The second messenger hydrogen peroxide is required for optimal activation of numerous signal transduction pathways, particularly those mediated by protein tyrosine kinases. One mechanism by which hydrogen peroxide regulates cellular processes is the transient inhibition of protein tyrosine phosphatases through the reversible oxidization of their catalytic cysteine, which suppresses protein dephosphorylation. Here we describe a structural analysis of the redox-dependent regulation of protein tyrosine phosphatase 1B (PTP1B), which is reversibly inhibited by oxidation after cells are stimulated with insulin and epidermal growth factor. The sulphenic acid intermediate produced in response to PTP1B oxidation is rapidly converted into a previously unknown sulphenyl-amide species, in which the sulphur atom of the catalytic cysteine is covalently linked to the main chain nitrogen of an adjacent residue. Oxidation of PTP1B to the sulphenyl-amide form is accompanied by large conformational changes in the catalytic site that inhibit substrate binding. We propose that this unusual protein modification both protects the active-site cysteine residue of PTP1B from irreversible oxidation to sulphonic acid and permits redox regulation of the enzyme by promoting its reversible reduction by thiols.

The 25-kDa Family 4 uracil-DNA glycosylase (UDG) from Pyrobaculum aerophilum has been expressed and purified in large quantities for structural analysis. In the process we observed it to be colored and subsequently found that it contained iron. Here we demonstrate that P. aerophilum UDG has an iron-sulfur center with the EPR characteristics typical of a 4Fe4S high potential iron protein. Interestingly, it does not share any sequence similarity with the classic iron-sulfur proteins, although four cysteines (which are strongly conserved in the thermophilic members of Family 4 UDGs) may represent the metal coordinating residues. The conservation of these residues in other members of the family suggest that 4Fe4S clusters are a common feature. Although 4Fe4S clusters have been observed previously in Nth/MutY DNA repair enzymes, this is the first observation of such a feature in the UDG structural superfamily. Similar to the Nth/MutY enzymes, the Family 4 UDG centers probably play a structural rather than a catalytic role.Uracil-DNA glycosylases are ubiquitous DNA repair enzymes responsible for the excision of uracil bases from DNA as the first step in a base excision repair pathway. Uracil arises in DNA either as a result of the hydrolytic deamination of cytosine residues in G:C base pairs (1) or from incorporation of deoxyuridine monophosphate (instead of thymidine monophosphate) opposite adenine during DNA replication (2). If left uncorrected the former process would cause G:C to A:T transition mutations (1), whereas the latter may result in the disruption of specific regulatory DNA-protein interactions (3).Hyperthermophilic organisms are at especially high risk of DNA damage by cytosine deamination, which is significantly enhanced by elevated temperature (4). Because hyperthermophiles do not exhibit any greater susceptibility to this type of damage they presumably possess more effective repair enzymes (5). However, despite the detection of UDG 1 activity in several hyperthermophiles (6) no sequences homologous to the archetypal Escherichia coli ung-encoded enzyme were initially apparent in archaeal genomes. Subsequently, UDGs were identified in hyperthermophilic Eubacteria and Archaea (7-9) with more obvious homology to a second family of uracil base excision repair enzymes typified by the human thymine DNA glycosylase (TDG) (10) and the bacterial MUG (11). These G:T/U mismatch-specific enzymes (Family 2) are structurally and mechanistically related to the UNG-type UDGs (Family 1) (12, 13), and they unite the UNG-type and thermophile enzymes (Family 4) into a uracil-DNA glycosylase superfamily (14). Pyrobaculum aerophilum is a hyperthermophilic archaeon isolated from a boiling marine water hole and growing optimally at 100°C and pH 7.0 (15). A fosmid-based genomic map of the 1.7-Mb P. aerophilum genome was constructed and used to identify 474 putative genes (16), but no homologues of the UNG or MUG/TDG UDG families were initially identified. Following the identification of Tm-UDG (a novel UDG weakly related to E....

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An immunodominant peptide (p185(378 -394)) derived from the c-erbB2 gene product, was recognized by an anti-DNA antibody, B3, and importantly by two classical DNA-binding proteins, Tgo polymerase and Pa-UDG. These reactivities were inhibited by DNA, confirming that the peptide mimicked DNA. BALB/c mice immunized with p185(378 -394) developed significant titers of IgG anti-dsDNA antibodies. Screening of 39 human lupus sera revealed that 5% of these sera possessed reactivity toward p185(378 -394). Representative mouse and human sera with anti-p185(378 -394) reactivity bound intact p185, and this binding was inhibited by dsDNA. This is the first demonstration of a naturally occurring autoantigen mimotope. The present study identifies a potential antigenic stimulus that might trigger systemic lupus erythematosus in a subset of patients.

Summary While immunoglobulin G (IgG) antibodies to double‐stranded (ds)DNA are serological markers of systemic lupus erythematosus (SLE), not all antibodies to DNA (anti‐DNA) are able to cause tissue damage to a similar extent. It has been proposed that anti‐DNA‐induced renal damage could be linked to differences in the fine specificity of the antibodies. In an attempt to gain insight into their fine binding properties, we investigated the cross‐reactivity of two human lupus monoclonal IgG anti‐dsDNA (B3 and RH14) to a recently described Escherichia coli PolIV (a DNA polymerase). These autoantibodies possess distinct pathogenic properties in severe combined immunodeficient (SCID) mice. Although both antibodies cause proteinuria, only RH14 induces early histological features of lupus nephritis. Both RH14 and B3 bound PolIV; however, they exhibited a marked difference in their reactivity to the PolIV–dsDNA complex. Alhough RH14 exhibited significant activity to the complex, the binding of B3 to PolIV complexed with dsDNA was almost abolished. Furthermore, there was a significant difference in the way the lupus sera recognized naked dsDNA and that presented on PolIV. Although 67% of lupus sera bound naked dsDNA, ≈ 90% of these sera (93% calf thymus DNA; 90% synthetic oligonucleotide) reacted to the complex when dsDNA was presented on PolIV. Thus, the IgG anti‐dsDNA likely to exist in lupus patients may be distinguished into those that recognize dsDNA in the context of PolIV and those which do not. This difference in binding ability may help to distinguish those dsDNA antibodies that are more pathogenic.