| 1. |
- Carnero Corrales, Marjorie A., et al.
(författare)
-
Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin
- 2021
-
Ingår i: Cell Chemical Biology. - : Elsevier. - 2451-9456 .- 2451-9448. ; 28:12, s. 1750-1757.e5
-
Tidskriftsartikel (refereegranskat)abstract
- Signaling pathways are frequently activated through signal-receiving membrane proteins, and the discovery ofsmall molecules targeting these receptors may yield insights into their biology. However, due to their intrinsicproperties,membrane protein targets often cannot be identified bymeans of established approaches, in particularaffinity-based proteomics, calling for the exploration of new methods. Here, we report the identification ofindophagolin as representative member of an indoline-based class of autophagy inhibitors through a targetagnosticphenotypic assay. Thermal proteome profiling and subsequent biochemical validation identified thepurinergic receptor P2X4 as a target of indophagolin, and subsequent investigations suggest that indophagolintargets further purinergic receptors. These results demonstrate that thermal proteome profiling may enable thede novo identification of membrane-bound receptors as cellular targets of bioactive small molecules.
|
|
| 2. |
- Konstantinidis, Georgios, et al.
(författare)
-
Identification of novel autophagy inhibitors via cell-based high-content screening
- 2019
-
Ingår i: Autophagy in differentiation and tissue maintenance. - New York, NY : Humana Press. - 9781493987474 - 9781493993826 - 9781493987481 ; , s. 187-195
-
Bokkapitel (refereegranskat)abstract
- Autophagy is a fundamental cellular catabolic pathway mediating the recycling of cellular components. Autophagy has been implicated in pathogenesis of diverse diseases such as neurodegeneration and cancer. Due to the therapeutic potential, the autophagy-modulating agents have profoundly enriched the spectrum of tools used to investigate autophagy. However, many of these compounds have additional off-target effects that may confound elucidation of autophagy in certain contexts. There remains high demand for highly specific and novel chemotypes that can be used to study the regulation mechanism of autophagy and contribute novel pharmacophores for therapeutic purposes. Here, we describe a cell-based quantitative high-content screening (HCS) for autophagy inhibitors using a human breast adenocarcinoma MCF7 cell line stably expressing EGFP-LC3, a bona fide marker of autophagy.
|
|
| 3. |
- Konstantinidis, Georgios, et al.
(författare)
-
Regulation of Myosin Light Chain Function by BMP Signaling Controls Actin Cytoskeleton Remodeling
- 2011
-
Ingår i: Cellular Physiology and Biochemistry. - : S. Karger AG. - 1015-8987 .- 1421-9778. ; 28:5, s. 1031-1044
-
Tidskriftsartikel (refereegranskat)abstract
- Background/Aims: Actin cytoskeleton dynamics support and coordinate signaling events that control cell proliferation, differentiation and migration. Growth factors provide essential signals that act on multi-protein complexes that regulate actin assembly with myosin. We previously analyzed the action of the transforming growth factor β (TGF-β) and now extend our studies to the bone morphogenetic protein (BMP) 7, an important regulator of stem cell function and bone differentiation. Methods: Using a well-established cell model of actin dynamics, Swiss3T3 fibroblasts, we applied cell biological and biochemical approaches to monitor the pathway that links the BMP-7 receptors to the acto-myosin complex. Results: We demonstrate that BMP-7 induces actin and focal adhesion remodeling in starved fibroblasts as potently as TGF-β. BMP-7 mediates changes of actin dynamics via the kinase ROCK1 and induces rapid activation of RhoA and RhoB with concomitant inactivation of Cdc42. These molecular events correlate well with induction of phosphorylation on Ser19 of the myosin light chain, but not with LIMK1 kinase activation. Depletion of endogenous myosin light chain inhibits actin remodeling induced by BMP-7. This novel pathway regulates fibroblast migration without affecting cell proliferation. Conclusion: We establish a BMP-Rho-ROCK1 pathway, which targets myosin light chain to control actin remodeling in fibroblasts.
|
|
| 4. |
- Konstantinidis, I., et al.
