A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for RME-8 in opioid receptor regulation
mardi 11 octobre 2022 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2022.10.11.511646v1?rss=1
Authors: Novy, B., Adoff, H., De Maria, M., Kampmann, M., Tsvetanova, N., Von Zastrow, M., Lobingier, B.
Abstract:
G protein-coupled receptors (GPCRs) are the largest family of membrane-bound signaling molecules. Activity of these receptors is critically regulated by their trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-protein interactions. Here we present a chemical biology fluorescence-based technique to interrogate GPCR trafficking. We show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). We used this approach to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety, depression, and pain. The screen identified 492 genes including known- and novel-regulators of DOR expression and trafficking. We demonstrate that one of the novel genes, RME-8, localizes to early endosomes and plays a critical role in regulating DOR trafficking to the lysosome. Together, our data demonstrate that GPCR-APEX2/AUR is a flexible and highly sensitive chemical biology platform for genetic interrogation of receptor trafficking.
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FBXL4 deficiency promotes mitophagy by elevating NIX.
mardi 11 octobre 2022 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2022.10.11.511735v1?rss=1
Authors: Elcocks, H., Brazel, A. J., McCarron, K. R., Kaulich, M., Husnjak, K., Mortiboys, H. J., Clague, M. J., Urbe, S.
Abstract:
The selective autophagy of mitochondria is linked to mitochondrial quality control and is critical to a healthy organism. We have conducted a CRISPR/Cas9 screen of human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and following acute mitochondrial depolarisation. We identify two Cullin RING ligases, VHL and FBXL4 as the most profound negative regulators of basal mitophagy. We show that these converge through control of the mitophagy adaptors BNIP3 and BNIP3L/NIX through different mechanisms. FBXL4 suppression of BNIP3 and NIX levels is mediated via direct interaction and protein destabilisation rather than suppression of HIF1-mediated transcription. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study enables a full understanding of the aetiology of early onset mitochondrial encephalomyopathy that is supported by analysis of a disease associated mutation. We further show that the compound MLN4924, which globally interferes with Cullin RING ligase activity, is a strong inducer of mitophagy providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.
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Endosomal Trafficking of Two Pore K+ Efflux Channel TWIK2 to Plasmalemma Mediates NLRP3 Inflammasome Activation and Inflammatory Injury
mercredi 12 octobre 2022 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2022.10.12.511914v1?rss=1
Authors: Di, A., Huang, L. S., Zhou, B., Toth, P. T., Krishnan, Y., Malik, A. B.
Abstract:
Potassium efflux via the two pore K+ channel TWIK2 is a requisite step for the activation of the NLRP3 inflammasome, however it is unclear how the efflux is activated in response to cues. Here we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in extrusion of K+ ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP ligated purinergic receptor P2X7 prevented endosomal fusion with the plasmalemma and K+ efflux and NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-deleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 abundance and activity on the cell surface and downstream activation of the NLRP3 inflammasome. Endosomal trafficking of TWIK2 to the plasmalemma is therefore a potential therapy target in acute or chronic inflammatory states.
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LSR Targets YAP to Modulate Intestinal Paneth Cell Differentiation
lundi 31 octobre 2022 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2022.10.30.514401v1?rss=1
Authors: An, Y., Wang, C., Fan, B., Li, Y., Kong, F., Zhou, C., Cao, Z., Liu, J., Wang, M., Sun, H., Zhao, S., Gong, Y.
Abstract:
Lipolysis-stimulated lipoprotein receptor (LSR) is a multi-functional protein that is best known for its roles in assembly of epithelial tricellular tight junctions and hepatic clearance of lipoproteins. Here, we investigate the function of LSR in intestine biology. By using multiple conditional deletion mouse models and ex vivo cultured organoids, we find that LSR elimination in intestinal stem cells results in disappearance of Paneth cell without affecting the differentiation of other cell lineages. Mechanistic studies reveal that LSR deficiency increase abundance and nuclear localization of YAP by modulating its phosphorylation and proteasomal degradation. Intestinal LSR-deficient mice are susceptible to development of necrotizing enterocolitis. In addition, LSR can sense and interpret fatty acid signals derived from dietary lipids and transduce into inactivation of YAP. Thus, this study identifies LSR as an upstream negative regulator of YAP activity and part of the mechanism mediating connection between fat diet and YAP signaling in intestine.
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Morphogen-driven human iPSCs differentiation in 3D in vitro models of gastrulation is precluded by physical confinement.
mercredi 29 mars 2023 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.03.29.534685v1?rss=1
Authors: Alsehli, H. S., Roy, E., Williams, T., Kuziola, A., Guo, Y., Green, J. B., Gentleman, E., Danovi, D.
Abstract:
In early human development, gastrulation is tightly associated with lineage specification. The interplay between mechanical forces and biochemical signals during these processes is poorly understood. Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model of gastrulation that uses spheroids formed from human induced pluripotent stem cells (hiPSCs). First, we compare self-renewing versus differentiating media conditions in free-floating cultures, and observe the emergence of organised tri-germ layers. In these unconfined cultures, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead. Our study combines high-content imaging, synthetic hydrogels and hiPSCs-derived models of early development to define the drivers causing changes in shape and emergence of germ layers.
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Structural insights into ligand recognition and selectivity of the human hydroxycarboxylic acid receptor HCAR2
mercredi 29 mars 2023 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.03.28.534513v1?rss=1
Authors: pan, x., ye, f., Ning, P., Zhang, Z., Zhang, B., Chen, G., Gao, W., Qiu, C., Wu, Z., Gong, K., Li, J., Xia, J., Du, Y.
