Mirta M. L. Sousa

Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage

Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1‐methyladenine and 3‐methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2‐oxoglutarate (2OG) at 1.5 Å resolution and analyse key site‐directed mutants. The hABH3 structure reveals the β‐strand jelly‐roll fold that coordinates a catalytically active iron centre by a conserved His1‐X‐Asp/Glu‐Xn‐His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG‐dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds‐DNA discrimination, and reveal self‐hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.

Cell cycle‐specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non‐replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non‐catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin‐dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c‐myc‐like phosphodegron. The enhanced catalytic turnover of UNG2 p‐S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation‐induced deaminase‐generated uracil at Ig loci in B cells.

Identification of a novel, widespread, and functionally important PCNA-binding motif

AlkB PCNA-interacting motif (APIM) is present in >200 proteins and may mediate PCNA binding during genotoxic stress.

Uracil in DNA and its processing by different DNA glycosylases.

Uracil in DNA may result from incorporation of dUMP during replication and from spontaneous or enzymatic deamination of cytosine, resulting in U:A pairs or U:G mismatches, respectively. Uracil generated by activation-induced cytosine deaminase (AID) in B cells is a normal intermediate in adaptive immunity. Five mammalian uracil-DNA glycosylases have been identified; these are mitochondrial UNG1 and nuclear UNG2, both encoded by the UNG gene, and the nuclear proteins SMUG1, TDG and MBD4. Nuclear UNG2 is apparently the sole contributor to the post-replicative repair of U:A lesions and to the removal of uracil from U:G contexts in immunoglobulin genes as part of somatic hypermutation and class-switch recombination processes in adaptive immunity. All uracil-DNA glycosylases apparently contribute to U:G repair in other cells, but they are likely to have different relative significance in proliferating and non-proliferating cells, and in different phases of the cell cycle. There are also some indications that there may be species differences in the function of the uracil-DNA glycosylases.

AID expression in B-cell lymphomas causes accumulation of genomic uracil and a distinct AID mutational signature

The most common mutations in cancer are C to T transitions, but their origin has remained elusive. Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many common cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism. Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows significantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancer cell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AID mRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdown significantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergo class switch recombination displayed a several-fold increase in total genomic uracil, indicating that B cells may undergo widespread cytosine deamination after stimulation. In line with this, we found that clustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carry AID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil with uracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion, AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutations in B-cell malignancies.

A kinetic-based approach to understanding heterologous mevalonate pathway function in E. coli.

To aid in debugging efforts to increase yield, titer, and productivity of engineered metabolic pathways, computational models are increasingly needed to predict how changes in experimentally manipulable variables such as enzyme expression map to changes in pathway flux. Here, an ordinary differential equation model is developed for a heterologous mevalonate pathway in E. coli using kinetic parameters culled from literature and enzyme concentrations derived from Selective Reaction Monitoring Mass Spectrometry (SRM‐MS). To identify parameters most important to further experimental investigation, a global sensitivity analysis was performed, which pointed to amorphadiene synthase activity as the main limiting factor for amorphadiene production. Furthermore, the model predicted that in this local enzyme expression regime, the overall pathway flux is insensitive to farnesyl pyrophosphate (FPP)‐mediated inhibition of mevalonate kinase, not supporting a hypothesis that had previously been posited to be limiting amorphadiene production. To test these predictions experimentally, two strains were constructed: (1) a strain containing a homologous mevalonate kinase with weaker feedback inhibition, and (2) a strain with greater amorphadiene synthase expression. The experimental results validate the qualitative model hypotheses and accurately match the predicted productivities for the two strains, particularly when an in vivo‐derived kcat for amorphadiene synthase was substituted for the literature value. These results demonstrate the utility of using kinetic representations of engineered metabolic pathways parameterized with experimentally derived protein concentrations and enzyme kinetic constants to predict productivities and test hypotheses about engineering strategies. Biotechnol. Bioeng. 2015;112: 111–119.

A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins

Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (www.addgene.org). To facilitate high sample throughput, we developed a fast, analytical-flow chromatography method using a 5.5-min gradient (10 min total run time). Overall this toolkit provides an invaluable resource for metabolic engineering by increasing sample throughput, minimizing development time and providing peptide standards for absolute quantification of E. coli proteins.

Modulation of Cell Metabolic Pathways and Oxidative Stress Signaling Contribute to Acquired Melphalan Resistance in Multiple Myeloma Cells

Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. Melphalan is the most commonly used chemotherapeutic agent against multiple myeloma. However, despite a 70–80% initial response rate, virtually all patients eventually relapse due to the emergence of drug-resistant tumour cells. By using global proteomic and transcriptomic profiling on melphalan sensitive and resistant RPMI8226 cell lines followed by functional assays, we discovered changes in cellular processes and pathways not previously associated with melphalan resistance in multiple myeloma cells, including a metabolic switch conforming to the Warburg effect (aerobic glycolysis), and an elevated oxidative stress response mediated by VEGF/IL8-signaling. In addition, up-regulated aldo-keto reductase levels of the AKR1C family involved in prostaglandin synthesis contribute to the resistant phenotype. Finally, selected metabolic and oxidative stress response enzymes were targeted by inhibitors, several of which displayed a selective cytotoxicity against the melphalan-resistant cells and should be further explored to elucidate their potential to overcome melphalan resistance.

Photodynamic therapy with hexyl aminolevulinate induces carbonylation, posttranslational modifications and changed expression of proteins in cell survival and cell death pathways

Photodynamic therapy (PDT) using blue light and the potent precursor for protoporphyrin IX, hexyl aminolevulinate (HAL), has been shown to induce apoptosis and necrosis in cancer cells, but the mechanism remains obscure. In the present study, we examined protein carbonylation, expression levels and post-translational modifications in rat bladder cells (AY-27) after PDT with HAL. Altered levels of expression and/or post-translational modifications induced by PDT were observed for numerous proteins, including proteins required for cell mobility, energy supply, cell survival and cell death pathways, by using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Moreover, 10 carbonylated proteins associated with cytoskeleton, transport, oxidative stress response, protein biosynthesis and stability, and DNA repair were identified using immunoprecipitation, two-dimensional gel electrophoresis and MS. Overall, the results indicate that HAL-mediated PDT triggers a complex cellular response involving several biological pathways. Our findings may account for the elucidation of mechanisms modulated by PDT, paving the way to improve clinic PDT-efficacy.

An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells.

Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.