonsense variant in GRIN2B was identified in an ASD case adding to the growing evidence that this is an important risk gene for the disorder. These data support synapse formation and maintenance as key molecular mechanisms for SZ and ASD.
RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster
(eLife Sciences Publications, 2019-11-05) Mills, Wilbur Kyle; Lee, Yuh Chwen G.; Kochendoerfer, Antje M.; Dunleavy, Elaine M.; Karpen, Gary H.; Science Foundation Ireland; Health Research Board; National Institutes of Health; Wellcome Trust
Tandemly-repeated DNAs, or satellites, are enriched in heterochromatic regions of eukaryotic genomes and contribute to nuclear structure and function. Some satellites are transcribed, but we lack direct evidence that specific satellite RNAs are required for normal organismal functions. Here, we show satellite RNAs derived from AAGAG tandem repeats are transcribed in many cells throughout Drosophila melanogaster development, enriched in neurons and testes, often localized within heterochromatic regions, and important for viability. Strikingly, we find AAGAG transcripts are necessary for male fertility, and that AAGAG RNA depletion results in defective histone-protamine exchange, sperm maturation and chromatin organization. Since these events happen late in spermatogenesis when the transcripts are not detected, we speculate that AAGAG RNA in primary spermatocytes 'primes' post-meiosis steps for sperm maturation. In addition to demonstrating essential functions for AAGAG RNAs, comparisons between closely related Drosophila species suggest that satellites and their transcription evolve quickly to generate new functions.
Determination of the number of RAD51 molecules in different human cell lines
(Taylor & Francis, 2019-11-15) Foertsch, Franziska; Kache, Tom; Drube, Sebastian; Biskup, Christoph; Nasheuer, Heinz-Peter; Melle, Christian
Knowledge about precise numbers of specific molecules is necessary for understanding and verification of biological pathways. The RAD51 protein is central in the repair of DNA double-strand breaks (DSBs) by homologous recombination repair and understanding its role in cellular pathways is crucial to design mechanistic DNA repair models. Here, we determined the number of RAD51 molecules in several human cell lines including primary fibroblasts. We showed that between 20000 to 100000 of RAD51 molecules are available per human cell that theoretically can be used for simultaneously loading at least 7 DSBs. Interestingly, the amount of RAD51 molecules does not significantly change after the induction of DNA damage using bleomycin or gamma-irradiation in cells but an accumulation of RAD51 on the chromatin occurs. Furthermore, we generated an EGFP-RAD51 fusion under the control of HSV thymidine kinase promoter sequences yielding moderate protein expression levels comparable to endogenously expressed RAD51. Initial characterizations suggest that these low levels of ectopically expressed RAD51 are compatible with cell cycle progression of human cells. Hence, we provide parameters for the quantitative understanding and modeling of RAD51-involving processes.
ATPase activity of human binding immunoglobulin protein (BiP) variants is enhanced by signal sequence and physiological concentrations of Mn2+
(Wiley Open Access, 2019-08-29) Bandla, Sravanthi; Diaz, Suraya; Nasheuer, Heinz-Peter; FitzGerald, Una; Hardiman Research Scholarship, National University of Ireland Galway; Foundation Office, National University of Ireland Galway; Else Kröner‐Fresenius‐Stiftung
B-cell immunoglobulin binding protein (BiP) is an essential endoplasmic reticulum (ER) chaperone normally found in the ER lumen. However, BiP also has other extracellular and intracellular functions. As it is unclear whether peripheral BiP has a signal and/or ER retention sequence, here we produced and biochemically characterised four variants of BiP. The variants differed depending on the presence or the absence of signal and ER retention peptides. Proteins were purified using nickel affinity chromatography, and variant size and quality were confirmed using SDS/PAGE gels. The thermal denaturing temperature of these proteins was found to be 46-47 degrees C. In addition, we characterised nucleotide binding properties in the absence and the presence of divalent cations. Interestingly, in the absence of cations, ADP has a higher binding affinity to BiP than ATP. The presence of divalent cations results in a decrease of the K-d values of both ADP and ATP, indicating higher affinities of both nucleotides for BiP. ATPase assays were carried out to study the enzyme activity of these variants and to characterise the kinetic parameters of BiP variants. Variants with the signal sequence had higher specific activities than those without. Both Mg2+ and Mn2+ efficiently stimulated the ATPase activity of these variants at low micromolar concentrations, whereas calcium failed to stimulate BiP ATPase. Our novel findings indicate the potential functionality of BiP variants that retain a signal sequence, and also reveal the effect of physiological concentrations of cations on the nucleotide binding properties and enzyme activities of all variants.
RIP2 enhances cell survival by activation of NF-ĸB in triple negative breast cancer cells
(Elsevier, 2018-02-06) Jaafar, Rola; Mnich, Katarzyna; Dolan, Sarah; Hillis, Jennifer; Almanza, Aitor; Logue, Susan E.; Samali, Afshin; Gorman, Adrienne M.; Breast Cancer Now; Health Research Board; Belgium Grant; Science Foundation Ireland; Irish Research Council; Clinical Research Development Ireland; Enterprise Ireland; College of Science, NUI Galway; Thomas Crawford Hayes Fund, NUI Galway
Receptor-interacting protein 2 (RIP2) is an essential mediator of inflammation and innate immunity, but little is known about its role outside the immune system. Recently, RIP2 has been linked to chemoresistance of triple negative breast cancer (TNBC), the most aggressive breast cancer subtype for which there is an urgent need for targeted therapies. In this study we show that high expression of RIP2 in breast tumors correlates with a worse prognosis and a higher risk of recurrence. We also demonstrate that RIP2 confers TNBC cell resistance against paclitaxel and ceramide-induced apoptosis. Over expression of RIP2 lead to NF-kappa B activation, which contributed to higher expression of pro-survival proteins and cell survival. Conversely, RIP2 knockdown inhibited NF-kappa B signaling, reduced levels of anti-apoptotic proteins and sensitized cells to drug treatment. Together, these data show that RIP2 promotes survival of breast cancer cells through NF-kappa B activation and that targeting RIP2 may be therapeutically beneficial for treatment of TNBC. (C) 2018 Published by Elsevier Inc.
Stress management at the ER: regulators of ER stress-induced apoptosis.
(Elsevier, 2012-02-17) Gorman, Adrienne M.; Healy, Sandra J.M.; Jäger, Richard; Samali, Afshin; Science Foundation Ireland; Health Research Board; Breast Cancer Campaign
The endoplasmic reticulum (ER) is an elaborate cellular organelle essential for cell function and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins which are detected by ER transmembrane receptors that initiate the unfolded protein response (UPR) to restore normal ER function. If the ER stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, particularly because ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review we aim to shed light on the proteins that are not core components of the UPR signaling pathway but which can influence the course of the ER stress response by regulating the switch from the adaptive phase to apoptosis.

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