The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. ...Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation, whereas FGF-10 promotes receptor recycling and cell migration. By combining mass-spectrometry-based quantitative proteomics with fluorescence microscopy and biochemical methods, we find that FGF-10 specifically induces the rapid phosphorylation of tyrosine (Y) 734 on FGFR2b, which leads to PI3K and SH3BP4 recruitment. This complex is crucial for FGFR2b recycling and responses, given that FGF-10 stimulation of either FGFR2b_Y734F mutant- or SH3BP4-depleted cells switches the receptor endocytic route to degradation, resulting in decreased breast cancer cell migration and the inhibition of epithelial branching in mouse lung explants. Altogether, these results identify an intriguing ligand-dependent mechanism for the control of receptor fate and cellular outputs that may explain the pathogenic role of deregulated FGFR2b, thus offering therapeutic opportunities.
•Functional proteomics is a powerful tool to uncover ligand-dependent cellular outcomes•FGF-7 and FGF-10 generate opposite FGFR2b trafficking and cellular responses•FGF-10-induced Y734-phosphorylated FGFR2b recruits PI3K and SH3BP4•PI3K and SH3BP4 control FGFR2b recycling and cell migration in response to FGF-10
Abstract The Structural Genomics Consortium is an international open science research organization with a focus on accelerating early-stage drug discovery, namely hit discovery and optimization. We, ...as many others, believe that artificial intelligence (AI) is poised to be a main accelerator in the field. The question is then how to best benefit from recent advances in AI and how to generate, format and disseminate data to enable future breakthroughs in AI-guided drug discovery. We present here the recommendations of a working group composed of experts from both the public and private sectors. Robust data management requires precise ontologies and standardized vocabulary while a centralized database architecture across laboratories facilitates data integration into high-value datasets. Lab automation and opening electronic lab notebooks to data mining push the boundaries of data sharing and data modeling. Important considerations for building robust machine-learning models include transparent and reproducible data processing, choosing the most relevant data representation, defining the right training and test sets, and estimating prediction uncertainty. Beyond data-sharing, cloud-based computing can be harnessed to build and disseminate machine-learning models. Important vectors of acceleration for hit and chemical probe discovery will be (1) the real-time integration of experimental data generation and modeling workflows within design-make-test-analyze (DMTA) cycles openly, and at scale and (2) the adoption of a mindset where data scientists and experimentalists work as a unified team, and where data science is incorporated into the experimental design.
Members of the IL-6 family, IL-6 and ciliary neurotrophic factor (CNTF), have been shown to increase glucose uptake and fatty acid oxidation in skeletal muscle. However, the metabolic effects of ...another family member, leukemia inhibitory factor (LIF), are not well characterized. Effects of LIF on skeletal muscle glucose uptake and palmitate oxidation and signaling were investigated in ex vivo incubated mouse soleus and EDL muscles from muscle-specific AMPKα2 kinase-dead, muscle-specific SOCS3 knockout, and lean and high-fat-fed mice. Inhibitors were used to investigate involvement of specific signaling pathways. LIF increased muscle glucose uptake in dose (50-5,000 pM/l) and time-dependent manners with maximal effects at the 30-min time point. LIF increased Akt Ser(473) phosphorylation (P) in soleus and EDL, whereas AMPK Thr(172) P was unaffected. Incubation with parthenolide abolished LIF-induced glucose uptake and STAT3 Tyr(705) P, whereas incubation with LY-294002 and wortmannin suppressed both basal and LIF-induced glucose uptake and Akt Ser(473) P, indicating that JAK and PI 3-kinase signaling is required for LIF-stimulated glucose uptake. Incubation with rapamycin and AZD8055 indicated that mammalian target of rapamycin complex (mTORC)2, but not mTORC1, also is required for LIF-stimulated glucose uptake. In contrast to CNTF, LIF stimulation did not alter palmitate oxidation. LIF-stimulated glucose uptake was maintained in EDL from obese insulin-resistant mice, whereas soleus developed LIF resistance. Lack of SOCS3 and AMPKα2 did not affect LIF-stimulated glucose uptake. In conclusion, LIF acutely increased muscle glucose uptake by a mechanism potentially involving the PI 3-kinase/mTORC2/Akt pathway and is not impaired in EDL muscle from obese insulin-resistant mice.
Soluble expression of proteins in a relevant form for functional and structural investigations still often remains a challenge. Although many biochemical factors are known to affect solubility, a ...thorough investigation of yield-limiting factors is normally not feasible in high-throughput efforts. Here we present a screening strategy for expression of biomedically relevant proteins in
Escherichia coli using a panel of six different genetic variations. These include engineered strains for rare codon supplementation, increased disulfide bond formation in the cytoplasm and novel vectors for secretion to the periplasm or culture medium. Combining these variants with expression construct truncations design, we report on parallel cloning and expression of more than 300 constructs representing 24 selected proteins; including full-length variants of human growth factors, interleukins and growth factor binding proteins. This rapid screening approach appears highly suitable for high-throughput efforts targeting either large sets of proteins or more focused investigations regarding individual high-profile targets.
