Uncovering key steps in FGF12 cellular release reveals a common mechanism for unconventional FGF protein secretion.

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.

FGF homologous factors are secreted from cells to induce FGFR-mediated anti-apoptotic response.

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.

Intracellular FGF1 protects cells from apoptosis through direct interaction with p53.

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.

Receptor clustering by a precise set of extracellular galectins initiates FGFR signaling.

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.

FGF12: biology and function.

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 ß-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.

Short report galectins use N-glycans of FGFs to capture growth factors at the cell surface and fine-tune their signaling.

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. Video Abstract.

Stability and Resistance to Proteolysis of Enterotoxins SEC and SEL Produced by Staphylococcus epidermidis and Staphylococcus aureus.

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.

FGF12 is a novel component of the nucleolar NOLC1/TCOF1 ribosome biogenesis complex.

Among the FGF proteins, the least characterized superfamily is the group of fibroblast growth factor homologous factors (FHFs). To date, the main role of FHFs has been primarily seen in the modulation of voltage-gated ion channels, but a full picture of the function of FHFs inside the cell is far from complete. In the present study, we focused on identifying novel FGF12 binding partners to indicate its intracellular functions. Among the identified proteins, a significant number were nuclear proteins, especially RNA-binding proteins involved in translational processes, such as ribosomal processing and modification. We have demonstrated that FGF12 is localized to the nucleolus, where it interacts with NOLC1 and TCOF1, proteins involved in the assembly of functional ribosomes. Interactions with both NOLC1 and TCOF1 are unique to FGF12, as other FHF proteins only bind to TCOF1. The formation of nucleolar FGF12 complexes with NOLC1 and TCOF1 is phosphorylation-dependent and requires the C-terminal region of FGF12. Surprisingly, NOLC1 and TCOF1 are unable to interact with each other in the absence of FGF12. Taken together, our data link FHF proteins to nucleoli for the first time and suggest a novel and unexpected role for FGF12 in ribosome biogenesis. Video Abstract.

FHF1 is a bona fide fibroblast growth factor that activates cellular signaling in FGFR-dependent manner.

Fibroblast growth factors (FGFs) via their receptors (FGFRs) transduce signals from the extracellular space to the cell interior, modulating pivotal cellular processes such as cell proliferation, motility, metabolism and death. FGF superfamily includes a group of fibroblast growth factor homologous factors (FHFs), proteins whose function is still largely unknown. Since FHFs lack the signal sequence for secretion and are unable to induce FGFR-dependent cell proliferation, these proteins were considered as intracellular proteins that are not involved in signal transduction via FGFRs. Here we demonstrate for the first time that FHF1 directly interacts with all four major FGFRs. FHF1 binding causes efficient FGFR activation and initiation of receptor-dependent signaling cascades. However, the biological effect of FHF1 differs from the one elicited by canonical FGFs, as extracellular FHF1 protects cells from apoptosis, but is unable to stimulate cell division. Our data define FHF1 as a FGFR ligand, emphasizing much greater similarity between FHFs and canonical FGFs than previously indicated. Video Abstract. (MP4 38460 kb).

High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1.

Fibroblast growth factor receptor 1 (FGFR1) is a plasma membrane protein that transmits signals from the extracellular environment, regulating cell homeostasis and function. Dysregulation of FGFR1 leads to the development of human cancers and noncancerous diseases. Numerous tumors overproduce FGFR1, making this receptor a perspective target for cancer therapies. Antibody-drug conjugates (ADCs) are highly potent and selective anticancer agents. ADCs are composed of antibodies (targeting factors) fused to highly cytotoxic drugs (warheads). The efficiency of ADC strategy largely depends on the internalization of cytotoxic conjugate into cancer cells. Here, we have studied an interplay between affinity of anti-FGFR1 antibodies and efficiency of their cellular uptake. We have developed a unique set of engineered anti-FGFR1 antibodies that bind the same epitope in the extracellular part of FGFR1, but with different affinities. We have demonstrated that these antibodies are effectively taken up by cancer cells in the FGFR1-dependent manner. Interestingly, we have found that efficiency, defined as rate and level of antibody internalization, largely depends on the affinity of engineered antibodies towards FGFR1, as high affinity antibody displays fastest internalization kinetics. Our data may facilitate design of therapeutically relevant targeting molecules for selective treatment of FGFR1 overproducing cancers.

