Fibroblast growth factor 1 (FGF1), a well-characterized member of the FGF family, effectively lowers blood glucose levels in animal models of type 2 diabetes by stimulating glucose uptake. However, its significant mitogenic potential poses a major challenge for clinical application. Here, we present engineered variants of FGF1 designed to dissociate its potent glucose-lowering effects from its undesired proliferative activity, aiming for a future therapeutic agent for type 2 diabetes. Through a series of rational mutations focused on modulating receptor binding and heparan interactions, coupled with enhanced thermodynamic stability, we developed two lead FGF1 variants. Comprehensive in vitro studies confirmed that these variants exhibit significantly reduced mitogenic potential across various cell types compared to wild-type FGF1. Specifically, one variant showed profound loss of proliferation due to disrupted FGFR binding, while the other displayed attenuated mitogenicity linked to decreased heparin affinity. Critically, both fully maintained potent glucose-lowering properties in db/db mice without inducing hypoglycemia or changes in body weight. Furthermore, these engineered proteins demonstrate superior thermodynamic stability and markedly improved pharmacokinetic profile, critical attributes for drug development. Our findings highlight a successful strategy to uncouple the therapeutic benefits of FGF1 from its mitogenic side effects, offering promising, stable, and safe protein-based drug candidates for type 2 diabetes treatment.

Fibroblast growth factor 2 (FGF2) is a canonical member of the fibroblast growth factor family with essential roles in embryonic development, tissue regeneration, and stem cell biology, where it supports pluripotency and high proliferative capacity. Consequently, FGF2 is widely used in experimental models of regeneration and stem cell maintenance; however, its rapid thermal degradation at physiological temperatures limits its practical utility and therapeutic potential. Despite the availability of stabilized variants, developing FGF2 analogs that combine exceptional thermodynamic resilience with favorable pharmacokinetic properties remains a critical challenge for stem cell research and regenerative medicine. Using a combination of consensus sequence analysis and structure-guided protein engineering, we generated novel FGF2 variants with unprecedented thermodynamic stability and optimized extracellular matrix interactions. To enhance bioavailability, we introduced mutations that reduced affinity for heparan sulfate proteoglycans, thereby limiting sequestration within the extracellular matrix. The most stable variant exhibited a denaturation temperature increase of more than 27 °C relative to wild-type FGF2 while fully retaining mitogenic and migratory activity after 5 days of incubation at 70 °C. This exceptional thermal stability was accompanied by markedly increased resistance to proteolytic degradation. In functional cellular models, including adipocyte differentiation and stem cell culture, an optimized long-acting variants demonstrated superior metabolic efficacy and sustained signaling, enabling a reduction in dosing frequency from daily administration to once every three days. We report the development of highly stable, highly bioactive FGF2 variants that retain full receptor specificity and binding affinity even under extreme conditions. By overcoming the intrinsic instability of the wild-type protein, these engineered FGF2s may enable future stem cell expansion and regenerative therapy applications.

Heparan sulphate proteoglycans (HSPGs) are cell surface and extracellular matrix proteoglycan family members, playing a key role in diverse biological activities including signal transduction or endocytosis. To date, many HSPG ligands have been identified, including growth factors interacting via sulfated domains of heparan sulphate (HS) chains. Due to significant overexpression of HSPGs in various cancers, discovering novel biomolecules capable of HSPGs recognition, which may act as HSPGs biosensors or drug carriers targeting HSPGs, is essential. Fibroblast growth factors (FGFs), mainly paracrine FGFs, are natural ligands of membrane-bound HSPGs, forming ternary signaling complex with HSPGs and fibroblast growth factor receptors (FGFRs). Here we employed the natural capability of FGF1 to bind HSPGs to develop HSPGs-specific proteinaceous probe. We identified three point mutations within FGF1 that elevate its affinity for heparin—S116R, S17K and L72R, named B, C and D respectively. Together with substitutions increasing FGF1 stability and abolishing FGF1 binding to FGFRs, we have generated eight HSPGs-specific variants. Among tested mutants, FGF1HSBCD exhibits the highest affinity for heparin and HS/HSPGs, showing over 20-fold increase in affinity for glypican-4 and requiring 0.23 M higher salt concentration for elution from heparin column compared to the initial FGF1HS molecule. Finally, we demonstrated FGF1HSBCD potential to act as a molecular sensor of HSPGs level in cancer cell lines overproducing HSPGs, implicating that FGF1HSBCD can be used as HSPGs biosensor or as a drug delivery carrier in protein-drug conjugates (PDC) targeting HSPGs.

