Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

International audienceDNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity

PARP-1 Expression is Increased in Colon Adenoma and Carcinoma and Correlates with OGG1

<div><p>The ethiology of colon cancer is largely dependent on inflammation driven oxidative stress. The analysis of 8-oxodeoxyguanosine (8-oxodGuo) level in leukocyte DNA of healthy controls (138 individuals), patients with benign adenomas (AD, 137 individuals) and with malignant carcinomas (CRC, 169 individuals) revealed a significant increase in the level of 8-oxodGuo in leukocyte DNA of AD and CRC patients in comparison to controls. The counteracting mechanism is base excision repair, in which OGG1 and PARP-1 play a key role. We investigated the level of PARP-1 and OGG1 mRNA and protein in diseased and marginal, normal tissues taken from AD and CRC patients and in leukocytes taken from the patients as well as from healthy subjects. In colon tumors the PARP-1 mRNA level was higher than in unaffected colon tissue and in polyp tissues. A high positive correlation was found between PARP-1 and OGG1 mRNA levels in all investigated tissues. This suggests reciprocal influence of PARP-1 and OGG1 on their expression and stability, and may contribute to progression of colon cancer. PARP-1 and OGG1 proteins level was several fold higher in polyps and CRC in comparison to normal colon tissues. Individuals bearing the <i>Cys326Cy</i>s genotype of OGG1 were characterized by higher PARP-1 protein level in diseased tissues than the <i>Ser326Cys</i> and <i>Ser326Se</i>r genotypes. Aforementioned result may suggest that the diseased cells with polymorphic OGG1 recruit more PARP protein, which is necessary to remove 8-oxodGuo. Thus, patients with decreased activity of OGG1/polymorphism of the OGG1 gene and higher 8-oxodGuo level may be more susceptible to treatment with PARP-1 inhibitors.</p></div

Correlation between PARP-1 and OGG1 mRNA levels in leukocytes of healthy volunteers (A), adenoma patients (B) and carcinoma patients (C), as well as in polyp tissue (D), normal colon (E) and tumor (F).

<p>Correlation between PARP-1 and OGG1 mRNA levels in leukocytes of healthy volunteers (A), adenoma patients (B) and carcinoma patients (C), as well as in polyp tissue (D), normal colon (E) and tumor (F).</p

Comparison of the level of PARP-1 protein expression in relation to OGG1polymorphism.

<p>Center mark in the box indicates medians for samples. The length of each box (IQR, interquartile range) represents the range within which the central 50% of the values fell, with the vertical edges placed at the first and third quartiles. Whiskers show variability outside the upper and lower quartiles. <i>P</i> was obtained with the Mann-Whitney test.</p

Level of PARP-1 (A) and OGG1 (B) mRNA in normal colon tissue (n = 60), polyp (n = 24) and cancer tissue (n = 60).

<p>Center mark in the box indicates the medians of the samples. The length of each boxes (IQR, interquartile range) represents the range within which the central 50% of the values fell, with the vertical edges placed at the first and third quartiles. Whiskers show variability outside the upper and lower quartiles. <i>P</i> was obtained with the Mann-Whitney test.</p

Comparison of the expression of OGG1 (A) and PARP-1 (B) protein in normal colon tissue, polyp and cancer tissue of adenoma (AD, n = 68) and carcinoma (CRC, n = 103) patients.

<p>Immunohistochemical detection in paraffin embedded sections stained with hematoxylin and eosin. Center mark in the box indicates the medians of the samples. The length of each box (IQR, interquartile range) represents the range within which the central 50% of the values fell, with the vertical edges placed at the first and third quartiles. Whiskers show variability outside the upper and lower quartiles. <i>P</i> was obtained with the Mann-Whitney test. Representative examples of the levels of PARP-1 protein in tissues of CRC patients determined by Western analysis. The analysis was performed on tumor and normal tissues of 41 CRC patients (C).</p

A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactiva�tion of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified cor�relation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors