Dual-site recognition of different extracellular matrix components by anti-angiogenic/neurotrophic serpin,PEDF

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) superfamily, possesses anti-angiogenic and neurotrophic activities. PEDF has been reported to bind to extracellular matrix (ECM) components such as collagens and glycosaminoglycans (GAGs). In this study, to determine the binding sites for collagens and GAGs, we analyzed the interaction of recombinant mouse PEDF (rPEDF) with collagen I and heparin. By utilizing residue-specific chemical modification and site-directed mutagenesis techniques, we revealed that the acidic amino acid residues on PEDF (Asp(255), Asp(257), and Asp(299)) are critical to collagen binding, and three clustered basic amino acid residues (Arg(145), Lys(146), and Arg(148)) are necessary for heparin binding. Mapping of these residues on the crystal structure of human PEDF (Simonovic, M., Gettins, P. G. W., and Volz, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 11131-11135) demonstrated that the collagen-binding site is oriented toward the opposite side of the highly basic surface where the heparin-binding site is localized. These results indicate that PEDF possesses dual binding sites for different ECM components, and this unique localization of ECM-binding sites implies that the binding to ECM components could regulate PEDF activities.     

Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation-induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with beta-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.     

Clostridium perfringens epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes

Clostridium perfringens epsilon-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled epsilon-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-beta-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When epsilon-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.     

Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage

Osteoarthritis is a degenerative joint disorder characterized by breakdown of articular cartilage. Degradation of aggrecan, which together with type II collagen provides cartilage with its unique characteristics of compressibility and elasticity, is an early and sustained feature of osteoarthritis. The present work was set up to identify the enzyme(s) responsible for aggrecan breakdown in osteoarthritis. We found that the two cartilage aggrecanases, ADAM-TS4 and ADAM-TS5, are present in osteoarthritic cartilage and that they are responsible for aggrecan degradation without the participation of matrix metalloproteinases. This is based on 1) neoepitopes found on aggrecan fragments in osteoarthritis (OA) cartilage explants in vitro, 2) aggrecan fragments detected in synovial fluid of OA patients, 3) the observation that an aggrecanase inhibitor, BB-16, blocked aggrecan degradation in OA cartilage in vitro, whereas the matrix metalloproteinase inhibitor XS309 did not, and 4) the presence of mRNA and protein for ADAM-TS4 and ADAM-TS5 in OA cartilage. These results suggest that ADAM-TS4 and ADAM-TS5 represent a potential target for the treatment of osteoarthritis.     

Mitochondrial cytochrome c oxidase as a target site for cephalosporin antibiotics in renal epithelial cells (LLC-PK(1)) and renal cortex

We reported previously that treatment of the pig kidney proximal tubular epithelial cell line LLC-PK(1) with cephaloridine (CLD) decreased the activity of cytochrome c oxidase in the mitochondria of the cells followed by increases in lipid peroxidation and cell necrosis. In this study, we investigated the effects of CLD on the activity of cytochrome c oxidase in mitochondria isolated from LLC-PK(1) cells and purified the enzyme from mitochondria of the rat renal cortex. The activity of cytochrome c oxidase in the isolated mitochondria from LLC-PK(1) cells was significantly decreased from 1 h after addition of 1 mM CLD. Other cephalosporin antibiotics, cefazolin and cefalotin, also decreased the activity of cytochrome c oxidase in the isolated mitochondria. The activity of cytochrome c oxidase purified from the mitochondria of the rat renal cortex was also decreased from 2 h after addition of 1 mM CLD in a non-competitive manner. These results suggest that the direct inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain by cephlosporins may result from the observed nephrotoxicity.     

Identification of the regulatory elements of the human von Willebrand factor for binding to platelet GPIb. Importance of structural integrity of the regions flanked by the CYS1272-CYS1458 disulfide bond

In vitro platelet glycoprotein Ib (GPIb) binding of the human von Willebrand factor (VWF) increases markedly by exogenous modulators such as ristocetin or botrocetin, and the binding does not occur in normal circulation. GPIb binding sites have been assigned in the VWF A1 domain, which consists of a disulfide loop Cys1272(509)-Cys1458(695) where amino acid residues are numbered from the starting methionine as +1. The previous numbering from the N-terminal Ser of the mature processed VWF is indicated in parentheses. In contrast, several gain-of-function mutations have been found in two regions comprised of the disulfide loop and its N- and C-terminal flanking regions. In this study, Cys1222(459)-Tyr1271(508), Gln1238(475)-Tyr1271(508), Glu1260(497)-Tyr1271(508), and Asp1459(696)-Asp1472(709) were sequentially deleted of full-length multimeric recombinant VWF. Deletions at either side resulted in normal GPIb binding, indicating that the flanking regions are not GPIb binding sites. However, the addition of a mutation at Arg1308(545) on each deletion mutant resulted in spontaneous GPIb binding without requiring modulators, suggesting that both regions are important for the inhibition of GPIb binding. Spontaneous binding was completely inhibited by monoclonal antibodies that recognize the GPIb binding sites. Interestingly, mutant proteins with N-terminal but not C-terminal deletions lost binding to monoclonal antibodies B328, B710, and 23C7, which selectively inhibit ristocetin-induced GPI binding. Their epitopes were found at His1268(505) or Asp1269(506). The crystallographic structure of the A1 domain suggests that GPIb binding is influenced by the molecular interface between the two regions and that the antibody binding to the interface inhibits binding.     

Novel variant isoform of G-CSF receptor involved in induction of proliferation of FDCP-2 cells: relevance to the pathogenesis of myelodysplastic syndrome

Recent studies have shown that point mutations in granulocyte colony-stimulating factor receptor (G-CSFR) are involved in the pathogenesis of severe congenital neutropenia (SCN) and in the transformation of SCN to acute myelogenous leukemia (AML). It is reasonably speculated that the abnormalities in the signal transduction pathways for G-CSF could be partly responsible for the pathogenesis and the development to AML in patients with myelodysplastic syndromes (MDS). Therefore, we investigated the structural and functional abnormalities of the G-CSFR in 14 patients with MDS and 10 normal subjects. In in vitro colony forming assay, MDS samples showed reduced response to growth factors. However, G-CSF, but not GM-CSF and IL-3, enhanced clonal growth in three cases of high risk patients with MDS (RAEB, RAEB-t, and MDS having progressed to acute myeloid leukemia (AML)) and one low risk patient (RA). Eight out of 14 patients including above 4 patients demonstrated a common deletion of the G-CSFR cDNA; a deletion of three nucleotides (2128-2130) in the juxtamembrane domain of the G-CSFR, which resulted in a conversion of Asn(630)Arg(631) to Lys(630). To assess the functional activities of this deletion in the G-CSFR isoform, a mutant with the same three-nucleotide deletion was constructed by site-directed mutagenesis. FDCP-2 cells expressing the G-CSFR isoform responded to G-CSF, and exhibited proliferative responses than did those cells having wild-type G-CSFR. Moreover, these isoforms showed prolonged activation of STAT3 in response to G-CSF than did the wild-type. These results suggest that the deletion in the juxtamembrane domain of the G-CSFR gives a growth advantage to abnormal MDS clones and may contribute to the pathogenesis of MDS.