Position Statement on Atopic Dermatitis in Sub-Saharan Africa: current status and roadmap.

BACKGROUND: The first International Society of Atopic Dermatitis (ISAD) global meeting dedicated to atopic dermatitis (AD) in Sub-Saharan Africa (SSA) was held in Geneva, Switzerland in April 2019. A total of 30 participants were present at the meeting, including those from 17 SSA countries, representatives of the World Health Organization (WHO), the International Foundation for Dermatology (IFD) (a committee of the International League of Dermatological Societies, ILDS www.ilds.org), the Fondation pour la Dermatite Atopique, as well as specialists in telemedicine, artificial intelligence and therapeutic patient education (TPE). RESULTS: AD is one of the most prevalent chronic inflammatory skin diseases in SSA. Besides neglected tropical diseases (NTDs) with a dermatological presentation, AD requires closer attention from the WHO and national Departments of Health. CONCLUSIONS: A roadmap has been defined with top priorities such as access to essential medicines and devices for AD care, in particular emollients, better education of primary healthcare workers for adequate triage (e.g. better educational materials for skin diseases in pigmented skin generally and AD in particular, especially targeted to Africa), involvement of traditional healers and to a certain extent also patient education, bearing in mind the barriers to effective healthcare faced in SSA countries such as travel distances to health facilities, limited resources and the lack of dermatological expertise. In addition, several initiatives concerning AD research in SSA were discussed and should be implemented in close collaboration with the WHO and assessed at follow-up meetings, in particular, at the next ISAD meeting in Seoul, South Korea and African Society of Dermatology and Venereology (ASDV) meeting in Nairobi, Kenya, both in 2020.

Potential double-flipping mechanism by E. coli MutY.

To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted. MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation. However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency. Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A. The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY. Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy. The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase. Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix.

Repair of hydantoins, one electron oxidation product of 8-oxoguanine, by DNA glycosylases of Escherichia coli.

8-oxoguanine (8-oxoG), induced by reactive oxygen species and arguably one of the most important mutagenic DNA lesions, is prone to further oxidation. Its one-electron oxidation products include potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) because of their mispairing with A or G. All three oxidized base-specific DNA glycosylases of Escherichia coli, namely endonuclease III (Nth), 8-oxoG-DNA glycosylase (MutM) and endonuclease VIII (Nei), excise Gh and Sp, when paired with C or G in DNA, although Nth is less active than the other two. MutM prefers Sp and Gh paired with C (kcat/K(m) of 0.24-0.26 min(-1) x nM(-1)), while Nei prefers G over C as the complementary base (k(cat)/K(m) - 0.15-0.17 min(-1) x nM(-1)). However, only Nei efficiently excises these paired with A. MutY, a 8-oxoG.A(G)-specific A(G)-DNA glycosylase, is inactive with Gh(Sp).A/G-containing duplex oligonucleotide, in spite of specific affinity. It inhibits excision of lesions by MutM from the Gh.G or Sp.G pair, but not from Gh.C and Sp.C pairs. In contrast, MutY does not significantly inhibit Nei for any Gh(Sp) base pair. These results suggest a protective function for MutY in preventing mutation as a result of A (G) incorporation opposite Gh(Sp) during DNA replication.

MutY catalytic core, mutant and bound adenine structures define specificity for DNA repair enzyme superfamily.

The DNA glycosylase MutY, which is a member of the Helix-hairpin-Helix (HhH) DNA glycosylase superfamily, excises adenine from mispairs with 8-oxoguanine and guanine. High-resolution crystal structures of the MutY catalytic core (cMutY), the complex with bound adenine, and designed mutants reveal the basis for adenine specificity and glycosyl bond cleavage chemistry. The two cMutY helical domains form a positively-charged groove with the adenine-specific pocket at their interface. The Watson-Crick hydrogen bond partners of the bound adenine are substituted by protein atoms, confirming a nucleotide flipping mechanism, and supporting a specific DNA binding orientation by MutY and structurally related DNA glycosylases.

Cloning, overexpression, and biochemical characterization of the catalytic domain of MutY.

Proteolysis of MutY with trypsin indicated that this DNA mismatch repair enzyme could exist as two modules and that the N-terminal domain (Met1-Lys225), designated as p26, could serve as the catalytic domain [Manuel et al. (1996) J. Biol. Chem. 271, 16218-16226]. In this study, the p26 domain has been cloned, overproduced, and purified to homogeneity. Synthetic DNA duplexes containing mismatches, generated with regular bases and nucleotide analogs containing altered functional groups, have been used to characterize the substrate specificity and mismatch repair efficiency of p26. In general, p26 recognized and cleaved most of the substrates which were catalyzed by the intact protein. However, p26 displayed enhanced specificity for DNA containing an inosine. guanine mismatch, and the specificity constant (Kcat/Km) was 2-fold higher. The truncated MutY was able to cleave DNA containing an abasic site with equal efficiency. Dissociation constants (Kd) were obtained for p26 on noncleavable DNA substrates containing a tetrahydrofuran (abasic site analog) or a reduced abasic site. p26 bound these substrates with high specificity, and the Kd values were 3-fold higher when compared to the intact MutY. p26 contains both DNA glycosylase and AP lyase activities, and we provide evidence for a reaction mechanism that proceeds through an imino intermediate. Thus, we have shown for the first time that deletion of 125 amino acids at the C-terminus of MutY generates a stable catalytic domain which retains the functional identity of the intact protein.

Identification of the structural and functional domains of MutY, an Escherichia coli DNA mismatch repair enzyme.

The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26- and 13-kDa fragments. NH2-terminal sequencing showed that the p13 domain begins at Gln226, indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A.G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2'-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met1-Trp216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.

The interaction of T4 endonuclease V E23Q mutant with thymine dimer- and tetrahydrofuran-containing DNA.

The interaction between endonuclease V, the cyclobutane pyrimidine dimer-specific N-glycosylase/abasic lyase from bacteriophage T4, and DNA was investigated by DNase I footprinting methods. The catalytically inactive mutant E23Q was found to interact with a smaller region of DNA at the abasic site analog, tetrahydrofuran, than at a thymine dimer site. Like the wild-type enzyme, the mutant contacted the DNA substrates primarily on the strand opposite the damage. The various complexes examined by footprinting techniques represent distinct points along the catalytic pathway of endonuclease V: before catalysis at a dimer, after N-glycosylase action but before abasic lyase action, and before catalysis at an abasic site. The differences between the footprints of the mutant and wild-type enzymes on both DNA substrates likely represent subtly different conformations within these complexes.

Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V.

Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the alpha-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino-terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.