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| 2. |
- Risso, V., et al.
(författare)
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Metalloprotease-mediated cleavage of CD95 ligand
- 2023
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Ingår i: FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 290:12, s. 3145-64
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Tidskriftsartikel (refereegranskat)abstract
- CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described.
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| 3. |
- Pelizzari-Raymundo, D., et al.
(författare)
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A novel IRE1 kinase inhibitor for adjuvant glioblastoma treatment
- 2023
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Ingår i: iScience. - 2589-0042. ; 26:5
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Tidskriftsartikel (refereegranskat)abstract
- Inositol-requiring enzyme 1 (IRE1) is a major mediator of the unfolded protein response (UPR), which is activated upon endoplasmic reticulum (ER) stress. Tumor cells experience ER stress due to adverse microenvironmental cues, a stress over-come by relying on IRE1 signaling as an adaptive mechanism. Herein, we report the discovery of structurally new IRE1 inhibitors identified through the structural exploration of its kinase domain. Characterization in in vitro and in cellular models showed that they inhibit IRE1 signaling and sensitize glioblastoma (GB) cells to the standard chemotherapeutic, temozolomide (TMZ). Finally, we demonstrate that one of these inhibitors, Z4P, permeates the blood-brain barrier (BBB), inhibits GB growth, and prevents relapse in vivo when administered together with TMZ. The hit compound disclosed herein satisfies an unmet need for targeted, non-toxic IRE1 inhibitors and our results support the attractiveness of IRE1 as an adjuvant therapeutic target in GB.
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| 4. |
- Amarasinghe, Kosala N., et al.
(författare)
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Sensor dimer disruption as a new mode of action to block the IRE1-mediated unfolded protein response
- 2022
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Ingår i: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 20, s. 1584-1592
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Tidskriftsartikel (refereegranskat)abstract
- The unfolded protein response (UPR) is activated to cope with an accumulation of improperly folded proteins in the Endoplasmic reticulum (ER). The Inositol requiring enzyme 1 alpha (IRE1 alpha) is the most evolutionary conserved transducer of the UPR. Activated IRE1 forms 'back-to-back'-dimers that enables the unconventional splicing of X-box Binding Protein 1 (XBP1) mRNA. The spliced XBP1 (XBP1s) mRNA is translated into a transcription factor controlling the expression of UPR target genes. Herein, we report a detailed in silico screening specifically targeting for the first time the dimer interface at the IRE1 RNase region. Using the database of FDA approved drugs, we identified four compounds (neomycin, pemetrexed, quercitrin and rutin) that were able to bind to and distort IRE1 RNase cavity. The activity of the compounds on IRE1 phosphorylation was evaluated in HEK293T cells and on IRE1 RNase activity using an in vitro fluorescence assay. These analyzes revealed sub-micromolar IC50 values. The current study reveals a new and unique mode of action to target and block the IRE1-mediated UPR signaling, whereby we may avoid problems associated with selectivity occurring when targeting the IRE1 kinase pocket as well as the inherent reactivity of covalent inhibitors targeting the RNase pocket. (C)& nbsp;2022 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.& nbsp;
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| 5. |
- 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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| 6. |
- Langlais, T., et al.
(författare)
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Structural and molecular bases to IRE1 activity modulation
- 2021
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 478:15, s. 2953-2975
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Tidskriftsartikel (refereegranskat)abstract
- The Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative, inflammatory and metabolic pathologies and cancer. As such several drugs altering IRE1 activity were developed that target either catalytic activity and showed some efficacy in preclinical pathological mouse models. In this review, we describe the different drugs identified to target IRE1 activity as well as their mode of action from a structural perspective, thereby identifying common and different modes of action. Based on this information we discuss on how new IRE1-targeting drugs could be developed that outperform the currently available molecules.
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| 7. |
- Mahdizadeh, Sayyed Jalil, et al.
