Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human acidic fibroblast growth factor (hFGF-1) is a potent mitogen and is involved in the regulation of key cellular process such as angiogenesis, differentiation, and morphogenesis. hFGF-1 is a signal peptide-less protein that is released into the extracellular compartment as a multiprotein complex consisting of S100A13, synaptotagmin (Syt1), and a hFGF-1 homodimer. Cu(2+) is known to play an important role in the formation of the multiprotein release complex. The source of Cu(2+) required for the formation of the multiprotein release complex is not clear. In this study, we show that the cytoplasmic C2A domain of synaptotagmin binds to Cu(2+) ions with high affinity. Results from the isothermal calorimetry (ITC), near-UV circular dichroism (CD), and absorption spectroscopy experiments suggest that four Cu(2+) ions bind per molecule of C2A domain. Far-UV CD and limited trypsin digestion analysis reveal that the C2A domain undergoes a mild conformational change upon binding to Cu(2+). Competition experiments monitored by ITC and fluorescence resonance energy transfer indicate that Cu(2+) and Ca(2+) ions share common binding sites on the C2A domain. Cu(2+) ions compete with and replace Ca(2+) ions bound to the C2A domain. Two-dimensional nuclear magnetic resonance spectroscopy data clearly show that Cu(2+) ions bind to the Ca(2+) binding sites in the loops (loops 1-3) located at the apex of the structure of the C2A domain. In addition, there is a unique Cu(2+) binding site located in the loop connecting beta-strands 7 and 8. It appears that the C2A domain provides the Cu(2+) ions required for the formation of the multiprotein FGF release complex.
 ...
PMID:The C2A domain of synaptotagmin exhibits a high binding affinity for copper: implications in the formation of the multiprotein FGF release complex. 1626 43

S100A13 is a member of the S100 protein family that is involved in the copper-dependent nonclassical secretion of signal peptideless proteins fibroblast growth factor 1 and interleukin 1 lpha. In this study, we investigate the effects of interplay of Cu2+ and Ca2+ on the structure of S100A13 using a variety of biophysical techniques, including multi-dimensional NMR spectroscopy. Results of the isothermal titration calorimetry experiments show that S100A13 can bind independently to both Ca2+ and Cu2+ with almost equal affinity (Kd in the micromolar range). Terbium binding and isothermal titration calorimetry data reveal that two atoms of Cu2+/Ca2+ bind per subunit of S100A13. Results of the thermal denaturation experiments monitored by far-ultraviolet circular dichroism, limited trypsin digestion, and hydrogen-deuterium exchange (using 1H-15N heteronuclear single quantum coherence spectra) reveal that Ca2+ and Cu2+ have opposite effects on the stability of S100A13. Binding of Ca2+ stabilizes the protein, but the stability of the protein is observed to decrease upon binding to Cu2+. 1H-15N chemical shift perturbation experiments indicate that S100A13 can bind simultaneously to both Ca2+ and Cu2+ and the binding of the metal ions is not mutually exclusive. The results of this study suggest that the Cu2+-binding affinity of S100A13 is important for the formation of the FGF-1 homodimer and the subsequent secretion of the signal peptideless growth factor through the nonclassical release pathway.
 ...
PMID:Copper binding affinity of S100A13, a key component of the FGF-1 nonclassical copper-dependent release complex. 1676 22

Acidic fibroblast growth factor (aFGF) is a signal peptide-less protein that is secreted into the extracellular compartment as part of a multiprotein release complex, consisting of aFGF, S100A13 (a calcium binding protein), and a 40 kDa (p40) form of synaptotagmin (Syt1), a protein that participates in the docking of a variety of secretory vesicles. p40 Syt1, and specifically its C2A domain, is believed to play a major role in the non-classical secretion of the aFGF release complex mediated by the interaction of aFGF and p40 Syt1with the phospholipids of the cell membrane inner leaflet. In the present study, we investigate the structural characteristics of aFGF and the C2A domain of p40 Syt1 under acidic conditions, using a variety of biophysical techniques including multidimensional NMR spectroscopy. Urea-induced equilibrium unfolding (at pH 3.4) of both aFGF and the C2A domain are non-cooperative and proceed with the accumulation of stable intermediate states. 1-Anilino-8-napthalene sulfonate (ANS) binding and size-exclusion chromatography results suggest that both aFGF and the C2A domain exist as partially structured states under acidic conditions (pH 3.4). Limited trypsin digestion analysis and 1H-15N chemical shift perturbation data reveal that the flexibility of certain portions of the protein backbone is increased in the partially structured state(s) of aFGF. The residues that are perturbed in the partially structured state(s) in aFGF are mostly located at the N- and C-terminal ends of the protein. In marked contrast, most of the interactions stabilizing the native secondary structure are preserved in the partially structured state of the C2A domain. Isothermal titration calorimetry data indicate that the binding affinity between aFGF and the C2A domain is significantly enhanced at pH 3.4. In addition, both aFGF and the C2A domain exhibit much higher lipid binding affinity in their partially structured states. The translocation of the multiprotein FGF release complex across the membrane appears to be facilitated by the formation of partially structured states of aFGF and the C2A domain of p40 Syt1.
 ...
PMID:Relevance of partially structured states in the non-classical secretion of acidic fibroblast growth factor. 1763 70

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca2+-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca2+ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.
 ...
PMID:S100A13-lipid interactions-role in the non-classical release of the acidic fibroblast growth factor. 1799 55

Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)-Golgi secretory pathway. FGF1 is released through a Cu(2+)-mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu(2+)-mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that the binding of Cu(2+) to the C2B domain is important for the release of FGF1 into the extracellular medium. In this study, using a variety of biophysical studies, Cu(2+) and lipid interactions of the C2B domain of Syt1 were characterized. Isothermal titration calorimetry (ITC) experiments reveal that the C2B domain binds to Cu(2+) in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb(3+) show that there are two Cu(2+)-binding pockets on the C2B domain, and one of these is also a Ca(2+)-binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that the C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, the binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu(2+). The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.
...
PMID:Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor. 2522 45