Fibroblast growth factors (FGFs) play a key role in lung development by mediating complex interactions between epithelial and mesenchymal cells, which are central to processes such as branching morphogenesis, epithelial differentiation, and alveolarization. The findings regarding this interplay highlight the complexity of FGF signaling, as different FGFs contribute to various aspects of lung formation and maturation. Understanding the role of FGF proteins in shaping the lung is crucial for gaining insight into the biology of its development. Furthermore, FGFs orchestrate complex signaling pathways that regulate lung regeneration in adulthood. Therapeutic strategies targeting FGF-dependent pathways appear promising for repairing and improving lung function in diverse pulmonary diseases. In this review, we describe the current perception of the role of FGF proteins in lung development and regeneration, together with an overview of emerging therapeutic strategies aiming at FGF signaling in lung-related disorders.

The FGF11 subfamily of FGF proteins also known as fibroblast growth factor homologous factors (FHFs) includes four proteins, FGF11, FGF12, FGF13, and FGF14. They are mainly expressed in excitable cells but are also present in fibroblasts or osteoclasts, where their function is much less understood. Each FGF11-14 protein has at least two isoforms formed by alternative splicing, which differ in both cellular localization and function. Until recently, only the short isoforms had been efficiently produced and purified in recombinant form. Here, we developed a protocol to produce in the bacterial expression system and efficiently purify the long "a" isoforms of FGF11, FGF12, FGF13 and FGF14. In addition, we characterized their biophysical and biological properties, demonstrating that they activate downstream signaling and, unlike short "b" isoforms, induce cell proliferation.

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.

FGF12 belongs to a subfamily of FGF proteins called FGF homologous factors (FHFs), which until recently were thought to be non-signaling intracellular proteins. Our recent studies have shown that although they lack a conventional signal peptide for secretion, they can reach the extracellular space, especially under stress conditions. Here, we unraveled that the long "a" isoform of FGF12 is secreted in a pathway involving the A1 subunit of Na(+)/K(+) ATPase (ATP1A1), Tec kinase and lipids such as phosphatidylinositol and phosphatidylserine. Further, we showed that the short "b" isoform of FGF12, which binds ATP1A1 and phosphatidylserine less efficiently, is not secreted from cells. We also indicated regions in the FGF12a protein sequence that are crucial for its secretion, including N-terminal fragment and specific residues, and proposed that liquid-liquid phase separation may be important in this process. Our results strongly suggest that the mechanism of this process is very similar for all unconventionally secreted FGF proteins.

Impaired fibroblast growth factor receptor (FGFR) signaling is associated with many human conditions, including growth disorders, degenerative diseases, and cancer. Current FGFR therapeutics are based on chemical inhibitors of FGFR tyrosine kinase activity (TKIs). However, FGFR TKIs are limited in their target specificity as they generally inhibit all FGFRs and other receptor tyrosine kinases. In the search for specific inhibitors of human FGFR1, we identified VZ23, a DNA aptamer that binds to FGFR1b and FGFR1c with a KD of 55 nM and 162 nM, respectively, but not to the other FGFR variants (FGFR2b, FGFR2c, FGFR3b, FGFR3c, FGFR4). In cells, VZ23 inhibited the activation of downstream FGFR1 signaling and FGFR1-mediated regulation of cellular senescence, proliferation, and extracellular matrix homeostasis. Consistent with the specificity toward FGFR1 observed in vitro, VZ23 did not inhibit FGFR2-4 signaling in cells. We show that the VZ23 inhibits FGFR1 signaling in the presence of cognate fibroblast growth factor (FGF) ligands and its inhibitory activity is linked to its capacity to form unusual G-quadruplex structure. Our data suggest that targeting FGFR1 with DNA aptamers could be an effective alternative to TKIs for treating impaired FGFR1 signaling in human craniosynostoses.

Fibroblast growth factor 12 (FGF12) belongs to the fibroblast growth factor homologous factors (FHF) subfamily, which is also known as the FGF11 subfamily. The human FGF12 gene is located on chromosome 3 and consists of four introns and five coding exons. Their alternative splicing results in two FGF12 isoforms - the shorter 'b' isoform and the longer 'a' isoform. Structurally, the core domain of FGF12, is highly homologous to that of the other FGF proteins, providing the classical tertiary structure of β-trefoil. FGF12 is expressed in various tissues, most abundantly in excitable cells such as neurons and cardiomyocytes. For many years, FGF12 was thought to be exclusively an intracellular protein, but recent studies have shown that it can be secreted despite the absence of a canonical signal for secretion. The best-studied function of FGF12 relates to its interaction with sodium channels. In addition, FGF12 forms complexes with signaling proteins, regulates the cytoskeletal system, binds to the FGF receptors activating signaling cascades to prevent apoptosis and interacts with the ribosome biogenesis complex. Importantly, FGF12 has been linked to nervous system disorders, cancers and cardiac diseases such as epileptic encephalopathy, pulmonary hypertension and cardiac arrhythmias, making it a potential target for gene therapy as well as a therapeutic agent.