S100A4 is a small calcium-binding protein that is commonly overexpressed in a range of different tumor types, and it is widely accepted that S100A4 has an important role in the process of cancer ...metastasis. In vitro binding assays has shown that S100A4 interacts with the tumor suppressor protein p53, indicating that S100A4 may have additional roles in tumor development. In the present study, we show that endogenous S100A4 and p53 interact in complex samples, and that the interaction increases after inhibition of MDM2-dependent p53 degradation using Nutlin-3A. Further, using proximity ligation assay, we show that the interaction takes place in the cell nucleus. S100A4 knockdown experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in stabilization of p53 protein, indicating that S100A4 is promoting p53 degradation. Finally, we demonstrate that S100A4 knockdown leads to p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus, our data add a new layer to the oncogenic properties of S100A4 through its inhibition of p53-dependent processes.
N-terminal extensions (“tags”) have proven valuable for producing peptides using high throughput recombinant expression technologies. However, the applicability is hampered by the limited options for ...specific and efficient proteases to release the fully native sequence without additional amino acids in the N-terminal. Here we describe the Escherichia coli (E. coli) expression, purification and characterization of engineered variants of Xaa-Pro dipeptidyl aminopeptidase (Xaa-Pro-DAP) derived from Lactococcus lactis for cleavage of Gly-Pro dipeptide extension in the N-terminal of glucagon and glucagon-like peptide 1 (GLP-1(7–37)). By single amino acid substitution in the Xaa-Pro-DAP protease, significantly higher product yields were achieved. The combination of HRV14 3C protease and engineered Xaa-Pro-DAP is suggested for obtaining native N-terminal of peptides.
•Engineered Xaa-Pro-DAP is a useful tool in recombinant protein production.•Mutations alter the catalytic properties of the enzyme.•Biophysical properties are largely unaffected.
High quality biological reagents are a prerequisite for pharmacological research. Herein a protein production screening approach, including quality assessment methods, for protein‐based discovery ...research is presented. Trends from 2895 expression constructs representing 253 proteins screened in mammalian and bacterial hosts—91% of which are successfully expressed and purified—are discussed. Mammalian expression combined with the use of solubility‐promoting fusion proteins is deemed suitable for most targets. Furthermore, cases utilizing stable cell line generation and choice of fusion protein for higher yield and quality of difficult‐to‐produce proteins (Leucine‐rich repeat‐containing G‐protein coupled receptor 4 (LGR4) and Neurturin) are presented and discussed. In the case of Neurturin, choice of fusion protein impacted the target binding 80‐fold. These results highlight the need for exploration of construct designs and careful Quality Control (QC) of difficult‐to‐produce protein reagents.
Graphical and Lay Summary
Over 2800 variants of over 250 different human proteins were expressed in bacterial and mammalian expression system. The highest success rate was found using large plasma proteins such as albumin as fusion proteins. The proteins were carefully characterized and stable cell line generated in a particular challenging case.
Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR ...signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (Z
EGFR:1907)
2-KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (Z
EGFR:1907)
2-KDEL resulted in 80% reduction of the cell surface level of EGFR, and fluorescent staining for EGFR and the (Z
EGFR:1907)
2-KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating a specific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate.
To combine the targeted gene delivery and cell-specific transcriptional targeting, a delivery system was designed based on Generation 5 polyamidoamine dendrimer (PAMAM), poly(ethylene glycol), and ...anti-HER2 (human epidermal growth factor receptor 2) variable domains of camelid heavy chain antibodies (single domain antibody, VHHs). Anti-HER2 VHHs were covalently attached to the distal ends of poly(ethylene glycols) in PAMAM-poly(ethylene glycol) structure. The prepared immunotargeted nanobiopolymer was then used to condense gene construct, encoding a transcriptionally targeted truncated-Bid killer gene under the control of the breast cancer–specific MUC1 (transmembrane glycoprotein mucin 1) promoter. Transfection and transcription efficiencies of the engineered dendriplexes were quantified by truncated-Bid gene expression measurements, using real-time polymerase chain reaction. The engineered dendriplexes exhibited desirable physicochemical characteristics evaluated by dynamic light scattering and agarose gel retardation assay. Flow cytometry results showed a significantly higher uptake of VHH-modified dendriplexes than untargeted particles in HER2-positive cell lines. In vitro gene expression studies using PAMAM-poly(ethylene glycol)-VHH in complex with a luciferase expressing plasmid resulted in 1.6- and 4.8-fold higher transfection efficiencies than intact PAMAM dendrimers in BT-474 and SK-BR-3 (both as HER2-positive cell lines), respectively. Transfection by PAMAM-poly(ethylene glycol)-VHH/pMUC1-truncated-Bid also increased the level of truncated-Bid gene expression at 2.2- and 3.6-folds in BT-474 and SK-BR-3 cell lines, respectively, compared to the untargeted PAMAM treatment. A remarkable cell death was also observed in case of HER2-positive cells transfected by the targeted dendriplexes, indicating not only an effective targeted delivery but also a better transcriptional targeting efficiency of truncated-Bid killer gene, when expressed under the control of MUC1 promoter.
To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the ...objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates.
EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation.
Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion.
Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-βH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.
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