Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.

• Klíčová slova
• computational design, embryonic stem cells, fibroblast growth factor, focused directed evolution, protein engineering, protein stability,
• MeSH
• bodová mutace MeSH
• design s pomocí počítače * MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• fibroblastový růstový faktor 2 chemie   genetika   metabolismus   MeSH
• lidé MeSH
• počítačová simulace MeSH
• proteinové inženýrství * MeSH
• řízená evoluce molekul MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH

Fibroblast growth factor-2 is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 occurs in several isoforms resulting from alternative initiations of traslation: an 18 kDa cytoplasmic isoform and four larger molecular weight nuclear isoforms (22, 22.5, 24 and 34 kDa). It acts mainly through a paracrine/autocrine mechanism involving high affinity transmembrane receptors and heparan sulfate proteoglycan low affinity receptors. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and plays an important role in mesoderm induction, stimulates the growth and development of the new blood vessels (angiogenesis), normal wound healing and tissue development. FGF-2 positively regulates hematopoiesis by acting on various cellular targets: stromal cells, early and committed hematopoietic progenitors and possibly some mature blood cells. FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.

• MeSH
• fibroblastový růstový faktor 2 chemie   farmakologie   fyziologie   MeSH
• fyziologická neovaskularizace fyziologie   MeSH
• hematopoéza fyziologie   MeSH
• lidé MeSH
• myeloproliferativní poruchy patofyziologie   MeSH
• receptory fibroblastových růstových faktorů metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH
• receptory fibroblastových růstových faktorů MeSH

Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF-2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF-2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular-mass isoforms of FGF-2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF-2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 --> FGFR3 --> FGFR4 --> FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF-2, extracellular signal-regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF-2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF-2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF-2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self-renewal or differentiation of hESCs.

• MeSH
• buněčná diferenciace MeSH
• embryo savčí cytologie   MeSH
• fibroblastový růstový faktor 2 biosyntéza   fyziologie   MeSH
• fosforylace MeSH
• kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• protein - isoformy MeSH
• pyrroly farmakologie   MeSH
• receptor fibroblastových růstových faktorů, typ 2 biosyntéza   fyziologie   MeSH
• receptory fibroblastových růstových faktorů biosyntéza   fyziologie   MeSH
• signální transdukce fyziologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• FGFR2 protein, human MeSH Prohlížeč
• fibroblastový růstový faktor 2 MeSH
• mitogenem aktivované proteinkinasy MeSH
• protein - isoformy MeSH
• pyrroly MeSH
• receptor fibroblastových růstových faktorů, typ 2 MeSH
• receptory fibroblastových růstových faktorů MeSH
• SU 5402 MeSH Prohlížeč

• MeSH
• fibroblastový růstový faktor 2 * MeSH
• fibroblasty MeSH
• hojení ran MeSH
• lidé MeSH
• popálení * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• Názvy látek
• fibroblastový růstový faktor 2 * MeSH

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.

• Klíčová slova
• Fibroblast Growth Factor 2, Unconventional protein secretion, biochemistry, biophysics, human, oligomerization, phosphoinositide, protein translocation across membranes, reconstitution with purified components, structural biology,
• MeSH
• fibroblastový růstový faktor 2 metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• heparitinsulfát metabolismus   MeSH
• membránové transportní proteiny metabolismus   MeSH
• multimerizace proteinu * MeSH
• sekreční vezikuly metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• heparitinsulfát MeSH
• membránové transportní proteiny MeSH

BACKGROUND: The purpose of dermal substitutes is to mimic the basic properties of the extracellular matrix of human skin. The application of dermal substitutes to the defect reduces the formation of hypertrophic scars and improves the scar quality. This study aims to develop an original dermal substitute enriched with stable fibroblast growth factor 2 (FGF2-STAB®) and test it in an animal model. METHODS: Dermal substitutes based on collagen/chitosan scaffolds or collagen/chitosan scaffolds with nanofibrous layer were prepared and enriched with FGF2-STAB® at concentrations of 0, 0.1, 1.0, and 10.0 µg ‧ cm-2. The performance of these dermal substitutes was tested in vivo on artificially formed skin defects in female swine. The outcomes were evaluated using cutometry at 3 and 6 months. In addition, visual appearance was assessed based on photos of the scars at 1-month, 3-month and 6-month follow-ups using Yeong scale and Visual Analog Scale. RESULTS: The dermal substitute was fully integrated into all defects and all wounds healed successfully. FGF2-STAB®-enriched matrices yielded better results in cutometry compared to scaffolds without FGF2. Visual evaluation at 1, 3, and 6 months follow-ups detected no significant differences among groups. The FGF2-STAB® effectiveness in improving the elasticity of scar tissues was confirmed in the swine model. This effect was independently observed in the scaffolds with nanofibres as well as in the scaffolds without nanofibres. CONCLUSION: The formation of scars with the best elasticity was exhibited by addition 1.0 µg ‧ cm-2of FGF2-STAB® into the scaffolds, although it had no significant effect on visual appearance at longer follow-ups. This study creates the basis for further translational studies of the developed product and its progression into the clinical phase of the research.