Abstract:
Hydroxycarboxylic acid receptor 2 (HCAR2) belongs to the family of class A G-protein-coupled receptors with key roles in regulating lipolysis and free fatty acid formation in humans. It is deeply involved in many pathophysiological processes and serves as an attractive target for the treatment of neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases. Here, we report four cryo-EM structures of human HCAR2-Gi1 complexes with or without agonists, including the drugs niacin and acipimox, and the highly subtype-specific agonist MK-6892. Combined with molecular docking and functional analysis, we have revealed the recognition mechanism of HCAR2 for different agonists and summarized the general pharmacophore features of HCAR2 agonists, which are based on three key residues R1113.36, S17945.52, and Y2847.43. Notably, the MK-6892-HCAR2 structure shows an extended binding pocket relative to other agonist-bound HCAR2 complexes. In addition, the key residues that determine the ligand selectivity between the HCAR2 and HCAR3 are also illuminated. Our findings provide structural insights into the ligand recognition, selectivity, activation, and G protein coupling mechanism of HCAR2, which sheds light on the design of new HCAR2-targeting drugs for greater efficacy, higher selectivity, and fewer or no side effects.
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The novel roles of bovine milk-derived exosomes on skin anti-aging
mercredi 29 mars 2023 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.03.23.532505v1?rss=1
Authors: Ge, X., Lu, L., Bai, W., Wang, M., Han, C., Du, H., Wang, N., Gao, M., Li, D., Dong, F.
Abstract:
Exosomes are small vesicles released from cells and present in various mammal biological fluids, such as bovine milk, which worked for skin care for many years besides dairy. In addition, Exosomes were regarded as a vehicle for intercellular communication. Therefore, we aimed to investigate the novel roles of bovine milk-derived exosomes (MK-Exo) on human skin anti-aging. Purified MK-Exo can be directly uptake by the keratinocytes and fibroblast in vitro and upregulate the expression of the natural factors related to skin moisturizing, including Filaggrin (FLG), Aquaporin 3 (AQP3), CD44 in the keratinocytes and hyaluronidase (HAS2) in the fibroblast, and MK-Exo promoted the cell migration of the fibroblast, while rescue its expression of type I collagen (Col I), type III collagen (Col III) after ultraviolet radiation. Furthermore, the phototoxicity test, photoallergy test, repeated skin irritation test, skin allergy test, and patch test confirm the safety of MK-Exo on the skin. Finally, the roles of MK-Exo in preserving moisture and anti-wrinkle were also identified in humans. Then, MK-Exo was smeared on the facial skin of 31 female volunteers twice a day for 28 days, and the functions were evaluated following the safety assessment in vivo. These studies reveal the novel roles of bovine milk-derived exosomes in human skin aging, which opens a new way of skin care.
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Single-cell transcriptome dataset of human and mouse in vitro adipogenesis models
mercredi 29 mars 2023 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.03.27.534456v1?rss=1
Authors: Li, J., Jin, C., Gustafsson, S., Rao, A., Wabitsch, M., Park, C. Y., Quertermous, T., Bielczyk-Maczynska, E., Knowles, J. W.
Abstract:
Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on human and mouse adipogenesis, as well as on cell-to-cell variability in gene expression during this process.
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A new ferrocene derivative blocks KRAS localization and function by oxidative modification at His95.
mercredi 29 mars 2023 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.03.28.534499v1?rss=1
Authors: Rehl, K. M., Selvakumar, J., Hoang, D., Arumugam, K., Gorfe, A., Cho, K.-J.
Abstract:
Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers (NSCLCs) harboring oncogenic mutant K-Ras. Here, we developed and tested a novel ferrocene derivative on the growth of human pancreatic ductal adenocarcinoma (PDAC) and NSCLC. Our compound inhibited the growth of K-Ras-dependent PDAC and NSCLC and abrogated the PM binding and signaling of K-Ras, but not other Ras isoforms. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified K-Ras His95 residue in the G-domain as being involved in the ferrocene-induced K-Ras PM dissociation via oxidative modification. Together, our studies demonstrate that the redox system directly regulates K-Ras PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced metastasis in K-Ras-driven lung cancers.
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Potential of Local Bacillus spp. Isolates as Wilt Disease Biocontrol Agents for Fusarium (Fusarium oxysporum f. sp. cepae) on Wakegi Onions (Allium x wakegi Araki)
mercredi 29 mars 2023 • Durée
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.03.28.534608v1?rss=1
Authors: Asrul, A.
Abstract:
The use of biological agents as a biocontrol against plant pathogens was often ineffective because it worked slowly. The objective of this research was to examine the potential of local isolates of Bacillus spp. as a biocontrol agent in suppressing Fusarium wilt disease (Fusarium oxysporum f. sp. cepae) on wakegi onions. The research was designed using a completely randomized design with the treatment of rhizosphere bacterial isolates. The treatments consisted of control (without isolate application), isolates KP17, KP5, DB9, DB12, DB18, DG4, and DG11 so that the number of treatments was eight. Each treatment was repeated 5 times and each replication consisted of 10 wakegi onion plants. This research was divided into 2 stages, namely laboratory research which included isolation, characterization of colony morphology of rhizosphere bacterial isolates, and in vitro testing of the inhibitory power of biocontrol agents against pathogens. The test in the greenhouse was in the form of a disease case suppression test. The results obtained seven candidate isolates of biocontrol from 46 isolates obtained from the rhizosphere of the wakegi onion. These isolates had similarities with Bacillus spp. based on colony morphology, physiology, and biochemistry characteristics. Among the isolates found, the DB12 isolate had the potential to be developed as a biocontrol agent compared to other isolates.
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