Numerous proteins, many essential for the DNA replication machinery, interact with proliferating cell nuclear antigen (PCNA) through the PCNA-interacting peptide (PIP) sequence called the PIP box. We have previously shown that the oxidative demethylase human AlkB homologue 2 (hABH2) colocalizes with PCNA in replication foci. In this study, we show that hABH2 interacts with a posttranslationally modified PCNA via a novel PCNA-interacting motif, which we term AlkB homologue 2 PCNA-interacting motif (APIM). We identify APIM in >200 other proteins involved in DNA maintenance, transcription, and cell cycle regulation, and verify a functional APIM in five of these. Expression of an APIM peptide increases the cellular sensitivity to several cytostatic agents not accounted for by perturbing only the hABH2–PCNA interaction. Thus, APIM is likely to mediate PCNA binding in many proteins involved in DNA repair and cell cycle control during genotoxic stress.
The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.
Heparan sulfate interacts with growth factors, matrix components, effectors and modulators of enzymatic catalysis as well as with microbial proteins via sulfated oligosaccharide domains. Although a number of such domains have been characterized, little is known about the regulation of their formation in vivo. Here we show that the structure of human aorta heparan sulfate is gradually modulated during aging in a manner that gives rise to markedly enhanced binding to isoforms of platelet-derived growth factor A and B chains containing polybasic cell retention sequences. By contrast, the binding to fibroblast growth factor 2 is affected to a much lesser extent. The enhanced binding of aorta heparan sulfate to platelet-derived growth factor is suggested to be due to an age-dependent increase of GlcN 6-O-sulfation, resulting in increased abundance of the trisulfated L-iduronic acid (2-OSO 3 )-GlcNSO 3 (6-OSO 3 ) disaccharide unit. Such units have been shown to hallmark the platelet-derived growth factor A chain-binding site in heparan sulfate.Interactions of the sulfated glycosaminoglycan (GAG) 1 heparan sulfate (HS) with various proteins affect the biological activity, tissue localization, and turnover of the protein ligands (1-3). Such interactions, generally electrostatic in nature, regularly involve specific oligosaccharide domains generated by an elaborate biosynthetic machinery in the Golgi apparatus. HS formation starts by assembly of an initial (GlcA-GlcNAc) n polymer. Parts of the nascent polymer are subsequently modified by Ndeacetylation/N-sulfation of GlcNAc residues, and further modifications, including C-5 epimerization of GlcA residues into IdceA residues as well as O-sulfation at various positions, occur mainly in the vicinity of the previously incorporated N-sulfate groups (3, 4). The O-sulfate groups are predominantly found at the C-2 position of IdceA residues and the C-6 position of GlcN residues. The protein-binding HS domains typically reside within the Nsulfated regions, their functional specificity being determined by the pattern of modification, particularly the positioning of sulfate groups. Although information regarding the structures of the recognition sites for individual proteins (5-11) and the general features of HS biosynthesis (3) is accumulating, the biological control of HS structure and function remains poorly understood. Nevertheless, HS species from various cells and tissues clearly differ in their structure and in some studies such differences have been correlated to differential protein-binding properties (12-15). These and other findings (discussed in Refs. 1 and 3) suggest that the biosynthesis of HS is subject to regulation during development or aging. Control of the appropriate expression of functional HS domains in given organs or at particular developmental stages would appear essential, whereas, conversely, perturbed regulation could contribute to various pathologies. In the present study we have explored the aging aortic wall as a model to gain insight into the control...
Cell surface heparan sulfates mediate primary attachment of herpes simplex virus type 1, the first step in virus invasion of the cells. Removal of the host cell heparan sulfate results in a significantly diminished susceptibility of the cell to virus infection. On the virus envelope, glycoprotein C has been identified as the major binding site for heparan sulfate in the primary attachment of the virus to host cells. Using selectively desulfated heparins and metabolically labeled host cell heparan sulfate, we have analyzed the structural requirements of heparan sulfate to provide binding sites for glycoprotein C and the whole virus. Employing glycoprotein C affinity chromatography and a virus binding assay, we subfractionated oligosaccharides derived from heparan sulfate and partially desulfated heparin into selectively bound and unbound pools. These were chemically depolymerized and analyzed at the disaccharide level. The shortest glycoprotein C-binding fragment consisted of 10 -12 monosaccharide units containing at least one 2-O-and one 6-O-sulfate group that have to be localized in a sequence-specific way, based on the finding that bound and unbound HS fragments do not differ in charge or composition. The binding sequence is found within N-sulfated blocks of heparan sulfate, although several N-acetyl groups can be tolerated within the minimal binding sequence. These minimal requirements for herpes simplex virus type 1 binding to heparan sulfate are clearly distinct from other identified protein binding sites.Herpes simplex virus type 1 (HSV-1) 1 infection is ubiquitous with a broad variation of clinical symptoms, from perioral lesions to encephalitis (for review see Ref. 1). The first step in viral infection is the attachment to the host cell that enables the virus to penetrate into the cell. Heparan sulfate (HS) on the host cell surface has been implicated in the adherence of numerous microbial agents (2) including human immunodeficiency virus (3), HSV-1 and HSV-2 (4, 5). That HS chains act as receptors for HSV-1 was demonstrated by inhibition of attachment to and infection of cells through exogenously added heparin (6) and by the finding that HSV-1 attaches poorly to HS-deficient mutant Chinese hamster ovary cells (5). Earlier work has shown that the interaction occurs via binding of HSV-1 envelope glycoproteins to cell surface HS. Studies with HSV-1 strains devoid of specific glycoproteins have revealed that glycoprotein C (gC) is the major mediator of the virus attachment (7). gC is a 120-kDa glycoprotein that is conserved within the simplex virus genus (8). Using synthetic peptides, it was previously shown that a cluster of basic amino acids (Arg-143, Arg-145, and Arg-147) as well as Gly-247, located in separate regions of the primary sequence of gC, are essential for the interaction (9). Besides gC, another HSV-1 glycoprotein designated gB, which is involved in virus penetration into the cell, also binds heparin (7). However, it is not known whether gB-HS interaction does indeed occur in wild-type gC pos...
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