T Cell Homeostatic Proliferation Promotes a Redox State That Drives Metabolic and Epigenetic Upregulation of Inflammatory Pathways in Lupus.

Significance: Numerous abnormalities in T cells have been described in patients with systemic lupus erythematosus (SLE), including lymphopenia, DNA demethylation, expression of endogenous retroviruses (ERVs), increased cell death, enlarged mitochondria, production of reactive oxygen species (ROS), and the appearance of unusual CD4-CD8- T cells. Our studies propose a model in which accelerated homeostatic proliferation of T cells promotes an epigenetic and metabolic program, leading to this cluster of abnormalities. Recent Advances: Growing knowledge of the innate immune disorders in SLE has included increased mitochondrial size and ROS production that induces oligomerization of the mitochondrial antiviral signaling (MAVS) protein and type I interferon production, as well as DNA demethylation, upregulation of inflammatory genes, and expression of certain ERVs in SLE peripheral blood mononuclear cells. All these events are part of the cellular program that occurs during homeostatic proliferation of T cells. Evidence from a murine model of SLE as well as in human SLE reveals that increased T cell homeostatic proliferation may be a driving factor in these processes. Critical Issues: Despite extensive knowledge of the myriad autoantibodies in SLE and other immune abnormalities, a cogent model has been lacking to link the numerous and seemingly disparate immune aberrations. This may partly explain the general lack of new drugs specifically for SLE in over 50 years. A more coherent model of SLE would not only unify the variety of immune abnormalities is SLE but would also suggest new therapies. Future Directions: The model of augmented homeostatic proliferation leading to increased mitochondrial mass, ROS, DNA demethylation, and upregulation of inflammatory genes suggests strategic new targets for SLE, including antioxidants and certain inhibitors of metabolism. Antioxid. Redox Signal. 36, 410-422.

Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues.

The base excision repair DNA glycosylases, EcoNth and hNTHL1, are homologous, with reported overlapping yet different substrate specificities. The catalytic amino acid residues are known and are identical between the two enzymes although the exact structures of the substrate binding pockets remain to be determined. We sought to explore the sequence basis of substrate differences using a phylogeny-based design of site-directed mutations. Mutations were made for each enzyme in the vicinity of the active site and we examined these variants for glycosylase and lyase activity. Single turnover kinetics were done on a subgroup of these, comparing activity on two lesions, 5,6-dihydrouracil and 5,6-dihydrothymine, with different opposite bases. We report that wild type hNTHL1 and EcoNth are remarkably alike with respect to the specificity of the glycosylase reaction, and although hNTHL1 is a much slower enzyme than EcoNth, the tighter binding of hNTHL1 compensates, resulting in similar kcat/Kd values for both enzymes with each of the substrates tested. For the hNTHL1 variant Gln287Ala, the specificity for substrates positioned opposite G is lost, but not that of substrates positioned opposite A, suggesting a discrimination role for this residue. The EcoNth Thr121 residue influences enzyme binding to DNA, as binding is significantly reduced with the Thr121Ala variant. Finally, we present evidence that hNTHL1 Asp144, unlike the analogous EcoNth residue Asp44, may be involved in resolving the glycosylase transition state.

Using shifts in amino acid frequency and substitution rate to identify latent structural characters in base-excision repair enzymes.

Protein evolution includes the birth and death of structural motifs. For example, a zinc finger or a salt bridge may be present in some, but not all, members of a protein family. We propose that such transitions are manifest in sequence phylogenies as concerted shifts in substitution rates of amino acids that are neighbors in a representative structure. First, we identified rate shifts in a quartet from the Fpg/Nei family of base excision repair enzymes using a method developed by Xun Gu and coworkers. We found the shifts to be spatially correlated, more precisely, associated with a flexible loop involved in bacterial Fpg substrate specificity. Consistent with our result, sequences and structures provide convincing evidence that this loop plays a very different role in other family members. Second, then, we developed a method for identifying latent protein structural characters (LSC) given a set of homologous sequences based on Gu's method and proximity in a high-resolution structure. Third, we identified LSC and assigned states of LSC to clades within the Fpg/Nei family of base excision repair enzymes. We describe seven LSC; an accompanying Proteopedia page (http://proteopedia.org/wiki/index.php/Fpg_Nei_Protein_Family) describes these in greater detail and facilitates 3D viewing. The LSC we found provided a surprisingly complete picture of the interaction of the protein with the DNA capturing familiar examples, such as a Zn finger, as well as more subtle interactions. Their preponderance is consistent with an important role as phylogenetic characters. Phylogenetic inference based on LSC provided convincing evidence of independent losses of Zn fingers. Structural motifs may serve as important phylogenetic characters and modeling transitions involving structural motifs may provide a much deeper understanding of protein evolution.

Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases.

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from gamma-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k(obs) of over 2500min(-1).

Clostridium acetobutylicum 8-oxoguanine DNA glycosylase (Ogg) differs from eukaryotic Oggs with respect to opposite base discrimination.

