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| 3. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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- Maurel, M., et al.
(författare)
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Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation
- 2019
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Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4676 .- 1757-4684. ; 11:6
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Tidskriftsartikel (refereegranskat)abstract
- Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.
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- 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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- 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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| 7. |
- Almanza, A., et al.
(författare)
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Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications
- 2019
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Ingår i: Febs Journal. - : Wiley. - 1742-464X. ; 286:2, s. 241-278
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Tidskriftsartikel (refereegranskat)abstract
- The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.
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| 8. |
- 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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| 9. |
- 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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| 10. |
- Fessart, D., et al.
(författare)
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Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties
- 2016
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Ingår i: Elife. - 2050-084X. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.
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