Correction: Serwotka-Suszczak, A. M. et al. A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells. Int. J. Mol. Sci. 2017, 18, 401.

It has been brought to our attention that the affiliation of Dr. Jerzy Pieczykolan at the time when he was responsible for the work described in the paper [...].

A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells.

Antibody-drug conjugates (ADCs) have recently emerged as efficient and selective cancer treatment therapeutics. Currently, alternative forms of drug carriers that can replace monoclonal antibodies are under intensive investigation. Here, a cytotoxic conjugate of an anti-HER2 (Human Epidermal Growth Factor Receptor 2) diaffibody with monomethyl-auristatin E (MMAE) is proposed as a potential anticancer therapeutic. The anti-HER2 diaffibody was based on the ZHER2:4 affibody amino acid sequence. The anti-HER2 diaffibody has been expressed as a His-tagged protein in E. coli and purified by Ni-nitrilotriacetyl (Ni-NTA) agarose chromatography. The molecule was properly folded, and the high affinity and specificity of its interaction with HER2 was confirmed by surface plasmon resonance (SPR) and flow cytometry, respectively. The (ZHER2:4)2DCS-MMAE conjugate was obtained by coupling the maleimide group linked with MMAE to cysteines, which were introduced in a drug conjugation sequence (DCS). Cytotoxicity of the conjugate was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide MTT assay and the xCELLigence Real-Time Cell Analyzer. Our experiments demonstrated that the conjugate delivered auristatin E specifically to HER2-positive tumor cells, which finally led to their death. These results indicate that the cytotoxic diaffibody conjugate is a highly potent molecule for the treatment of various types of cancer overexpressing HER2 receptors.

In vivo formation of Plasmodium falciparum ribosomal stalk - a unique mode of assembly without stable heterodimeric intermediates.

BACKGROUND: The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins. METHODS: We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods. RESULTS: All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)2form represents the most favorable architecture for parasite P-proteins. CONCLUSION: The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1-P2 heterodimeric intermediate. GENERAL SIGNIFICANCE: On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.

Design and characteristics of a stable protein scaffold for specific binding based on variable lymphocyte receptor sequences.

Variable lymphocyte receptors (VLRs) serve as antigen binding proteins in jawless vertebrates. Their relatively low molecular weight makes VLRs an interesting alternative to antibodies in biotechnological applications. A typical VLR comprises several unique motifs called leucine-rich repeats (LRRs). Using consensus approach we designed a novel VLR protein (called dVLR) containing six LRR repeats based on a sea lamprey receptor sequence. The designed protein was expressed in Escherichia coli in a soluble, native form and showed very favorable biophysical properties. Recombinant dVLR is monomeric in solution and preserves its secondary structure within the pH range 3.0 to 11.0 and tertiary structure between pH 4.0 and 10.0. It undergoes reversible thermal denaturation in a broad pH range (4.0 to 10.0). The maximal denaturation temperature of 73.9°C is observed at pH 6.0, 0.3M NaCl. Chemical denaturation of dVLR at pH 7.5 is a cooperative two-state process with a midpoint at 3.3M GdmCl and a very high free energy change of unfolding in the absence of denaturant equal to 14.1kcal/mol. The biophysical properties of dVLR make it highly suitable for biotechnological applications such as generation of specific ligand-binding molecules.

Biophysical properties of the eukaryotic ribosomal stalk.

The landing platform for the translational GTPases is located on the 60S ribosomal subunit and is referred to as a GTPase-associated center. The most distinctive feature of this center is an oligomeric complex, the stalk, responsible for the recruitment of translation factors and stimulation of translation factor-dependent GTP hydrolysis. In eukaryotes, the stalk has been investigated in vitro and in vivo, but most information available concerns its individual components only. In the present study, we provide an insight into the biophysical nature of the native stalk isolated from the yeast Saccharomyces cerevisiae. Using fluorescence, circular dichroism, and mass spectrometry analyses, we were able to characterize the natively formed yeast stalk, casting new light on the oligomeric properties of the complex and its quaternary topology, showing that folding and assembly are coupled processes. The pentameric stalk is an exceptionally stable structure with the protein core composed of P0, P1A, and P2B proteins and less tightly bound P1B and P2A capable of dissociating from the stalk core. We obtained also the whole picture of the posttranslational modifications at the logarithmic phase of yeast growth, using mass spectrometry approach, where P proteins are phosphorylated at a single serine residue, P0 may accept two phosphate groups, and P1A none. Additionally, only P1B undergoes N-terminal acetylation after prior methionine removal.

