PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways
P-selectin glycoprotein ligand-1 (PSGL-1) is a mucinlike ligand for P-and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P-and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Gal1-3Gal-NAc␣1-Ser/Thr); they lack core 2 O-linked glycans (Gal1-3(Gal1-4GlcNAc1-6)GalNAc␣1-Ser/Thr) because they do not express the core 2 1-6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack ␣1-3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P-and E-selectin only when it was co-expressed with both C2GnT and an ␣1-3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of -eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to Pselectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P-and E-selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin.
P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin on human leukocytes, mediates rolling of leukocytes on P-selectin expressed by activated platelets or endothelial cells under shear forces. PSGL-1 requires both tyrosine sulfate and O-linked glycans to bind P-selectin. Electron microscopy of rotary-shadowed PSGL-1 purified from human neutrophils indicated that it is a highly extended molecule with an extracellular domain that is -50 nm long. Both individual PSGL-1 molecules and rosettes composed of several molecules presumably attached at their transmembrane segments were observed. The extracellular domain of PSGL-1 has 318 residues, including a signal peptide from residues 1-18 and a propeptide from residues 19-41. Using bacterially expressed fusion proteins and synthetic peptides derived from the extracellular domain, we mapped the epitopes for two IgG anti-PSGL-1 monoclonal antibodies, PL1 and PL2. PL2 bound to a region within residues 188-235 that is located in a series of decameric consensus repeats. PL1, which blocks binding of PSGL-1 to P-selectin, recognized an epitope spanning residues 49-62. This sequence overlaps the tyrosine sulfation sites at residues 46, 48, and 51 that have been implicated in binding of PSGL-1 to P-selectin. Our results demonstrate that PSGL-1 is a long, extended molecule and suggest that the P-selectin binding site is located near the N terminus, well above the membrane. This location may facilitate interactions of PSGL-1 with P-selectin under shear stress.
Many of the protein-protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3-and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three Nterminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCHNck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor- was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2-and SH3-domain-containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways.
Polymeric micelles are emerging as an effective drug delivery system for hydrophobic photosensitizers in photodynamic therapy (PDT). The objective of this study was to investigate the formulation of hydrophobic protoporphyrin IX (PpIX) with MePEG(5000)-b-PCL(4100) [methoxy poly (ethylene glycol)-b-poly (caprolactone)] diblock copolymers and to compare their PDT response to that of free PpIX. The photophysical and photochemical properties of the polymeric PpIX micelles were studied by measuring absorbance and fluorescence spectra, PpIX-loading efficiency and stability, the micelle particle size and morphology, as well as singlet oxygen luminescence and lifetime. The spherical micelles have a high PpIX-loading efficiency of 82.4% and a narrow size distribution with a mean diameter of 52.2 +/- 6.4 nm. The cellular uptake of PpIX in RIF-1 cells using PpIX micelles was approximately two-fold higher than that for free PpIX. Free PpIX and PpIX formulated in micelles exhibited similar subcellular localization in or around the cellular plasma membrane, as demonstrated using fluorescence microscopy. In vitro PDT results showed that the PpIX micelles have markedly increased photocytotoxicity over that with free PpIX, by nearly an order of magnitude at the highest light dose used. The micelles alone had no evident phototoxicity or dark toxicity. These findings suggest that MePEG(5000)-b-PCL(4100) diblock copolymer micelles have great potential as a drug delivery system for hydrophobic photodynamic sensitizers.
We synthesized a rare (3,12)-connected zirconium metal–organic framework with a sky topology showing efficient iodine adsorption and pH sensing capacity.
A major goal of metal−organic framework (MOF) research is to control the structures and functions of materials in accordance with their specific applications. However, due to the flexible coordination modes between metal ions and organic linkers in MOFs, it is still challenging to rationally assemble a framework with deliberate structures and desired functional groups. Sometimes, two or more phases coexist in a one-pot reaction, making them difficult to separate. To this end, sieving and purification of MOF mixtures become vital for the following application. Herein, we demonstrate that the formation of zirconium-based MOFs (Zr-MOFs) can be regulated in a wider two-dimensional scale by thermodynamics using auxiliary linkers. The auxiliary linkers favor the formation of the targeted Zr-MOF by selectively binding to its coordination vacancies and therefore increasing its formation enthalpy to achieve the sieving of MOF mixture. Furthermore, the resulting mixed-linkers MOFs not only maintain porosities but also contain the installed auxiliary linkers as chemical handles to further incorporate functional groups, providing the possibility of introduction of active sites through postmodification. Finally, this synthetic strategy was applied to assemble a cooperative catalytic system in a MOF platform for CO 2 cycloaddition with epoxides. To the best of our knowledge, this is the first example of sieving and functionalization of MOFs through insertion and post-modification of auxiliary linkers.
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