The only staphylococcal enterotoxins produced by Staphylococcus epidermidis include SECepi and SELepi, whereas Staphylococcus aureus produces orthologous SECs and SEL having different sequences. We compared S. epidermidis and S. aureus SECs and SELs in terms of resistance to proteolysis and both, thermal and chemical stability. We show that SECepi and SELepi produced by S. epidermidis have similar resistance to proteolysis if compared with their respective orthologues produced by S. aureus. Studied S. epidermidis and S. aureus SEC variants incubated with pepsin at pH 2.0 were found to be more resistant to proteolysis than SELs. SELs turned out to be more resistant than SECs to proteolysis with trypsin at pH 8.0. SECepi was found to be more resistant to thermal denaturation if compared with its S. aureus orthologues. The S. epidermidis and S. aureus SEC variants were found to have higher thermal stability than SELs. Our data indicate that, due to their high stability, the enterotoxins SECepi and SELepi produced in food by S. epidermidis may pose a food safety risk comparable with that posed by S. aureus enterotoxins.

Fibroblast growth factor 1 (FGF1) acts by activating specific tyrosine kinase receptors on the cell surface. In addition to this classical mode of action, FGF1 also exhibits intracellular activity. Recently, we found that FGF1 translocated into the cell interior exhibits anti-apoptotic activity independent of receptor activation and downstream signaling. Here, we show that expression of FGF1 increases the survival of cells treated with various apoptosis inducers, but only when wild-type p53 is present. The p53-negative cells were not protected by either ectopically expressed or translocated FGF1. We also confirmed the requirement of p53 for the anti-apoptotic intracellular activity of FGF1 by silencing p53, resulting in loss of the protective effect of FGF1. In contrast, in p53-negative cells, intracellular FGF1 regained its anti-apoptotic properties after transfection with wild-type p53. We also found that FGF1 directly interacts with p53 in cells and that the binding region is located in the DBD domain of p53. We therefore postulate that intracellular FGF1 protects cells from apoptosis by directly interacting with p53.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute complex signaling hubs that are crucial for the development and homeostasis of the human body. Most of FGFs are released by cells using the conventional secretory pathway and are N-glycosylated, yet the role of FGFs glycosylation is largely unknown. Here, we identify N-glycans of FGFs as binding sites for a specific set of extracellular lectins, galectins − 1, -3, -7 and − 8. We demonstrate that galectins attract N-glycosylated FGF4 to the cell surface, forming a reservoir of the growth factor in the extracellular matrix. Furthermore, we show that distinct galectins differentially modulate FGF4 signaling and FGF4-dependent cellular processes. Using engineered variants of galectins with altered valency we demonstrate that multivalency of galectins is critical for the adjustment of FGF4 activity. Summarizing, our data reveal a novel regulatory module within FGF signaling, in which the glyco-code in FGFs provides previously unanticipated information differentially deciphered by multivalent galectins, affecting signal transduction and cell physiology.

FGF homologous factors (FHFs) are the least described group of fibroblast growth factors (FGFs). The FHF subfamily consists of four proteins: FGF11, FGF12, FGF13, and FGF14. Until recently, FHFs were thought to be intracellular, non‐signaling molecules, despite sharing structural and sequence similarities with other members of FGF family that can be secreted and activate cell signaling by interacting with surface receptors. Here, we show that despite lacking a canonical signal peptide for secretion, FHFs are exported to the extracellular space. Furthermore, we propose that their secretion mechanism is similar to the unconventional secretion of FGF2. The secreted FHFs are biologically active and trigger signaling in cells expressing FGF receptors (FGFRs). Using recombinant proteins, we demonstrated their direct binding to FGFR1, resulting in the activation of downstream signaling and the internalization of the FHF‐FGFR1 complex. The effect of receptor activation by FHF proteins is an anti‐apoptotic response of the cell.

FGF/FGFR signaling is critical for the development and homeostasis of the human body and imbalanced FGF/FGFR contributes to the progression of severe diseases, including cancers. FGFRs are N-glycosylated, but the role of these modifications is largely unknown. Galectins are extracellular carbohydrate-binding proteins implicated in a plethora of processes in heathy and malignant cells. Here, we identified a precise set of galectins (galectin-1, -3, -7, and -8) that directly interact with N-glycans of FGFRs. We demonstrated that galectins bind N-glycan chains of the membrane-proximal D3 domain of FGFR1 and trigger differential clustering of FGFR1, resulting in activation of the receptor and initiation of downstream signaling cascades. Using engineered galectins with controlled valency, we provide evidence that N-glycosylation-dependent clustering of FGFR1 constitutes a mechanism for FGFR1 stimulation by galectins. We revealed that the consequences of galectin/FGFR signaling for cell physiology are markedly different from the effects induced by canonical FGF/FGFR units, with galectin/FGFR signaling affecting cell viability and metabolic activity. Furthermore, we showed that galectins are capable of activating an FGFR pool inaccessible for FGF1, enhancing the amplitude of transduced signals. Summarizing, our data identify a novel mechanism of FGFR activation, in which the information stored in the N-glycans of FGFRs provides previously unanticipated information about FGFRs’ spatial distribution, which is differentially deciphered by distinct multivalent galectins, affecting signal transmission and cell fate.