(författare)
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QM/MM Well-Tempered Metadynamics Study of the Mechanism of XBP1 mRNA Cleavage by Inositol Requiring Enzyme 1 alpha RNase
- 2022
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 62:17, s. 4247-4260
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Tidskriftsartikel (refereegranskat)abstract
- A range of in silico methodologies were herein employed to study the unconventional XBP1 mRNA cleavage mechanism performed by the unfolded protein response (UPR) mediator Inositol Requiring Enzyme 1 alpha (IRE1). Using Protein- RNA molecular docking along with a series of extensive restrained/ unrestrained atomistic molecular dynamics (MD) simulations, the dynamical behavior of the system was evaluated and a reliable model of the IRE1/XBP1 mRNA complex was constructed. From a series of well-converged quantum mechanics molecular mechanics well-tempered metadynamics (QM/MM WT-MetaD) simulations using the Grimme dispersion interaction corrected semiempirical parametrization method 6 level of theory (PM6-D3) and the AMBER14SB-OL3 force field, the free energy profile of the cleavage mechanism was determined, along with intermediates and transition state structures. The results show two distinct reaction paths based on general acid-general base type mechanisms, with different activation energies that perfectly match observations from experimental mutagenesis data. The study brings unique atomistic insights into the cleavage mechanism of XBP1 mRNA by IRE1 and clarifies the roles of the catalytic residues H910 and Y892. Increased understanding of the details in UPR signaling can assist in the development of new therapeutic agents for its modulation.
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| 8. |
- Martin, S., et al.
(författare)
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SARS-CoV-2 integral membrane proteins shape the serological responses of patients with COVID-19
- 2021
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Ingår i: Iscience. - : Elsevier BV. - 2589-0042. ; 24:10
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Tidskriftsartikel (refereegranskat)abstract
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has elicited a unique mobilization of the scientific community to develop efficient tools to understand and combat the infection. Like other coronavirae, SARS-CoV-2 hijacks host cell secretory machinery to produce viral proteins that compose the nascent virions; including spike (S), envelope (E), and membrane (M) proteins, the most exposed transmembrane proteins to the host immune system. As antibody response is part of the anti-viral immune arsenal, we investigate the immunogenic potential of S, E, and M using a human cell-based system to mimic membrane insertion and N-glycosylation. Both S and M elicit specific Ig production in patients with SARS-CoV-2. Patients with moderate and severe diseases exhibit elevated Ig responses. Finally, reduced Ig binding was observed with spike G614 compared to D614 variant. Altogether, our assay points toward an unexpected immune response against M and represents a powerful tool to test humoral responses against actively evolving SARS-CoV-2 variants and vaccine effectiveness.
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| 9. |
- 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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| 10. |
- Pelizzari Raymundo, D., et al.
(författare)
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Structure-Based Drug Discovery of IRE1 Modulators
- 2022
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Ingår i: The Unfolded Protein Response. - New York, NY : Springer US. - 1064-3745.
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Bokkapitel (refereegranskat)abstract
- IRE1α (inositol-requiring enzyme 1 alpha, referred to IRE1 hereafter) is an Endoplasmic Reticulum (ER) resident transmembrane enzyme with cytosolic kinase/RNAse activities. Upon ER stress IRE1 is activated through trans-autophosphorylation and oligomerization, resulting in a conformational change of the RNase domain, thereby promoting two signaling pathways: i) the non-conventional splicing of XBP1 mRNA and ii) the regulated IRE1-dependent decay of RNA (RIDD). IRE1 RNase activity has been linked to diverse pathologies such as cancer or inflammatory, metabolic, and degenerative diseases and the modulation of IRE1 activity is emerging as an appealing therapeutic strategy against these diseases. Several modulators of IRE1 activity have been reported in the past, but none have successfully translated into the clinics as yet. Based on our expertise in the field, we describe in this chapter the approaches and protocols we used to discover novel IRE1 modulators and characterize their effect on IRE1 activity. © 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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