Cell communication systems based on polypeptide ligands use transmembrane receptors to transmit signals across the plasma membrane. In their biogenesis, receptors depend on the endoplasmic reticulum (ER)-Golgi system for folding, maturation, transport and localization to the cell surface. ER stress, caused by protein overproduction and misfolding, is a well-known pathology in neurodegeneration, cancer and numerous other diseases. How ER stress affects cell communication via transmembrane receptors is largely unknown. In disease models of multiple myeloma, chronic lymphocytic leukemia and osteogenesis imperfecta, we show that ER stress leads to loss of the mature transmembrane receptors FGFR3, ROR1, FGFR1, LRP6, FZD5 and PTH1R at the cell surface, resulting in impaired downstream signaling. This is caused by downregulation of receptor production and increased intracellular retention of immature receptor forms. Reduction of ER stress by treatment of cells with the chemical chaperone tauroursodeoxycholic acid or by expression of the chaperone protein BiP resulted in restoration of receptor maturation and signaling. We show a previously unappreciated pathological effect of ER stress; impaired cellular communication due to altered receptor processing. Our findings have implications for disease mechanisms related to ER stress and are particularly important when receptor-based pharmacological approaches are used for treatment.

• Keywords
• ER, Endoplasmic reticulum, Impaired, Receptor, Signaling, Stress, Transmembrane,
• MeSH
• Endoplasmic Reticulum Chaperone BiP MeSH
• Taurochenodeoxycholic Acid pharmacology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Receptors, Cell Surface * metabolism   MeSH
• Signal Transduction * drug effects   MeSH
• Endoplasmic Reticulum Stress * drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Endoplasmic Reticulum Chaperone BiP MeSH
• Taurochenodeoxycholic Acid MeSH
• Receptors, Cell Surface * MeSH
• ursodoxicoltaurine MeSH Browser

Fibroblast growth factors (FGFs) control organ morphogenesis during development as well as tissue homeostasis and repair in the adult organism. Despite their importance, many mechanisms that regulate FGF function are still poorly understood. Interestingly, the thermodynamic stability of 22 mammalian FGFs varies widely, with some FGFs remaining stable at body temperature for more than 24 h, while others lose their activity within minutes. How thermodynamic stability contributes to the function of FGFs during development remains unknown. Here we show that FGF10, an important limb and lung morphogen, exists as an intrinsically unstable protein that is prone to unfolding and is rapidly inactivated at 37 °C. Using rationally driven directed mutagenesis, we have developed several highly stable (STAB) FGF10 variants with a melting temperature of over 19 °C more than that of wildtype FGF10. In cellular assays in vitro, the FGF10-STABs did not differ from wildtype FGF10 in terms of binding to FGF receptors, activation of downstream FGF receptor signaling in cells, and induction of gene expression. In mouse embryonal lung explants, FGF10-STABs, but not wildtype FGF10, suppressed branching, resulting in increased alveolarization and expansion of epithelial tissue. Similarly, FGF10-STAB1, but not FGF10 wildtype, inhibited the growth of mouse embryonic tibias and markedly altered limb morphogenesis when implanted into chicken limb buds, collectively demonstrating that thermal instability should be considered an important regulator of FGF function that prevents ectopic signaling. Furthermore, we show enhanced differentiation of human iPSC-derived lung organoids and improved regeneration in ex vivo lung injury models mediated by FGF10-STABs, suggesting an application in cell therapy.