• Klíčová slova
• Animal experiment, Cutometry, Dermal substitute, Fibroblast grow factor 2, Scar, Visual assessment,
• MeSH
• chitosan * MeSH
• fibroblastový růstový faktor 2 * MeSH
• hojení ran účinky léků   MeSH
• jizva hypertrofická MeSH
• kolagen MeSH
• kůže MeSH
• modely nemocí na zvířatech MeSH
• nanovlákna terapeutické užití   MeSH
• popálení MeSH
• prasata MeSH
• pružnost * MeSH
• tkáňové podpůrné struktury MeSH
• umělá kůže * MeSH
• viskozita MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chitosan * MeSH
• fibroblastový růstový faktor 2 * MeSH
• kolagen MeSH

Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor family that possess broad mitogenic and cell survival activities and is involved in a variety of biological processes. We investigated possible genetic polymorphism in the promoter and 5' flanking region of the bFGF gene. Polymorphism was analysed by means of heteroduplex analysis, fragments with altered mobility were sequenced. Three novel substitutions (-553T/A, -834T/A and -921C/G) were identified in the promoter region. Allele frequencies in a sample of healthy Caucasian subjects (n=126) were determined by polymerase chain reactions followed by restriction analyses with specific endonucleases. The frequencies of the mutated alleles (-553A, -834A and -921G) were 0.04, 0.05 and 0.14, respectively. Newly identified variants in the bFGF gene promoter appear to be common polymorphisms in the Czech population.

• MeSH
• 5' přiléhající oblast DNA MeSH
• bodová mutace MeSH
• fibroblastový růstový faktor 2 genetika   MeSH
• genetická variace * MeSH
• lidé středního věku MeSH
• lidé MeSH
• promotorové oblasti (genetika) * MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH

Human embryonic stem cells (hESCs) are pluripotent stem cells with long-lasting capacity to self-renew and differentiate into various cell types of endodermal, ectodermal or mesodermal origin. Unlike mouse ESCs (mESCs), which can be maintained in an undifferentiated state simply by adding leukemia inhibitory factor (LIF) into the culture medium, hESCs are notorious for the sustained willingness to differentiate and not yet clearly defined signaling pathways that are crucial for their "stemness". Presently, our knowledge involves only limited number of growth factor signaling pathways that appear to be biologically relevant for stem cell functions in vitro. These include BMP, TGFbeta, Wnt, and FGF signaling pathway. The purpose of this review is to summarize recent data on the expression of FGFs and their receptors in hESCs, and critically evaluate the potential effects of FGF signals for their undifferentiated growth and/or differentiation in context with our current understanding of FGF/FGFR biology.

• MeSH
• biologické modely MeSH
• buněčná diferenciace fyziologie   MeSH
• embryo savčí cytologie   fyziologie   MeSH
• fibroblastový růstový faktor 2 * genetika   metabolismus   fyziologie   MeSH
• kmenové buňky cytologie   fyziologie   MeSH
• lidé MeSH
• receptory fibroblastových růstových faktorů * genetika   metabolismus   fyziologie   MeSH
• signální transdukce fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• fibroblastový růstový faktor 2 * MeSH
• receptory fibroblastových růstových faktorů * MeSH

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

• Klíčová slova
• FGFR, ICK, cilia length, fibroblast growth factor, intestinal cell kinase,
• MeSH
• buňky NIH 3T3 MeSH
• cilie metabolismus   MeSH
• CRISPR-Cas systémy MeSH
• fibroblastové růstové faktory metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• modely u zvířat MeSH
• myši knockoutované MeSH
• myši MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny hedgehog metabolismus   MeSH
• proteomika MeSH
• receptor fibroblastových růstových faktorů, typ 1 metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 4 metabolismus   MeSH
• receptory fibroblastových růstových faktorů genetika   metabolismus   MeSH
• signální transdukce MeSH
• simulace molekulového dockingu MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• CILK1 protein, human MeSH Prohlížeč
• Cilk1 protein, mouse MeSH Prohlížeč
• FGFR1 protein, human MeSH Prohlížeč
• FGFR3 protein, human MeSH Prohlížeč
• FGFR4 protein, human MeSH Prohlížeč
• fibroblastové růstové faktory MeSH
• protein-serin-threoninkinasy MeSH
• proteiny hedgehog MeSH
• receptor fibroblastových růstových faktorů, typ 1 MeSH
• receptor fibroblastových růstových faktorů, typ 3 MeSH
• receptor fibroblastových růstových faktorů, typ 4 MeSH
• receptory fibroblastových růstových faktorů MeSH

Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC-mediated FGF2 secretion is essential for hPSC self-renewal, and its lack mediates specific differentiation. Following both short hairpin RNA- and small interfering RNA-mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2-mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050-2059.

• Klíčová slova
• Cardiac differentiation, Embryonic stem cells, Fibroblast growth factor, Fibroblast growth factor 2, Neural differentiation, Pluripotent stem cells, Tyrosine kinase expressed in hepatocellular carcinoma,
• MeSH
• biologické markery metabolismus   MeSH
• buněčné linie MeSH
• buněčný rodokmen * účinky léků   MeSH
• down regulace účinky léků   MeSH
• fibroblastový růstový faktor 2 metabolismus   MeSH
• lidé MeSH
• pluripotentní kmenové buňky cytologie   enzymologie   MeSH
• proliferace buněk účinky léků   MeSH
• rekombinantní proteiny farmakologie   MeSH
• tyrosinkinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• fibroblastový růstový faktor 2 MeSH
• rekombinantní proteiny MeSH
• Tec protein-tyrosine kinase MeSH Prohlížeč
• tyrosinkinasy MeSH