Browsing by Author "Goodarzi, Aaron A."

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    Advancing Techniques of Structural Mass Spectrometry for Integrative Structural Modelling
    (2020-04-20) Ziemianowicz, Daniel S.; Schriemer, David C.; Goodarzi, Aaron A.; MacCallum, Justin L.; Fraser, Marie Elizabeth
    Proteins are the fundamental functional units underlying all cellular activities. Protein function emerges from structure. To understand cellular activity and the diseases that arise from protein dysfunction we require knowledge of protein structure and structural dynamics. The toolbox offered by mass spectrometry (MS) allows a wide range of perspectives on protein structure, enabled by the application of chemical reagents that can encode structural properties. Improvements in the performance of labelling chemistries in turn can enhance the data returned and the structural models that are ultimately produced. Photogenerated carbenes are one such high-performance chemistry that offer unbiased sampling of protein structure at timescales relevant to protein dynamics. On the other hand, no single method can offer the breadth of data necessary to produce a comprehensive model of protein structure and dynamics—protein systems span broad spatial and temporal scales that exceed the scope of any single technique. To resolve large and complex protein systems, the integration of multiple data sets from orthogonal techniques is necessary. Here, I evaluate and advance structural MS methods with the goal of improving the accuracy and precision of structural models produced by MS-driven integrative structural modelling. Of particular interest is the application of carbene-based crosslinking and covalent labelling reagents which are shown to produce data with greater sequence coverage and improved accuracy in representing the equilibrated conformational state. Novel analytical software routines are developed to overcome the complications that arise from the labelling of proteins with a non-specific chemistry such as ambiguity in localizing modifications. Structural models are produced with integrative modelling workflows, including the development of a novel modelling restraint based on crosslinking and hydrogen/deuterium exchange data. MS-driven integrative modelling is demonstrated on multiple systems, including large complexes and systems with substantial disorder.
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    An Analysis of Human Exposure to Alpha Particle Radiation
    (2018-12-17) Stanley, Fintan; Goodarzi, Aaron A.; Lees-Miller, Susan P.; Cobb, Jennifer A.
    High linear energy transfer (LET) ionizing radiation (IR) is the predominant source of IR humans are exposed to. Radon gas, which emits a high energy alpha-particle, represents the greatest single lifetime source, but also remains comparatively understudied versus low LET IR sources such as x-rays. The inhalation radon (222Rn) gas from indoor air exposes lung tissue to alpha particle radiation, damaging DNA and increasing the lifetime risk of lung cancer. Buildings can concentrate radioactive radon (222Rn) gas to harmful levels. To enable cancer prevention, I examined how Canadian Prairie radon exposure is modified by environmental design and human behavior and evaluated different radon test modalities. I also developed a high-throughput, benchtop alpha-particle irradiation system to facilitate future research into the biological consequences of high LET radiation exposure. Initially, I examined 90+ day radon test results from 2,382 residential homes from an area encompassing 82.5% of the Southern Alberta population. Remediated homes were retested to determine efficacy of radon reduction techniques in this region. Subsequently, 11,726 Alberta and Saskatchewan homes were radon tested, coupled to geographic, design and behavior metrics. Canadian Prairie homes contained 140 Bq/m3 average radon (min <15 Bq/m3; max 7,199 Bq/m3) and 17.8% were ≥ 200 Bq/m3. Geostatistical analysis indicates significant variation between regions. More recently constructed homes contain higher radon versus older. Finally, I also designed and validated a benchtop, 96 well plate-based 241Am irradiation system to expose cultured eukaryotic cells to alpha particles in a controlled environment. My validation of this novel setup includes quantification of nuclear alpha particle-induced DNA damage signalling (γH2AX) using a purpose-designed 3D analysis method, physical readouts of alpha particle-induced DNA damage by alkaline comet assay, and an investigation of cellular viability after alpha particle exposure. This method brings significant advances over existing techniques in its ease of setup and use, affordability, accessibility and flexibility and should enable future alpha particle radiation biology. Collectively, my work demonstrates that radon is a genuine public health concern in the Canadian Prairies, legitimatizes efforts to understand the consequences of radon exposure to the public, and suggest that radon testing and mitigation is likely to be an impactful cancer prevention strategy.
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    ATM and the cellular response to ionizing radiation-induced DNA damage
    (2005) Goodarzi, Aaron A.; Lees-Miller, Susan P.
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    CHD6 and the oxidative stress-induced DNA damage response
    (2018-06-21) Moore, Shaun; Goodarzi, Aaron A.; Lees-Miller, Susan; Cobb, Jennifer A.; Chan, Jennifer A. W.; Downs, Jessica A.; Kurz, Ebba U.
