| 1. |
- Papaioannou, A., et al.
(författare)
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Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs
- 2022
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Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:5
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Tidskriftsartikel (refereegranskat)abstract
- ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs. © 2022 Papaioannou et al.
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| 2. |
- Chintha, C., et al.
(författare)
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Molecular modeling provides a structural basis for PERK inhibitor selectivity towards RIPK1
- 2020
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 10:1, s. 367-375
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Tidskriftsartikel (refereegranskat)abstract
- Protein kinases are crucial drug targets in cancer therapy. Kinase inhibitors are promiscuous in nature due to the highly conserved nature of the kinase ATP binding pockets. PERK has emerged as a potential therapeutic target in cancer. However, PERK inhibitors GSK2606414 and GSK2656157 also target RIPK1 whereas AMG44 is more specific to PERK. To understand the structural basis for the selectivity of PERK ligands to RIPK1 we have undertaken a detailed in silico analysis using molecular docking followed by molecular dynamics simulations to explore the selectivity profiles of the compounds. Although the binding sites of PERK and RIPK1 are similar, their binding response to small molecules is different. The docking models revealed a common binding mode for GSK2606414 and GSK2656157 in the RIPK1 binding site, similar to its cognate ligand. In contrast, AMG44 had a strikingly different predicted binding profile in the RIPK1 binding site with both rigid docking and induced fit docking settings. Our study shows a molecular mechanism responsible for dual targeting by the GSK ligands. More broadly, this work illustrates the potential of molecular docking to correctly predict the binding towards different kinase structures, and will aid in the design of selective PERK kinase inhibitors.
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| 3. |
- Doultsinos, D., et al.
(författare)
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Peptidomimetic-based identification of FDA-approved compounds inhibiting IRE1 activity
- 2021
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Ingår i: Febs Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 288:3, s. 945-960
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Tidskriftsartikel (refereegranskat)abstract
- Inositol-requiring enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease that is a major mediator of the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. Tumour cells experience ER stress due to adverse environmental cues such as hypoxia or nutrient shortage and high metabolic/protein-folding demand. To cope with those stresses, cancer cells utilise IRE1 signalling as an adaptive mechanism. Here, we report the discovery of the FDA-approved compounds methotrexate, cefoperazone, folinic acid and fludarabine phosphate as IRE1 inhibitors. These were identified through a structural exploration of the IRE1 kinase domain using IRE1 peptide fragment docking and further optimisation and pharmacophore development. The inhibitors were verified to have an impact on IRE1 activityin vitroand were tested for their ability to sensitise human cell models of glioblastoma multiforme (GBM) to chemotherapy. We show that all molecules identified sensitise glioblastoma cells to the standard-of-care chemotherapy temozolomide (TMZ).
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| 4. |
- Leclerc, D., et al.
(författare)
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Gene Editing Corrects In Vitro a G > A GLB1 Transition from a GM1 Gangliosidosis Patient
- 2023
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Ingår i: Crispr Journal. - : Mary Ann Liebert Inc. - 2573-1599 .- 2573-1602. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced beta-galactosidase (beta-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of beta-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient's cells, except a bystander edit without consequences on beta-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.
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| 5. |
- Mnich, K., et al.
(författare)
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The stressosome, a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner
- 2021
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Ingår i: Journal of Cellular and Molecular Medicine. - : Wiley. - 1582-1838 .- 1582-4934. ; 25:18, s. 8809-8820
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Tidskriftsartikel (refereegranskat)abstract
- Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the 'stressosome'. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1 beta-converting enzyme-inhibitory protein (cFLIP(L)), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.
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| 6. |
- Moncan, M., et al.
(författare)
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Insights into the structure and function of the RNA ligase RtcB
- 2023
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Ingår i: Cellular and Molecular Life Sciences. - 1420-682X. ; 80:12
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Tidskriftsartikel (refereegranskat)abstract
- To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5 ' -3 ' and 3 '-5 ' RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3 '-5 ' RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3 '-5 ' RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer.
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| 7. |
- Noori, A. R., et al.