• Keywords
• Development, FGF10, Fibroblast growth factor, Lung, Morphogen, Stability,
• MeSH
• Fibroblast Growth Factor 10 * metabolism   genetics   chemistry   MeSH
• Humans MeSH
• Mice MeSH
• Lung metabolism   embryology   MeSH
• Receptors, Fibroblast Growth Factor metabolism   MeSH
• Signal Transduction * MeSH
• Protein Stability MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fgf10 protein, mouse MeSH Browser
• Fibroblast Growth Factor 10 * MeSH
• Receptors, Fibroblast Growth Factor MeSH

Fibroblast growth factor 2 (FGF2) is a signaling protein that plays a significant role in tissue development and repair. FGF2 binds to fibroblast growth factor receptors (FGFRs) alongside its co-factor heparin, which protects FGF2 from degradation. The binding between FGF2 and FGFRs induces intracellular signaling pathways such as RAS-MAPK, PI3K-AKT, and STAT. FGF2 has strong potential for application in cell culturing, wound healing, and cosmetics but the potential is severely limited by its low protein stability. The thermostable variant FGF2-STAB was constructed by computer-assisted protein engineering to overcome the natural limitation of FGF2. Previously reported characterization of FGF2-STAB revealed an enhanced ability to induce MAP/ERK signaling while having a lower dependence on heparin when compared with FGF2-wt. Here we report the crystal structure of FGF2-STAB solved at 1.3 Å resolution. Protein stabilization is achieved by newly formed hydrophobic interactions, polar contacts, and one additional hydrogen bond. The overall structure of FGF2-STAB is similar to FGF2-wt and does not reveal information on the experimentally observed lower dependence on heparin. A noticeable difference in flexibility in the receptor binding region can explain the differences in signaling between FGF2-STAB and its wild-type counterpart. Our structural analysis provided molecular insights into the stabilization and unique biological properties of FGF2-STAB.

• Keywords
• Protein flexibility, Stabilized fibroblast growth factor 2, X-ray structural analysis,
• Publication type
• Journal Article MeSH

FGF21 is an endocrine signaling protein belonging to the family of fibroblast growth factors (FGFs). It has emerged as a molecule of interest for treating various metabolic diseases due to its role in regulating glucogenesis and ketogenesis in the liver. However, FGF21 is prone to heat, proteolytic, and acid-mediated degradation, and its low molecular weight makes it susceptible to kidney clearance, significantly reducing its therapeutic potential. Protein engineering studies addressing these challenges have generally shown that increasing the thermostability of FGF21 led to improved pharmacokinetics. Here, we describe the computer-aided design and experimental characterization of FGF21 variants with enhanced melting temperature up to 15 °C, uncompromised efficacy at activation of MAPK/ERK signaling in Hep G2 cell culture, and ability to stimulate proliferation of Hep G2 and NIH 3T3 fibroblasts cells comparable with FGF21-WT. We propose that stabilizing the FGF21 molecule by rational design should be combined with other reported stabilization strategies to maximize the pharmaceutical potential of FGF21.

• Keywords
• Fibroblast growth factor 21, Protein engineering, Protein stabilization,
• Publication type
• Journal Article MeSH

The FGF system is the most complex of all receptor tyrosine kinase signaling networks with 18 FGF ligands and four FGFRs that deliver morphogenic signals to pattern most embryonic structures. Even when a single FGFR is expressed in the tissue, different FGFs can trigger dramatically different biological responses via this receptor. Here we show both quantitative and qualitative differences in the signaling of one of the FGF receptors, FGFR1c, in response to different FGFs. We provide an overview of the recent discovery that FGFs engage in biased signaling via FGFR1c. We discuss the concept of ligand bias, which represents qualitative differences in signaling as it is a measure of differential ligand preferences for different downstream responses. We show how FGF ligand bias manifests in functional data in cultured chondrocyte cells. We argue that FGF-ligand bias contributes substantially to FGF-driven developmental processes, along with known differences in FGF expression levels, FGF-FGFR binding coefficients and differences in FGF stability in vivo.

