The human glucocorticoid receptor as an RNA-binding protein: global analysis of glucocorticoid receptor-associated transcripts and identification of a target RNA motif.

Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation. We investigated the role of the human GC receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells. Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t(1/2) = 8 ± 1 min versus 62 ± 17 min in DMSO-treated cells) and CCL7 mRNA (t(1/2) = 15 ± 4 min versus 114 ± 37 min), but not that of CCL5 mRNA (t(1/2)=231 ± 8 min versus 266 ± 5 min) in the BEAS-2B cell line. This effect was inhibited by preincubation with an anti-GR Ab, indicating that GR itself plays a role in the turnover of these transcripts. Coimmunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells. Association of the GR was localized to the 5' untranslated region of CCL2 mRNA and further mapped to nt 44-60. The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR. Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro. This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein

Translational control of gene expression via interacting feedback loops

Translation is a key step in the synthesis of proteins. Accordingly, cells have evolved an intricate array of control mechanisms to regulate this process. By constructing a multi-component mathematical framework for translation we uncover how translation may be controlled via interacting feedback loops. Our results reveal that this interplay gives rise to a remarkable range of protein synthesis dynamics, including oscillations, step-change and bistability. This suggests that cells may have recourse to a much richer set of control mechanisms than was previously understood.Comment: Supplementary Material Available on Reques

Chemical Cascading Between Polymersomal Nanoreactor Populations

[EN] Harnessing interactions of functional nano-compartments to generate larger particle assemblies allows studying diverse biological behaviors based on their population states and can lead to the development of smart materials. Herein, thiol-functionalized polymersome nanoreactors are utilized as responsive organelle-like nano-compartments-with inherent capacity to associate into larger aggregates in response to change in the redox state of their environment-to study the kinetics of cascade reactions and explore functions of their collective under different population states. Two nanoreactor populations, glucose oxidase- and horseradish peroxidase-loaded polymersomes, are prepared, and the results of their cascading upon addition of glucose are investigated. The kinetics of resorufin production in associated polymersomes and non-associated polymersome populations are compared, observing a decreased rate upon association. For the associated populations, faster chemical cascading is found when the two types of nanoreactors are associated in a concerted step, as compared to sequential association. The addition of competing agents such as catalase impacts the communication between non-associated polymersomes, whereas such an effect is less pronounced for the associated ones. Altogether, the results showcase the impact of collective associations on enzymatic cascading between organelle-like nanoreactors.Y.A. and A.L.-L. contributed equally to this work. The authors would like to acknowledge the support from the Dutch Ministry of Education, Culture, and Science (Gravitation program 024.001.035 and Spinoza premium) and the ERC Advanced Grant (Artisym 694120).A.L.-L. acknowledges support from the MSCA Cofund project oLife, which has received funding from the European Union's Horizon 2020 research and innovation program under the Grant Agreement 847675; and the Maria Zambrano Program from the Spanish Government funded by NextGenerationEU from the European Union. Dr. Imke Pijpers is thanked for cryo-TEM imaging. Dr. Pascal Welzen is acknowledged for advice and useful discussion on polymer and polymersome preparation.Altay, Y.; Llopis-Lorente, A.; Abdelmohsen, LKEA.; Van Hest, JC. (2023). Chemical Cascading Between Polymersomal Nanoreactor Populations. Macromolecular Chemistry and Physics. 224(1):1-5. https://doi.org/10.1002/macp.20220026915224

Cucurbit-Like Polymersomes with Aggregation-Induced Emission Properties Show Enzyme-Mediated Motility

Polymersomes that incorporate aggregation-induced emission (AIE) moieties are attractive inherently fluorescent nanoparticles with biomedical application potential for cell/tissue imaging and tracking, as well as phototherapeutics. An intriguing feature that has not been explored yet is their ability to adopt a range of asymmetric morphologies. Structural asymmetry allows nanoparticles to be exploited as active (motile) systems. Here, we present the design and preparation of AIE fluorophore integrated (AIEgenic) cucurbit-shaped polymersome nanomotors with enzyme-powered motility. The cucurbit scaffold was constructed via morphology engineering of biodegradable fluorescent AIE-polymersomes, followed by functionalization with enzymatic machinery via a layer-by-layer (LBL) self-assembly process. Because of the enzyme-mediated decomposition of chemical fuel on the cucurbit-like nanomotor surface, enhanced directed motion was attained, when compared with the spherical counterparts. These cucurbit-shaped biodegradable AIE-nanomotors provide a promising platform for the development of active delivery systems with potential for biomedical applications

Formation of Well-Defined, Functional Nanotubes via Osmotically Induced Shape Transformation of Biodegradable Polymersomes

Contains fulltext : 161997.pdf (publisher's version ) (Open Access

Engineering transient dynamics of artificial cells by stochastic distribution of enzymes

Here the authors develop a coacervate micromotor that can display autonomous motion as a result of stochastic distribution of propelling units. This stochastic-induced mobility is validated and explained through experiments and theory. Random fluctuations are inherent to all complex molecular systems. Although nature has evolved mechanisms to control stochastic events to achieve the desired biological output, reproducing this in synthetic systems represents a significant challenge. Here we present an artificial platform that enables us to exploit stochasticity to direct motile behavior. We found that enzymes, when confined to the fluidic polymer membrane of a core-shell coacervate, were distributed stochastically in time and space. This resulted in a transient, asymmetric configuration of propulsive units, which imparted motility to such coacervates in presence of substrate. This mechanism was confirmed by stochastic modelling and simulations in silico. Furthermore, we showed that a deeper understanding of the mechanism of stochasticity could be utilized to modulate the motion output. Conceptually, this work represents a leap in design philosophy in the construction of synthetic systems with life-like behaviors

Guidelines for the use and interpretation of assays for monitoring autophagy (2nd edition)

In 2008 we published the first set of guidelines for standardiz- ing 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 tech- nologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in differ- ent 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..

Design, synthesis and biological assessment of novel N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines and 3-substituted 2,6-dioxopiperidines for TNF-α inhibitory activity

Eight novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithiocarbamates 9 and 10, N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines 11-14 and 3-substituted 2,6-dioxopiperidines 16 and 18 were synthesized as tumor necrosis factor-α (TNF-α) synthesis inhibitors. Synthesis involved utilization of a novel condensation approach, a one-pot reaction involving addition, iminium rearrangement and elimination, to generate the phthalimidine ring required for the creation of compounds 9-14. Agents were, thereafter, quantitatively assessed for their ability to suppress the synthesis on TNF-α in a lipopolysaccharide (LPS)-challenged mouse macrophage-like cellular screen, utilizing cultured RAW 264.7 cells. Whereas compounds 9, 14 and 16 exhibited potent TNF-α lowering activity, reducing TNF-α by up to 48% at 30 μM, compounds 12, 17 and 18 presented moderate TNF-α inhibitory action. The TNF-α lowering properties of these analogs proved more potent than that of revlimid (3) and thalidomide (1). In particular, N-dithiophthalimidomethyl-3-(phthalimidin-2-yl)-2,6-dioxopiperidine 14 not only possessed the greatest potency of the analogs to reduce TNF-α synthesis, but achieved this with minor cellular toxicity at 30 μM. The pharmacological focus of the presented compounds is towards the development of well-tolerated agents to ameliorate the neuroinflammation, that is, commonly associated with neurodegenerative disorders, epitomized by Alzheimer's disease and Parkinson's disease