| 1. |
- Bai, Ming, et al.
(författare)
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ARFGAP1 promotes AP-2-dependent endocytosis
- 2011
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Ingår i: Nature Cell Biology. - : Springer Science and Business Media LLC. - 1465-7392 .- 1476-4679. ; 13:5, s. 559-U144
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Tidskriftsartikel (refereegranskat)abstract
- COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.
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| 2. |
- Bräutigam, Lars, et al.
(författare)
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Hypoxic Signaling and the Cellular Redox Tumor Environment Determine Sensitivity to MTH1 Inhibition
- 2016
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 76:8, s. 2366-2375
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Tidskriftsartikel (refereegranskat)abstract
- Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1 alpha response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance.
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| 3. |
- Carter, Megan, et al.
(författare)
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Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2
- 2015
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.
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| 4. |
- Gad, Helge, et al.
(författare)
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MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool
- 2014
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 508:7495, s. 215-221
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Tidskriftsartikel (refereegranskat)abstract
- Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
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| 5. |
- Gad, Helge
(författare)
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Synaptic vesicle endocytosis studied in a living synapse
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neurons in the central nervous system communicate by transmitting electrical signals at special sites of contact called synapses. Synapses contain neurotransmitter-filled synaptic vesicles, which fuse with the presynaptic plasma membrane. The neurotransmitter diffuses to the neighboring neuron and triggers a new electrical signal. In order to maintain synaptic transmission, the synaptic vesicle membrane must be retrieved from the presynaptic plasma membrane to be reused for another round of transmitter release. In one proposed model, the synaptic vesicles fuse completely with the plasma membrane and the vesicle membrane is retrieved through clathrin-coated buds. In an alternative model, the synaptic vesicle connects transiently to the plasma membrane and clathrin is not involved in the membrane retrieval. Clathrin-mediated endocytosis depends on a number of accessory proteins that appear to assist the clathrin-coated intermediate at different steps in the process. The aim of this thesis was to investigate the role of clathrin-coated buds in synaptic vesicle retrieval. The function of the accessory proteins endophilin, dynamin, amphiphysin and synaptojanin in clathrin-mediated endocytosis was also examined. For this purpose, the giant reticulospinal synapse in lamprey was employed. The organization of this synapse allows acute perturbations of synaptic vesicle recycling. The following conclusions were drawn from the experiments presented in this thesis: * Impairment of several clathrin-associated proteins led to an inhibition of synaptic vesicle endocytosis and accumulation of clathrin coated intermediates. This suggests that the main pathway for synaptic vesicle retrieval is through clathrin-mediated endocytosis. * After temporally dissociating synaptic vesicle release from endocytosis, clathrin-mediated retrieval could be initiated by readding low micromolar concentrations of extracellular Ca2+, without additional action potential stimulation. The results indicate that the synaptic vesicle membrane incorporated in the plasma membrane during exocytosis is a sufficient trigger of synaptic vesicle endocytosis. * Antibody-mediated disruption of endophilin led to a massive accumulation of shallow clathrin-coated pits on the presynaptic plasma membrane. This suggests that endophilin is required for the invagination of the shallow coated pit, possibly by altering the lipid composition of the coated membrane bud. * Microinjection of compounds that disrupt the binding of dynamin to amphiphysin and endophilin blocked clathrin-mediated retrieval at the stage of deeply invaginated coated buds with a constricted neck. These data indicate that dynamin and its interaction with amphiphysin and endophilin is essential in the fission of the neck of the coated pit. * Injection of a peptide blocking the endophilin-synaptojanin interaction and antibodies to the proline-rich domain of synaptojanin induced accumulation of free clathrin-coated vesicles. These results suggest that synaptojanin is recruited to the free coated vesicle by the interaction with endophilin to facilitate the removal of the clathrin coat. Endophilin may be a part of a molecular switch that couples the fission reaction to uncoating and imparts a vectorality to synaptic vesicle endocytosis. * Dynamin was shown to be associated with both early and late stages of coated pits. Antibody-mediated disruption of dynamin blocked synaptic vesicle endocytosis at a stage preceding clathrin coat formation. These results indicate that dynamin participates at early stages of endocytosis in addition to its role in fission. The results presented in this thesis have increased the understanding of how synaptic vesicles are retrieved and the molecular mechanisms that govern clathrin-mediated endocytosis, a process important in all animal cells to regulate their response to the external environment.
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| 6. |
- Pudelko, Linda, et al.
(författare)
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Glioblastoma and glioblastoma stem cells are dependent on functional MTH1
- 2017
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Ingår i: Oncotarget. - : Impact Journals LLC. - 1949-2553. ; 8:49, s. 84671-84684
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Tidskriftsartikel (refereegranskat)abstract
- Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease.
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| 7. |
- Sanjiv, Kumar, et al.
(författare)
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MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia
- 2021
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Ingår i: Cancer Research. - : American Association For Cancer Research (AACR). - 0008-5472 .- 1538-7445. ; 81:22, s. 5733-5744
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Tidskriftsartikel (refereegranskat)abstract
- Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin−CD34+CD38−), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial.
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| 8. |
- Visnes, Torkild, et al.
(författare)
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Targeting OGG1 arrests cancer cell proliferation by inducing replication stress
- 2020
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:21, s. 12234-12251
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Tidskriftsartikel (refereegranskat)abstract
- Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
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