Browsing by Subject "ATM"

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Characterization of proteostasis loss in neurodegeneration using a proteomics approach
(2021-05-11) Ryu, Seung Woo; Paull, Tanya T.; Huibregtse, Jon M.; Johnson, Arlen W.; Matouschek, Andreas; Wilk, Claus O.; Marcotte, Edward M.
Chaperones such as HSP70s are the key proteins in the maintenance of protein homeostasis. They maintain protein balance from translation of nascent protein to degradation of misfolded proteins and aggregates. Therefore, chaperone–client interactions provide insights into protein homeostasis within cells. Here I show that chaperone-client interactions can be deciphered using ubiquitin-activated interaction traps (UBAITs). Using LC/MS label-free proteomics together with HSP/HSC70 UBAITs, I have identified both chaperone clients and their co-chaperones with higher confidence compared to traditional IP-LC/MS. The results demonstrated that HSP70/HSC70, despite their similarity, have non-overlapping sets of clients. Unstable subunits of a large protein complex are often recognized by either HSP70 or HSC70 but not both. Most importantly, HSP70/HSC70 UBAITs were sensitive enough to monitor change in chaperone-client interaction landscape caused by the presence of neurotoxic protein SOD1(A4V), demonstrating their application in the future studies of neurodegenerative disorders. Neurodegenerative disorders are a collection of diseases that are characterized by progressive death of neurons where, for most cases, the causes are still unknown. However, loss of protein homeostasis and an increase in protein aggregation are common hallmarks for neurodegenerative disorders. Ataxia-telangiectasia (A-T) is a rare neurodegenerative disorder that is caused by a mutation in the ATM gene. A-T patients suffer from a loss of cerebellum-specific neurons, causing early-onset ataxia. The A-T neurodegenerative disease, which was previously thought to be unrelated to other age-related neurodegenerative disorders, also has a similar protein homeostasis loss. Similar to other forms of neurodegenerative disorders, the cerebellum of A-T patients also shows widespread protein aggregation in cerebellum tissues. The loss of protein homeostasis in A-T is driven by an increase in poly-(ADP- ribosylation), caused by an increase in transcription-dependent single-strand DNA damage (SSBs) via an increase in formation of R-loops in the absence of ATM. Additionally, the protein homeostasis loss was limited to the cerebellum, further demonstrating that protein homeostasis loss correlates with neurodegeneration. These findings provide new methods that could be used to further our understanding of a relationship between protein homeostasis loss and neurodegeneration while providing important insights into how protein aggregation occurs in A-T neurodegenerative disorders.
Homeodomain proteins directly regulate ATM kinase activity
(2018-02-07) Johnson, Tanya Ellen; Paull, Tanya T.; Huibregtse, Jon; Lambowitz, Alan; Russell, Rick; Iyer, Vishy
The ataxia-telangiectasia mutated (ATM) kinase is a master regulator involved in the detection and repair of DNA double-strand breaks (DSBs). The Mre11/Rad50/Nbs1 (MRN) complex recruits and activates ATM at DSBs, causing a signaling cascade to initiate cell-cycle checkpoint arrest, DNA repair, and apoptosis. Alternatively, ATM can be activated by direct oxidation and may act as a redox sensor in the cellular response to oxidative stress. Loss of functional ATM results in ataxia-telangiectasia, a genomic instability disorder characterized by neurodegeneration, DNA repair defects, and predisposition to cancer. Understanding how ATM kinase activity is regulated is critical to understanding its function in the DNA damage and oxidative stress responses. Recently, ATM kinase activity was shown to be stimulated directly by a homeodomain protein, NKX3.1, in prostate cells. NKX3.1 is thought to be the gatekeeper to prostate tumor suppression and, although normally expressed solely in prostate cells, as many as 80% of prostate tumors exhibit loss of NKX3.1 protein. One mechanism of tumor suppression by NKX3.1 may be modulating the DNA damage response via ATM. In in vitro kinase assays, NKX3.1 protein was sufficient to stimulate ATM both in the MRN-mediated and oxidative pathways. Thus, NKX3.1 acts as a tissue-specific modulator of ATM function. Since the conserved homeodomain was critical for the stimulation of ATM by NKX3.1, it poses the question whether this is a general mechanism of ATM regulation by all homeodomain proteins. Here, I address this question by identifying other tissue-specific regulators of ATM activity within the homeodomain family of proteins. Five homeodomain proteins (TTF1, HOXB7, NKX2.5, NKX2.2, and CDX2) were tested for the ability to regulate ATM activity. In in vitro kinase assays, all of the homeodomain proteins tested stimulated ATM kinase activity to varying degrees, suggesting that the homeodomain itself may act as a conserved regulator of ATM function. I show that CDX2 modulates ATM function in mammalian cell lines with global effects on the DNA damage response. Together, these data support the hypothesis that homeodomain proteins regulate ATM function in a tissue-specific manner.
