ABSTRACT
Purpose: Abnormal blood vessel formation is a defining feature of many blinding eye diseases. Targeting abnormal angiogenesis by inhibiting VEGF has revolutionized the treatment of many ocular angiogenic diseases over the last decade. However, a substantial number of patients are refractory to anti-VEGF treatment or may develop resistance over time. The objective of this study was to determine the efficacy and the mechanism of action of Apratoxin S4 in ocular angiogenesis. Methods: Retinal vascular cell proliferation, migration, and the ability to form tube-like structure were studied in vitro. Ex vivo aortic ring, choroid, and metatarsal assays were used to study Apratoxin S4's impact on vessel outgrowth in a multicellular environment. Apratoxin S4 was also tested in mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), and in a rabbit model of persistent retinal neovascularization (PRNV). Western blot and ELISA were used to determine the expression of key angiogenic regulators after Apratoxin S4 treatment. Results: Apratoxin S4 strongly inhibits retinal vascular cell activation by suppressing multiple angiogenic pathways. VEGF-activated vascular cells and angiogenic vessels are more susceptible to Apratoxin S4 treatment than quiescent vascular cells and vessels. Both intraperitoneal and intravitreal delivery of Apratoxin S4 are able to impede ocular neovascularization in vivo. Apratoxin S4 specifically attenuates pathological ocular angiogenesis and exhibits a combinatorial inhibitory effect with standard-of-care VEGF inhibitor drug (aflibercept). Conclusions: Apratoxin S4 is a potent antiangiogenic drug that inhibits the activation of retinal endothelial cells and pericytes through mediating multiple angiogenic pathways.
Subject(s)
Depsipeptides/administration & dosage , Retinal Neovascularization/drug therapy , Retinal Vessels/pathology , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Female , Humans , Intravitreal Injections , Male , Mice , Mice, Inbred C57BL , Rabbits , Retinal Neovascularization/pathology , Retinal Vessels/drug effects , Treatment OutcomeABSTRACT
EcoP1I DNA MTase (M.EcoP1I), an N(6)-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5'-AGACC-3' and methylates the internal adenine. M.EcoP1I requires Mg(2+) for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn(2+) and Ca(2+) other than Mg(2+) with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [(358)ID(x)(n) ExK(401) and (600)DxDxD(604) motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.
ABSTRACT
The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin Nup153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and Nup153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or Nup153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor.
Subject(s)
Cell Nucleus/metabolism , Conserved Sequence , Nuclear Pore Complex Proteins/metabolism , Retroelements/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Animals , Epigenesis, Genetic , Humans , Molecular Sequence Data , Mutation/genetics , Nuclear Pore Complex Proteins/chemistry , Peptides/chemistry , Protein Structure, Tertiary , Rabbits , Repetitive Sequences, Amino Acid , Schizosaccharomyces/cytology , Schizosaccharomyces pombe Proteins/chemistry , Sequence Homology, Amino Acid , Structure-Activity Relationship , alpha Karyopherins/metabolism , beta Karyopherins/metabolismABSTRACT
Restriction-modification (R-M) enzymes are classified into type I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. While the role of S-Adenosyl-L-methionine (AdoMet) as the methyl group donor in the methylation reaction is undisputed, its requirement in DNA cleavage reaction has been subject to intense study. AdoMet is a prerequisite for the DNA cleavage by most type I enzymes known so far, with the exception of R.EcoR124I. A number of new type II restriction enzymes belonging to the type IIB and IIG family were found to show AdoMet dependence for their cleavage reaction. The type III enzymes have been found to require AdoMet for their restriction function. AdoMet functions as an allosteric effector of the DNA cleavage reaction and has been shown to bring about conformational changes in the protein upon binding.
Subject(s)
DNA Restriction Enzymes/metabolism , S-Adenosylmethionine/physiology , DNA Modification Methylases/metabolism , DNA Restriction Enzymes/chemistry , DNA Restriction Enzymes/classification , Molecular Structure , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolismABSTRACT
EcoP1I methyltransferase (M.EcoP1I) belongs to the type III restriction-modification system encoded by prophage P1 that infects Escherichia coli. Binding of M.EcoP1I to double-stranded DNA and single-stranded DNA has been characterized. Binding to both single- and double-stranded DNA could be competed out by unlabeled single-stranded DNA. Metal ions did not influence DNA binding. Interestingly, M.EcoP1I was able to methylate single-stranded DNA. Kinetic parameters were determined for single- and double-stranded DNA methylation. This feature of the enzyme probably functions in protecting the phage genome from restriction by type III restriction enzymes and thus could be considered as an anti-restriction system. This study describing in vitro methylation of single-stranded DNA by the type III methyltransferase EcoP1I allows understanding of the mechanism of action of these enzymes and also their role in the biology of single-stranded phages.