(författare)
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Loss of WD2 subdomain of Apaf-1 forms an apoptosome structure which blocks activation of caspase-3 and caspase-9
- 2021
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Ingår i: Biochimie. - : Elsevier BV. - 0300-9084. ; 180, s. 23-29
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Tidskriftsartikel (refereegranskat)abstract
- Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1's nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1 's second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1-921) on Apaf-1 interactions and caspase activation. Apaf-1 1-921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
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| 8. |
- Nunez, J. C. B., et al.
(författare)
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Footprints of natural selection at the mannose-6-phosphate isomerase locus in barnacles
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 117:10, s. 5376-5385
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Tidskriftsartikel (refereegranskat)abstract
- The mannose-6-phosphate isomerase (Mpi) locus in Semibalanus balanoides has been studied as a candidate gene for balancing selection for more than two decades. Previous work has shown that Mpi allozyme genotypes (fast and slow) have different fre-quencies across Atlantic intertidal zones due to selection on post-settlement survival (i.e., allele zonation). We present the complete gene sequence of the Mpi locus and quantify nucleotide polymor-phism in S. balanoides, as well as divergence to its sister taxon Semibalanus cariosus. We show that the slow allozyme contains a derived charge-altering amino acid polymorphism, and both allo-zyme classes correspond to two haplogroups with multiple internal haplotypes. The locus shows several footprints of balancing selec-tion around the fast/slow site: an enrichment of positive Tajima's D for nonsynonymous mutations, an excess of polymorphism, and a spike in the levels of silent polymorphism relative to silent diver-gence, as well as a site frequency spectrum enriched for midfre-quency mutations. We observe other departures from neutrality across the locus in both coding and noncoding regions. These in-clude a nonsynonymous trans-species polymorphism and a recent mutation under selection within the fast haplogroup. The latter suggests ongoing allelic replacement of functionally relevant amino acid variants. Moreover, predicted models of Mpi protein structure provide insight into the functional significance of the putatively selected amino acid polymorphisms. While footprints of selection are widespread across the range of S. balanoides, our data show that intertidal zonation patterns are variable across both spatial and temporal scales. These data provide further evidence for heteroge-neous selection on Mpi.
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| 9. |
- Phuyal, S., et al.
(författare)
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Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1
- 2022
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Ingår i: Embo Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 41:18
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Tidskriftsartikel (refereegranskat)abstract
- Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.
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| 10. |
- Chatgilialoglu, C., et al.
(författare)
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Oxygen Dependent Purine Lesions in Double-Stranded Oligodeoxynucleotides: Kinetic and Computational Studies Highlight the Mechanism for 5 ',8-Cyclopurine Formation
- 2020
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 142:12, s. 5825-5833
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Tidskriftsartikel (refereegranskat)abstract
- The reaction of HO center dot radical with DNA is intensively studied both mechanistically and analytically for lesions formation. Several aspects related to the reaction paths of purine moieties with the formation of 5',8-cyclopurines (cPu), 8-oxopurines (8-oxo-Pu), and their relationship are not well understood. In this study, we investigated the reaction of HO center dot radical with a 21-mer double-stranded oligodeoxynucleotide (ds-ODNs) in gamma-irradiated aqueous solutions under various oxygen concentrations and accurately quantified the six purine lesions (i.e., four cPu and two 8-oxo-Pu) by LC-MS/MS analysis using isotopomeric internal standards. In the absence of oxygen, 8-oxo-Pu lesions are only similar to 4 times more than cPu lesions. By increasing oxygen concentration, the 8-oxo-Pu and the cPu gradually increase and decrease, respectively, reaching a gap of similar to 130 times at 2.01 x 10(-4) M of O-2. Kinetic treatment of the data allows to estimate the C5' radical competition between cyclization and oxygen trapping in ds-ODNs, and lastly the rate constants of the four cyclization steps. Tailored computational studies by means of dispersion-corrected DFT calculations were performed on the CGC and TAT in their double-strand models for each cPu diastereoisomer along with the complete reaction pathways of the cyclization steps. Our findings reveal unheralded reaction mechanisms that resolve the long-standing issues with C5' radical cyclization in purine moieties of DNA sequences.
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