• Keywords
• Bias, FGF, FGFR, Signaling,
• MeSH
• Chondrocytes metabolism   MeSH
• Fibroblast Growth Factors * metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Receptor, Fibroblast Growth Factor, Type 1 * metabolism   MeSH
• Receptors, Fibroblast Growth Factor * metabolism   MeSH
• Signal Transduction * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Fibroblast Growth Factors * MeSH
• Ligands MeSH
• Receptor, Fibroblast Growth Factor, Type 1 * MeSH
• Receptors, Fibroblast Growth Factor * MeSH

Fat grafting, a key regenerative medicine technique, often requires repeat procedures due to high-fat reabsorption and volume loss. Addressing this, a novel drug delivery system uniquely combines a thermosensitive, FDA-approved hydrogel (itaconic acid-modified PLGA-PEG-PLGA copolymer) with FGF2-STAB, a stable fibroblast growth factor 2 with a 21-day stability, far exceeding a few hours of wild-type FGF2's stability. Additionally, the growth factor was encapsulated in "green" liposomes prepared via the Mozafari method, ensuring pH protection. The system, characterized by first-order FGF2-STAB release, employs green chemistry for biocompatibility, bioactivity, and eco-friendliness. The liposomes, with diameters of 85.73 ± 3.85 nm and 68.6 ± 2.2% encapsulation efficiency, allowed controlled FGF2-STAB release from the hydrogel compared to the unencapsulated FGF2-STAB. Yet, the protein compromised the carrier's hydrolytic stability. Prior tests were conducted on model proteins human albumin (efficiency 80.8 ± 3.2%) and lysozyme (efficiency 81.0 ± 2.7%). This injectable thermosensitive system could advance reconstructive medicine and cosmetic procedures.

• MeSH
• Fibroblast Growth Factor 2 * MeSH
• Hydrogels chemistry   MeSH
• Drug Delivery Systems MeSH
• Humans MeSH
• Liposomes * MeSH
• Drug Carriers chemistry   MeSH
• Polyethylene Glycols chemistry   MeSH
• Polyglactin 910 chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fibroblast Growth Factor 2 * MeSH
• Hydrogels MeSH
• Liposomes * MeSH
• Drug Carriers MeSH
• poly(lactic-glycolic acid)-poly(ethyleneglycol) copolymer MeSH Browser
• polyethylene glycol-poly(lactide-co-glycolide) MeSH Browser
• Polyethylene Glycols MeSH
• Polyglactin 910 MeSH

Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching. Pharmacological inhibition of ROCK or non-muscle myosin II, or fibroblast-specific knock-out of Myh9 abrogate fibroblast-induced epithelial branching. The process of fibroblast-induced branching requires epithelial proliferation and is associated with distinctive epithelial patterning of yes associated protein (YAP) activity along organoid branches, which is dependent on fibroblast contractility. Moreover, we provide evidence for the in vivo existence of contractile fibroblasts specifically surrounding terminal end buds (TEBs) of pubertal murine mammary glands, advocating for an important role of fibroblast contractility in branching in vivo. Together, we identify fibroblast contractility as a novel stromal factor driving mammary epithelial morphogenesis. Our study contributes to comprehensive understanding of overlapping but divergent employment of mechanically active fibroblasts in developmental versus tumorigenic programs.

• MeSH
• Epithelial Cells * metabolism   MeSH
• Fibroblasts metabolism   MeSH
• Coculture Techniques MeSH
• Mammary Glands, Animal * metabolism   MeSH
• Morphogenesis physiology   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The fibroblast growth factors (FGF) family holds significant potential for addressing chronic diseases. Specifically, recombinant FGF18 shows promise in treating osteoarthritis by stimulating cartilage formation. However, recent phase 2 clinical trial results of sprifermin (recombinant FGF18) indicate insufficient efficacy. Leveraging our expertise in rational protein engineering, we conducted a study to enhance the stability of FGF18. As a result, we obtained a stabilized variant called FGF18-E4, which exhibited improved stability with 16 °C higher melting temperature, resistance to trypsin and a 2.5-fold increase in production yields. Moreover, the FGF18-E4 maintained mitogenic activity after 1-week incubation at 37 °C and 1-day at 50 °C. Additionally, the inserted mutations did not affect its binding to the fibroblast growth factor receptors, making FGF18-E4 a promising candidate for advancing FGF-based osteoarthritis treatment.

