Detection of Protein–Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway

Ristić, Marko; Brockly, Frédérique; Piechaczyk, Marc; Bossis, Guillaume

doi:10.1007/978-1-4939-3756-1_17

Cited by 26 publications

(24 citation statements)

References 15 publications

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“…To visualise the interaction between endogenous NLRP3 and SUMO-2/-3 in intact WT BMDMs, we applied in situ proximity ligation assay (PLA). In addition to detecting protein-protein interactions 27 and inflammasome activation 28 – 30 , this method has been described for the study of PTMs, specifically sumoylation 31 . Using primary antibodies against NLRP3 and SUMO-2/-3, which in turn were recognised by oligonucleotide-coupled secondary antibodies, we obtained discrete proximity labelling of NLRP3 and SUMO-2/-3 under unstimulated conditions (Fig.…”

Section: Resultsmentioning

confidence: 99%

SUMO-mediated regulation of NLRP3 modulates inflammasome activity

et al. 2018

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The NLRP3 inflammasome responds to infection and tissue damage, and rapidly escalates the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. How the NLRP3 inflammasome is negatively regulated is poorly understood. Here we show that NLRP3 inflammasome activation is suppressed by sumoylation. NLRP3 is sumoylated by the SUMO E3-ligase MAPL, and stimulation-dependent NLRP3 desumoylation by the SUMO-specific proteases SENP6 and SENP7 promotes NLRP3 activation. Defective NLRP3 sumoylation, either by NLRP3 mutation of SUMO acceptor lysines or depletion of MAPL, results in enhanced caspase-1 activation and IL-1β release. Conversely, depletion of SENP7 suppresses NLRP3-dependent ASC oligomerisation, caspase-1 activation and IL-1β release. These data indicate that sumoylation of NLRP3 restrains inflammasome activation, and identify SUMO proteases as potential drug targets for the treatment of inflammatory diseases.

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Section: Resultsmentioning

confidence: 99%

SUMO-mediated regulation of NLRP3 modulates inflammasome activity

et al. 2018

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“…To validate the findings that SUMO placental isoforms interact with cytokeratin-7 (Ct-7), placental JAR cells were subjected to a proximity ligation assay (Ristic et al 2016). JAR cells were grown overnight on chamber slides (Lab Tek, Thermo Fisher Scientific) to 50% confluency.…”

Section: Proximity Ligation Assaymentioning

confidence: 99%

Spatiotemporal distribution of small ubiquitin‐like modifiers during human placental development and in response to oxidative and inflammatory stress

Baczyk

Audette

Coyaud

et al. 2018

The Journal of Physiology

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Key points The post‐translational modification of target proteins by SUMOylation occurs in response to stressful stimuli in a variety of organ systems.Small ubiquitin‐like modifier (SUMO) isoforms 1–4 have recently been identified in the human placenta, and are upregulated in the major obstetrical complication of pre‐eclampsia.This is the first study to characterize the spatiotemporal distribution of SUMO isoforms and their targets during placental development across gestation and in response to stress induced by pre‐eclampsia and chorioamnionitis.Keratins were identified as major targets of placental SUMOylation. The interaction with SUMOs and cytoskeletal filaments provides evidence for SUMOylation possibly contributing to underlying dysfunctional trophoblast turnover, which is a hallmark feature of pre‐eclampsia.Further understanding the role of individual SUMO isoforms and SUMOylation underlying placental dysfunction may provide a target for a novel therapeutic candidate as an approach for treating pre‐eclampsia complicated with placental pathology. AbstractSUMOylation is a dynamic, reversible post‐translational modification that regulates cellular protein stability and localization. SUMOylation occurs in response to various stressors, including hypoxia and inflammation, features common in the obstetrical condition of pre‐eclampsia. SUMO isoforms 1–4 have recently been identified in the human placenta, but less is known about their role in response to pre‐eclamptic stress. We hypothesized that SUMOylation components have a unique spatiotemporal distribution during placental development and that their subcellular localization can be further modulated by extra‐cellular stressors. Placental SUMO expression was examined across gestation. First‐trimester human placental explants and JAR cells were subjected to hypoxia or TNF‐α cytokine, and subcellular translocation of SUMOs was monitored. SUMOylation target proteins were elucidated using mass spectrometry and proximity ligation assay. Placental SUMO‐1 and SUMO‐4 were restricted to villous cytotrophoblast cells in first trimester and syncytium by term, while SUMO‐2/3 staining was evenly distributed throughout the trophoblast across gestation. In placental villous explants, oxidative stress induced hyperSUMOylation of SUMO‐1 and SUMO‐4 in the syncytial cytoplasm, whereas SUMO‐2/3 nuclear expression increased. Oxidative stress also upregulated cytoplasmic SUMO‐1 and SUMO‐4 protein expression (P < 0.05), similar to pre‐eclamptic placentas. Keratins were identified as major targets of placental SUMOylation. Oxidative stress increased the cytokeratin‐7 to SUMO‐1 and SUMO‐4 interactions, while inflammatory stress increased its interaction with SUMO‐2/3. Overall, SUMOs display a unique spatiotemporal distribution in normal human placental development. Our data indicate SUMOylation in pre‐eclampsia, which may impair the stability of cytoskeleton filaments and thus promote trophoblast shedding into the maternal circulation in this condition.

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“…The amplified DNA copies can be further detected through hybridization with fluorescence‐labeled nucleotides and observed using fluorescence microscopy . PLA is an attractive proposition for the identification of PPIs, exhibiting an ability for in situ detection under near‐natural conditions, and it has been applied to investigate the SUMO (small ubiquitin‐like modifier) pathway and in situ detection of inflammasomes . For experimental PPI detection, the major problems are the relatively long time scale and inconsistency between different approaches.…”

Section: Modulation Of Ppismentioning

confidence: 99%

Emerging roles of allosteric modulators in the regulation of protein‐protein interactions (PPIs): A new paradigm for PPI drug discovery

Zhang

2019

Medicinal Research Reviews

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Protein‐protein interactions (PPIs) are closely implicated in various types of cellular activities and are thus pivotal to health and disease states. Given their fundamental roles in a wide range of biological processes, the modulation of PPIs has enormous potential in drug discovery. However, owing to the general properties of large, flat, and featureless interfaces of PPIs, previous attempts have demonstrated that the generation of therapeutic agents targeting PPI interfaces is challenging, rendering them almost “undruggable” for decades. To date, rapid progress in chemical and structural biology techniques has promoted the exploitation of allostery as a novel approach in drug discovery. By attaching to allosteric sites that are topologically and spatially distinct from PPI interfaces, allosteric modulators can achieve improved physiochemical properties. Thus, allosteric modulators may represent an alternative strategy to target intractable PPIs and have attracted intense pharmaceutical interest. In this review, we first briefly introduce the characteristics of PPIs and then present different approaches for investigating PPIs, as well as the latest methods for modulating PPIs. Importantly, we comprehensively review the recent progress in the development of allosteric modulators to inhibit or stabilize PPIs. Finally, we conclude with future perspectives on the discovery of allosteric PPI modulators, especially the application of computational methods to aid in allosteric PPI drug discovery.

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Detection of Protein–Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway

Cited by 26 publications

References 15 publications

SUMO-mediated regulation of NLRP3 modulates inflammasome activity

SUMO-mediated regulation of NLRP3 modulates inflammasome activity

Spatiotemporal distribution of small ubiquitin‐like modifiers during human placental development and in response to oxidative and inflammatory stress

Emerging roles of allosteric modulators in the regulation of protein‐protein interactions (PPIs): A new paradigm for PPI drug discovery

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