    Oxidative stress-induced DNA damage is a threat to the health and survival of a cell. Oxidative DNA damage responses involve nucleosome displacement, exchange or removal by ATP-dependent chromatin remodeling enzymes to promote DNA repair and transcriptional events. The CHD6 (Chromodomain, Helicase, DNA-binding 6) chromatin remodeling enzyme has been identified as an interactor of NFE2-related factor 2 (NRF2), a key transcription factor in the oxidative stress response. Several human ataxias have linkage map regions that encompass the CHD6 gene locus (20q11.1-12), while catalytically-inactive CHD6 mutant mice exhibit motor coordination defects most consistent with a cerebellar neuron disorder. Here, I describe a role for CHD6 in the response to oxidative stress-induced DNA damage. CHD6 relocates rapidly to DNA damage caused by microirradiation or KillerRed-induced oxidative stress, but not to enzyme-induced DNA double strand breaks. CHD6 interacts with poly ADP-ribose (PAR), and retention at laser microirradiation-induced DNA damage sites is PAR polymerase-dependent and prolonged by PAR Glycohydrolase depletion. I have narrowed down the PAR-interaction region of CHD6 to the extreme N-terminus and, using laser microirradiation, I have demonstrated the importance of the double chromodomain and a putative DNA-binding domain for normal recruitment to DNA-damage tracks. CHD6 protein levels are stabilized following H2O2 exposure via suppressed proteolytic degradation. Ablation of CHD6 in A549 cells using CRISPR-Cas9 led to elevated reactive oxygen species levels, created an impaired antioxidant response and a reduced ability to survive or proliferate following chronic H2O2 exposure. CHD6-deleted cells displayed elevated γH2AX and 53BP1 foci, increased ATM auto-phosphorylation and a hypersensitive G2/M checkpoint after exposure to ionizing radiation or H2O2. In conclusion, my data identify CHD6 as a novel responder to oxidative stress-induced DNA damage.
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    Exploring brain cancer pathogenesis in a novel mouse model of glioblastoma (GBM)
    (2018-06-08) Bohm, Alexandra Katerina; Cairncross, J. Gregory; Goodarzi, Aaron A.; Gallo, Marco; Cobb, Jennifer A.
    The molecular architecture of glioblastoma (GBM) has been well documented, but the cause of the disease, especially the primary adult form, remains elusive. Although it has been hypothesized that amplification of platelet-derived growth factor A (PDGFA) and p53 mutations are early alterations, how these orchestrate tumour initiation has yet to be discerned. Considering the poor prognosis and minimal benefit received from current treatment, understanding the early events of tumour initiation may be the only way to find more effective or preventative treatment strategies. It is well known that p53 has multiple functions as a tumour suppressor, but the outcome of its loss and the role this aberration plays in GBM remains unknown. Similarly, as a growth factor, PDGFA is hypothesized to promote rapid proliferation leading to cellular transformation, but this has yet to be demonstrated or modelled in GBM. Using a mouse model of GBM developed in the Cairncross lab, I show that, unlike most growth factors, subventricular zone (SVZ) cells struggle to proliferate in PDGFA. When p53 is present, PDGFA-cultured SVZ cells undergo apoptosis, eventually resulting in a total loss of cell culture viability. When p53 is absent, cells undergo a crisis-like period, and eventually transform. Detailed characterization of the crisis period in PDGFA demonstrates the cells undergo abnormal mitosis, likely due to abnormal centrosome numbers, and sustain gross chromosomal alterations that lead to alterations in pathways similar to those observed in human GBM. These analyses clarify how PDGFA and the absence of p53 promote gliomagenesis: PDGFA supports attenuated proliferation concurrent to inducing chromosomal instability, while the absence of p53 enables cells to evade apoptosis and continue through the crisis period until transformation is achieved.
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    Investigating radiosensitivity and DNA repair in different neural cell types
    (2020-05-12) Berger, Nelson Daniel; Chan, Jennifer A. W.; Goodarzi, Aaron A.; Lees-Miller, Susan P.; Yong, Voon Wee
    Cranial radiotherapy (CRT) is an important and effective treatment for childhood brain cancers, but is also strongly associated with persistent neurocognitive decline, a heightened risk of secondary radiation-induced malignancies and altered white matter developmental trajectory. These late effects of CRT increase in severity the younger a patient is treated. Despite substantial advances in radiotherapy planning and treatment, late effects remain an issue to the ever-increasing group of childhood cancer survivors. The cellular and molecular mechanisms of these late effects are largely unknown, and the subtleties of how a developing, pediatric brain interacts with the ionizing radiation (IR) requires further investigation. This study aimed to characterize how distinct neural lineages present in a developing brain respond to IR, and how these cells repair DNA damage incurred by IR. Cells of the oligodendrocyte lineage, and particularly, oligodendrocyte progenitor cells (OPCs), exhibit marked radiosensitivity relative to other neural cell types, both post-mitotic and replicative. These OPCs are sensitive to a single dose of IR as low as 0.25 Gy, and display impaired DNA double-strand break (DSB) repair kinetics with a high baseline level of accumulated DSBs. While high in reactive oxygen species, modulation of the oxidative stress levels of OPCs is ineffective in rescuing radiosensitivity or DSB levels at rest. Interestingly, OPCs are profoundly sensitive to small molecules that compromise replication fork stability, and have other indicators consistent with aberrant collapsed replication fork processing and repair. Though the reason for this replication phenomenon remains elusive, it may be related to a baseline difficulty engaging HR-directed fork repair and restart mechanisms. Altogether, these data provide cellular mechanisms for the sensitivity of the developing brain to IR. Highly radiosensitive OPCs, more abundant and proliferative in the pediatric brain, might be ablated by IR at a young age. This may contribute in part to the clinical late effects of pediatric cranial radiotherapy. Further understanding of this sensitive cell type might provide an opportunity to protect the developing brain from IR, decrease the severity of late effects of childhood CRT, and provide the safest, most effective treatment for childhood brain cancers.