• Keywords
• Computer-assisted stabilization, FGF-18, Fibroblast growth factor, Improved yield, Protease, Resistance to, Thermostability,
• Publication type
• Journal Article MeSH

Given its highly innovative character and potential socioeconomic impact, Synthetic Biology is often ranked among prominent research areas and national research priorities in developed countries. The global evolution of this field is proceeding by leaps and bounds but its development at the level of individual states varies widely. Despite their current satisfactory economic status, the majority of 13, mostly post-communist, countries that entered the European Union family in and after 2004 (EU13) have long overlooked the blossoming of Synthetic Biology. Their prioritized lines of research have been directed elsewhere or "Synthetic Biology" did not become a widely accepted term to encompass their bioengineering and biotechnology domains. The Czech Republic is not an exception. The local SynBio mycelium already exists but is mainly built bottom-up through the activities of several academic labs, iGEM teams, and spin-off companies. In this article, we tell their individual stories and summarize the prerequisites that allowed their emergence in the Czech academic and business environment. In addition, we provide the reader with a brief overview of laboratories, research hubs, and companies that perform biotechnology and bioengineering-oriented research and that may be included in a notional "shadow SynBio community" but have not yet adopted Synthetic Biology as a unifying term for their ventures. We also map the current hindrances for a broader expansion of Synthetic Biology in the Czech Republic and suggest possible steps that should lead to the maturity of this fascinating research field in our country.

• Keywords
• Biotechnology and bioengineering, Community, Czech Republic, EU13 countries, Public perception, Research landscape, Synthetic biology, iGEM,
• Publication type
• Journal Article MeSH

Many growth factors have been studied as additives accelerating lumbar fusion rates in different animal models. However, their low hydrolytic and thermal stability both in vitro and in vivo limits their workability and use. In the proposed work, a stabilized vasculogenic and prohealing fibroblast growth factor-2 (FGF2-STAB®) exhibiting a functional half-life in vitro at 37 °C more than 20 days was applied for lumbar fusion in combination with a bioresorbable scaffold on porcine models. An experimental animal study was designed to investigate the intervertebral fusion efficiency and safety of a bioresorbable ceramic/biopolymer hybrid implant enriched with FGF2-STAB® in comparison with a tricortical bone autograft used as a gold standard. Twenty-four experimental pigs underwent L2/3 discectomy with implantation of either the tricortical iliac crest bone autograft or the bioresorbable hybrid implant (BHI) followed by lateral intervertebral fixation. The quality of spinal fusion was assessed by micro-computed tomography (micro-CT), biomechanical testing, and histological examination at both 8 and 16 weeks after the surgery. While 8 weeks after implantation, micro-CT analysis demonstrated similar fusion quality in both groups, in contrast, spines with BHI involving inorganic hydroxyapatite and tricalcium phosphate along with organic collagen, oxidized cellulose, and FGF2- STAB® showed a significant increase in a fusion quality in comparison to the autograft group 16 weeks post-surgery (p = 0.023). Biomechanical testing revealed significantly higher stiffness of spines treated with the bioresorbable hybrid implant group compared to the autograft group (p < 0.05). Whilst histomorphological evaluation showed significant progression of new bone formation in the BHI group besides non-union and fibrocartilage tissue formed in the autograft group. Significant osteoinductive effects of BHI based on bioceramics, collagen, oxidized cellulose, and FGF2-STAB® could improve outcomes in spinal fusion surgery and bone tissue regeneration.

• Keywords
• FGF2, animal model, autograft, biomechanics, ceramic, collagen, histology, lumbar spinal fusion, micro-CT, tissue engineering,
• Publication type
• Journal Article MeSH