Increased protein stability of FGF1 can compensate for its reduced affinity for heparin.

Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1.fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1.FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.

Fructose 1,6-Bisphosphatase 2 Plays a Crucial Role in the Induction and Maintenance of Long-Term Potentiation.

Long-term potentiation (LTP) is a molecular basis of memory formation. Here, we demonstrate that LTP critically depends on fructose 1,6-bisphosphatase 2 (Fbp2)-a glyconeogenic enzyme and moonlighting protein protecting mitochondria against stress. We show that LTP induction regulates Fbp2 association with neuronal mitochondria and Camk2 and that the Fbp2-Camk2 interaction correlates with Camk2 autophosphorylation. Silencing of Fbp2 expression or simultaneous inhibition and tetramerization of the enzyme with a synthetic effector mimicking the action of physiological inhibitors (NAD+ and AMP) abolishes Camk2 autoactivation and blocks formation of the early phase of LTP and expression of the late phase LTP markers. Astrocyte-derived lactate reduces NAD+/NADH ratio in neurons and thus diminishes the pool of tetrameric and increases the fraction of dimeric Fbp2. We therefore hypothesize that this NAD+-level-dependent increase of the Fbp2 dimer/tetramer ratio might be a crucial mechanism in which astrocyte-neuron lactate shuttle stimulates LTP formation.

Structure of a highly stable mutant of human fibroblast growth factor 1.

Fibroblast growth factors (FGFs) are involved in diverse cellular processes such as cell migration, angiogenesis, osteogenesis, wound healing and embryonic and foetal development. Human acidic fibroblast growth factor (FGF-1) is the only member of the FGF family that binds with high affinity to all four FGF receptors and thus is considered to be the human mitogen with the broadest specificity. However, pharmacological applications of FGF-1 are limited owing to its low stability. It has previously been reported that the introduction of single mutations can significantly improve the stability of FGF-1 and its resistance to proteolytic degradation. Here, the structure of the Q40P/S47I/H93G triple mutant of FGF-1, which exhibits much higher stability, a prolonged half-life and enhanced mitogenic activity, is presented. Compared with the wild-type structure, three localized conformational changes in the stable triple mutant were observed, which is in agreement with the perfect energetic additivity of the single mutations described in a previous study. The huge change in FGF-1 stability (the denaturation temperature increased by 21.5 K, equivalent to DeltaDeltaG(den) = 24.3 kJ mol(-1)) seems to result from the formation of a short 3(10)-helix (position 40), an improvement in the propensity of amino acids to form beta-sheets (position 47) and the rearrangement of a local hydrogen-bond network (positions 47 and 93).

Low Stability of Integrin-Binding Deficient Mutant of FGF1 Restricts Its Biological Activity.

Fibroblast growth factor 1 (FGF1) has been shown to interact with integrin αvß3 through a specific binding site, involving Arg35 residue. The FGF1 mutant (R35E) with impaired integrin binding was found to be defective in its proliferative response, although it was still able to interact with FGF receptors (FGFR) and heparin and induce the activation of downstream signaling pathways. Here, we demonstrate that the lack of mitogenic potential of R35E mutant is directly caused by its decreased thermodynamic stability and susceptibility to proteolytic degradation. Introduction of three stabilizing mutations into R35E variant compensated the effect of destabilizing R35E mutation and restored the proliferation potential of FGF1. Moreover, the stabilized R35E variant regained both anti-apoptotic and wound healing activities, while remaining defective in binding to integrin αvß3. Our results suggest that the thermodynamic stability and resistance to degradation, rather than the interaction with integrin are required for mitogenic response of FGF1.