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    Profound DNA methylomic differences between single- and multi-fraction alpha irradiations of lung fibroblasts
    (2023-10-27) Vera-Chang, Marilyn N.; Danforth, John M.; Stuart, Marilyne; Goodarzi, Aaron A.; Brand, Marjorie; Richardson, Richard B.
    Abstract Background Alpha (α)-radiation is a ubiquitous environmental agent with epigenotoxic effects. Human exposure to α-radiation at potentially harmful levels can occur repetitively over the long term via inhalation of naturally occurring radon gas that accumulates in enclosed spaces, or as a result of a single exposure from a nuclear accident. Alterations in epigenetic DNA methylation (DNAm) have been implicated in normal aging and cancer pathogenesis. Nevertheless, the effects of aberrations in the methylome of human lung cells following exposure to single or multiple α-irradiation events on these processes remain unexplored. Results We performed genome-wide DNAm profiling of human embryonic lung fibroblasts from control and irradiated cells using americium-241 α-sources. Cells were α-irradiated in quadruplicates to seven doses using two exposure regimens, a single-fraction (SF) where the total dose was given at once, and a multi-fraction (MF) method, where the total dose was equally distributed over 14 consecutive days. Our results revealed that SF irradiations were prone to a decrease in DNAm levels, while MF irradiations mostly increased DNAm. The analysis also showed that the gene body (i.e., exons and introns) was the region most altered by both the SF hypomethylation and the MF hypermethylation. Additionally, the MF irradiations induced the highest number of differentially methylated regions in genes associated with DNAm biomarkers of aging, carcinogenesis, and cardiovascular disease. The DNAm profile of the oncogenes and tumor suppressor genes suggests that the fibroblasts manifested a defensive response to the MF α-irradiation. Key DNAm events of ionizing radiation exposure, including changes in methylation levels in mitochondria dysfunction-related genes, were mainly identified in the MF groups. However, these alterations were under-represented, indicating that the mitochondria undergo adaptive mechanisms, aside from DNAm, in response to radiation-induced oxidative stress. Conclusions We identified a contrasting methylomic profile in the lung fibroblasts α-irradiated to SF compared with MF exposures. These findings demonstrate that the methylome response of the lung cells to α-radiation is highly dependent on both the total dose and the exposure regimen. They also provide novel insights into potential biomarkers of α-radiation, which may contribute to the development of innovative approaches to detect, prevent, and treat α-particle-related diseases. Graphical abstract
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    The Role of Telomere Clustering and Compaction in Cellular Senescence
    (2020-06-02) Adam, Nanda Johanna; Riabowol, Karl T.; Beattie, Tara L.; Goodarzi, Aaron A.; Shutt, Timothy E.; Patel, Kamala D.; Autexier, Chantal
    Chronological age is highly correlated with the development of numerous diseases, including cancer. Thus, delaying the onset of cellular aging should increase healthspan and reduce the period of morbidity that is seen as one approaches maximal lifespan. One of the main contributors to age-related diseases is replicative senescence, which is induced by a telomere-specific DNA damage response. This highly regulated pathway is modulated by an interplay between telomere length and shelterin proteins, such as Telomere Repeat-binding Factor 2 (TRF2). Since telomeres shorten with each cell division, telomere length has been used as a biomarker for cellular aging. However, many cancer cells display short telomeres despite their continued replicative capability. This indicates that cells can extend their cellular lifespan with relatively short telomeres if other factors are in place. Here, we have developed a method for 3D quantitative immunofluorescence microscopy of telomeres in interphase cells that includes image acquisition, processing and analysis to accurately determine telomere count, length and volume. Using this technique, we observed that long telomeres in telomerase-immortalized cells as well as DNA damage positive-telomeres in replicatively senescent cells can cluster, resulting in lower telomere counts. Analysis of individual telomeres by super-resolution microscopy techniques showed that telomeres in senescent cells have significantly more damage foci and are less dense when compared to low passage replicating cells. Alternatively, cell strains with short telomeres, but elevated TRF2 levels, showed more telomere compaction which correlated with an absence of DNA damage responses and increased replicative lifespan. These results show that telomere compaction, rather than average telomere length, may be more indicative of both cellular senescence and immortality, and provides novel insights into the key mechanisms involved in the development of age-related diseases due to the accumulation of senescent cells.