Mechanisms and consequences of ATM activation
(2015-05) Mand, Michael Rodgers; Paull, Tanya T.; Dalby, Kevin; Huibregtse, Jon; Marcotte, Edward; Miller, Kyle
Mutations in the ataxia telangiectasia mutated (ATM) gene cause the disease ataxia-telangiectasia (A-T). Patients with this disease have multiple symptoms, including the eponymous ataxia and telangiectasia as well as immunodeficiency, radiation sensitivity, and increased cancer rates. The ATM protein is a kinase and is activated by multiple types of stress to affect many cellular processes. At sites of DNA double-strand breaks (DSBs), ATM is activated by the protein complex Mre11/Rad50/Nbs1 (MRN). As part of this complex, Rad50 binds and hydrolyzes ATP and causes large conformational changes in the complex. However, the importance of this enzymatic activity in the activation of ATM has been unknown. Here I show ATP binding by Rad50 is required for ATM activation while ATP hydrolysis is dispensable. ATM is also activated in the presence of oxidative stress. Separation-of-function mutations for the activation of ATM by DSBs and oxidative stress have been characterized in vitro. Here, the effects of expressing wild-type ATM or ATM with these different separation-of-function mutations in an ATM-deficient lymphoblast cell line have been characterized. Analysis of the proteomes of these cells and a control cell line revealed that non-functional ATM resulted in the loss of a large group of proteins by mass spectrometry. The levels of these proteins were similar in the cells, but in the presence of non-functional ATM they showed increased levels of aggregation. Thus my results suggest ATM may function to prevent aggregation in these conditions. Notably neurodegeneration is often associated with aggregation. In the phosphoproteomes of cells expressing the various ATM constructs, the parental cell line and cells with ATM unable to be activated by oxidative stress had lower levels of phosphopeptides predicted to be phosphorylated by CK2. This decrease in CK2 activity was also associated with increased aggregation, specifically a subunit of CK2 known as CK2β. This work provides insights into the mechanism of ATM activation by MRN and the potential involvement of ATM in the prevention of protein aggregation.
Regulation of DNA damage response by ATM and DNA-PKcs
(2015-12-09) Zhou, Yi, Ph. D.; Paull, Tanya T.; Finkelstein, Ilya J; Iyer, Vishwanath R; Miller, Kyle M; Lee, Seongmin
The 5’ strand resection of DNA double strand breaks (DSBs) initiates homologous recombination (HR) and is critical for genomic stability. To date there is no quantitative method to measure single-stranded DNA (ssDNA) intermediates of resection in mammalian cells. In this study I develop a quantitative PCR (qPCR)-based assay to quantitate ssDNA intermediates, specifically, the 3’ ssDNA product of resection at specific DSBs induced by AsiSI restriction enzyme in human cells. I protect the large mammalian genome from shearing by embedding the cells in low-gelling-point agar during genomic DNA extraction, and measure the levels of ssDNA intermediates by qPCR following restriction enzyme digestion. This assay is more quantitative and precise compared with existing protein foci-based methods. Using this assay I quantitatively measure ssDNA intermediates of resection in human cells and find that the 5' strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle dependent manner. Depletion of CtIP, Mre11, Exo1, or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit (DNA-PKcs) leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of BRCA1 does not, suggesting that the role of BRCA1 in HR is primarily to promote RAD51 filament formation, not to regulate end resection. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, I also show that DNA-PKcs, a classic non-homologous end joining (NHEJ) factor, antagonizes DSB resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. ATM kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11/Rad50/Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation. This work suggests that, in addition to its key role in NHEJ, DNA-PKcs also acts in concert with MRN and ATM to regulate resection and thus DNA repair pathway choice. In addition, I find that MRN strongly suppresses DNA Ligase IV/XRCC4-mediated end rejoining, whereas it dramatically promotes DNA end ligation by the DNA Ligase III/XRCC1 complex. The Ataxia-Telangiectasia mutated (ATM) protein is a key regulator of checkpoint activation and HR in response to DSBs. The MRN complex acts as a DSB sensor and is essential for ATM recruitment to broken DNA ends and ATM activation. However, the precise mechanism for ATM activation upon DNA damage and ATM inactivation after DNA repair has remained poorly understood. Phosphorylation of ATM has been suggested to play important roles in this process. Autophosphorylation of ATM at four sites (S1981, S367, S1893, and S2996) has been shown to be essential for ATM activation and function in response to DNA damage in human cells. However, mutations at these four sites do not affect ATM kinase activity in vitro or in mouse models, suggesting that there are other mechanisms for regulation of ATM activity. Previous studies show that CDK5-mediated phosphorylation of ATM at Ser794 and EGFR-mediated phosphorylation of ATM at Tyr370 both positively regulate ATM activation upon DNA damage. In this study, I further propose that DNA-dependent protein kinase (DNA-PK) negatively regulates ATM activity through phosphorylation of ATM at multiple sites. ATM is hyperactive when DNA-PK activity is blocked by DNA-PK specific inhibitor or when the DNA-PKcs gene is deleted in cells. Pre-incubation of ATM protein with DNA-PK significantly inhibits ATM kinase activity in vitro. Using mass spectrometry analysis and site-directed mutagenesis, I characterized three clusters of sites: S85/T86, T372/T373 and T1985/S1987/S1988. The phospho-mimetic mutations at these residues repress ATM activation both in vitro and in cells. Overexpression of phospho-mimetic ATM mutants in ATM-deficient cells fails to restore cell survival, DSB end resection and G2/M checkpoint activation in response to DNA damage. In addition, I have observed that the phospho-blocking ATM mutants, T1985A/S1987A/S1988A and T86A/T373A, are resistant to DNA-PK pre-incubation in vitro and that overexpression of ATM T1985A/S1987A/S1988A mutant fails to respond to DNA-PK inhibition in comparison to wild-type ATM in cells. Taken together, my data suggests that the NHEJ factor DNA-PK suppresses the catalytic activity of ATM through phosphorylation and that the phosphorylation of a group of Ser/Thr residues on ATM negatively regulates ATM signaling upon DNA damage. Since ATM is known to promote the HR pathway, this may provide a novel mechanism for DNA repair pathway choice upon DNA damage as well as ATM inactivation after DNA repair.