During repair of damaged DNA, the oxidized base 8-oxoguanine (8-oxoG) is removed by 8-oxoguanine-DNA glycosylase (Ogg) in eukaryotes and most archaea, whereas in most bacteria it is removed by formamidopyrimidine-DNA glycosylase (Fpg). We report the first characterization of a bacterial Ogg, Clostridium acetobutylicum Ogg (CacOgg). Like human OGG1 and Escherichia coli Fpg (EcoFpg), CacOgg excised 8-oxoguanine. However, unlike hOGG1 and EcoFpg, CacOgg showed little preference for the base opposite the damage during base excision and removed 8-oxoguanine from single-stranded DNA. Thus, our results showed unambiguous qualitative functional differences in vitro between CacOgg and both hOGG1 and EcoFpg. CacOgg differs in sequence from the eukaryotic enzymes at two sequence positions, M132 and F179, which align with amino acids (R154 and Y203) in human OGG1 (hOGG1) found to be involved in opposite base interaction. To address the sequence basis for functional differences with respect to opposite base interactions, we prepared three CacOgg variants, M132R, F179Y, and M132R/F179Y. All three variants showed a substantial increase in specificity for 8-oxoG.C relative to 8-oxoG.A. While we were unable to definitively associate these qualitative functional differences with differences in selective pressure between eukaryotes, Clostridia, and other bacteria, our results are consistent with the idea that evolution of Ogg function is based on kinetic control of repair.

Molecular heterogeneity of EmaA, an oligomeric autotransporter adhesin of Aggregatibacter (Actinobacillus) actinomycetemcomitans.

Adhesion of Aggregatibacter actinomycetemcomitans to extracellular matrix proteins is mediated by antennae-like surface structures composed of EmaA oligomers. EmaA is an outer-membrane protein orthologous to the autotransporter YadA, a virulence determinant of Yersinia. emaA was present in the 27 strains examined, covering the six serotypes of A. actinomycetemcomitans. Ten individual genotypes and three different forms of the protein (full-length, intermediate and truncated) were predicted. The prototypic, full-length EmaA (202 kDa) was only associated with serotypes b and c, which displayed antennae-like surface structures. These strains bound to collagen embedded in a 3D matrix. The intermediate form of EmaA (173 kDa) was exclusively associated with serotypes d and a, which contained a 279 aa in-frame deletion, as well as a different N-terminal head domain sequence. These differences modified the appearance of the EmaA structures on the cell surface but maintained collagen-binding activity. Strains containing the truncated form of EmaA had single or multiple substitutions, deletions or insertions in the sequences, which resulted in the absence of EmaA molecules on the outer membrane and loss of collagen-binding activity. Population structure analyses of this organism, based on emaA, indicated that serotypes b and c belonged to one subpopulation, which was independent of the other serotypes. The main divergence was found in the functional head domain. The conserved emaA genotype within serotypes suggests a stable clonal linkage between this autotransporter protein and other virulence determinants.

Potential regulatory relationship between the nested gene DDC8 and its host gene tissue inhibitor of metalloproteinase-2.

Nested genes are fairly common within the mammalian nervous system, yet few studies have examined whether the guest and host genes might be coordinately regulated. Tissue inhibitors of metalloproteinase (TIMPs) inhibit extracellular matrix proteolysis mediated by metzincin proteases. TIMP-2 is the only TIMP not nested within a synapsin gene. It does, however, serve as a host for differential display clone 8 (DDC8), a testis-specific gene whose expression is upregulated during spermatogenesis. Here, we demonstrate that DDC8 is not testis specific. Furthermore, DDC8 expression in nonneural and neural tissues mimics that of TIMP-2, including its upregulation in response to traumatic brain injury, suggesting a potential regulatory relationship. The most striking observation is that the TIMP-2 knockout mouse brain contains TIMP-2 mRNA encoding exons 2-5, which are downstream of DDC8, but not exon 1, which contains the signal sequence and cysteine residue required for MMP inhibition, indicating a functional knockout. That TIMP-2 transcripts in wild-type brain contain DDC8 sequence suggests alternative splicing between the two genes.

The CDKN2A database: Integrating allelic variants with evolution, structure, function, and disease association.

In this report, we introduce the CDKN2A Database, an online database of germline and somatic variants of the CDKN2A tumor suppressor gene recorded in human disease through the year 2002, annotated with evolutionary, structural, and functional information. The CDKN2A Database improves upon existing resources by: 1) including both somatic mutations and germline variants, thereby adding the perspective of somatic cell carcinogenesis to that of hereditary cancer predisposition; 2) including information that assists with the interpretation of allelic variants, such as other primary data (sequences, structures, alignments, functional measurements, and literature references) and annotations (extensive text, figures, and a tree-based phylogenetic classification); and 3) providing the information in a format that allows a user to either download the database or to easily manipulate it online. We describe the database structure, content, current uses, and potential implications (http://biodesktop.uvm.edu/perl/p16).