(författare)
-
Sources of tolerance towards corrupted politicians in Greece: the role of trade offs and individual benefits
- 2013
-
Ingår i: Crime Law and Social Change. - : Springer Science and Business Media LLC. - 0925-4994 .- 1573-0751. ; 60:5, s. 549-563
-
Tidskriftsartikel (refereegranskat)abstract
- Reelection of corrupted politicians points to a problem of democratic accountability. Voters do have the chance to 'throw the rascals out', but they do not take it. Employing a survey experiment, we test two popular explanations of why Greek voters fail to effectively sanction corrupt politicians. One is related to the distorting effects of psychological attachment to parties and the second to tradeoffs that seem to come into play when voters weigh the prevalence of corruption against other tangible benefits that they receive from governments and parties, such as lower taxes or clientelistic exchanges. Our findings suggest that collective benefits, such as cutting taxes, outweigh the costs of tolerating political corruption. On the contrary, exclusive provision of goods to specific voters, such as in the case of clientelistic exchanges, seems to be negatively related to support for a corrupt politician and therefore should rather not be regarded as a source of tolerance to corruption, at least not in present time Greece.
|
|
| 5. |
- Laraia, Luca, et al.
(författare)
-
Discovery of Novel Cinchona‐Alkaloid‐Inspired Oxazatwistane Autophagy Inhibitors
- 2017
-
Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 56:8, s. 2145-2150
-
Tidskriftsartikel (refereegranskat)abstract
- The cinchona alkaloids are a privileged class of natural products and are endowed with diverse bioactivities. However, for compounds with the closely‐related oxazatricyclo[4.4.0.0]decane (“oxazatwistane”) scaffold, which are accessible from cinchonidine and quinidine by means of ring distortion and modification, biological activity has not been identified. We report the synthesis of an oxazatwistane compound collection through employing state‐of‐the‐art C−H functionalization, and metal‐catalyzed cross‐coupling reactions as key late diversity‐generating steps. Exploration of oxazatwistane bioactivity in phenotypic assays monitoring different cellular processes revealed a novel class of autophagy inhibitors termed oxautins, which, in contrast to the guiding natural products, selectively inhibit autophagy by inhibiting both autophagosome biogenesis and autophagosome maturation.
|
|
| 6. |
- Laraia, Luca, et al.
(författare)
-
The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis
- 2019
-
Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 15:7, s. 710-720
-
Tidskriftsartikel (refereegranskat)abstract
- Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.
|
|
| 7. |
- Pantoom, Supansa, et al.
(författare)
-
RAB33B recruits the ATG16L1 complex to the phagophore via a noncanonical RAB binding protein
- 2021
-
Ingår i: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 17:9, s. 2290-2304
-
Tidskriftsartikel (refereegranskat)abstract
- Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12-ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12-ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12-ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation.
|
|
| 8. |
- Robke, Lucas, et al.
(författare)
-
Discovery of the novel autophagy inhibitor aumitin that targets mitochondrial complex I
- 2018
-
Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 9:11, s. 3014-3022
-
Tidskriftsartikel (refereegranskat)abstract
- Macroautophagy is a conserved eukaryotic process for degradation of cellular components in response to lack of nutrients. It is involved in the development of diseases, notably cancer and neurological disorders including Parkinson's disease. Small molecule autophagy modulators have proven to be valuable tools to dissect and interrogate this crucial metabolic pathway and are in high demand. Phenotypic screening for autophagy inhibitors led to the discovery of the novel autophagy inhibitor aumitin. Target identification and confirmation revealed that aumitin inhibits mitochondrial respiration by targeting complex I. We show that inhibition of autophagy by impairment of mitochondrial respiration is general for several mitochondrial inhibitors that target different mitochondrial complexes. Our findings highlight the importance of mitochondrial respiration for autophagy regulation.
|
|
| 9. |
- Robke, Lucas, et al.
(författare)
-
Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34
- 2017
-
Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 56:28, s. 8153-8157
-
Tidskriftsartikel (refereegranskat)abstract
- Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small‐molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy‐induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.
|
|
