Anthony T. Yeung

Biphasic Kinetics of the Human DNA Repair Protein MED1 (MBD4), a Mismatch-specific DNA N-Glycosylase

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.

Role of platinum-DNA adduct formation and removal in cisplatin resistance in human ovarian cancer cell lines

A series of cisplatin-resistant cell lines were used to examine the formation and removal of platinum-DNA adducts from the overall genome and the formation and removal of cisplatin-interstrand cross-links from specific genomic regions. Cisplatin accumulation and DNA platination levels, which correlated linearly, were similar in three of the resistant cell lines despite differences in their primary cisplatin resistance. Increased platinum removal from total genomic DNA was found to be associated with increased resistance. Interstrand cross-link levels were found to be 2- to 4-fold lower in the 28S ribosomal RNA gene and a non-coding genomic region of the resistant cell lines as compared with the parental A2780 cell line. In addition, 1.2- to 2.7-fold more cross-links were formed in the non-coding region than in the ribosomal RNA gene in all of the cell lines. Interstrand cross-links were removed more rapidly from both regions of the highly cisplatin-resistant C80 and C200 cells and from the ribosomal RNA gene only in the cell lines of lower resistance. The results support a role for DNA repair and alterations in interstrand cross-link formation in cisplatin resistance and provide evidence for heterogeneous interstrand cross-link formation in the genome.

Histone modifications silence the GATA transcription factor genes in ovarian cancer

Altered expression of GATA factors was found and proposed as the underlying mechanism for dedifferentiation in ovarian carcinogenesis. In particular, GATA6 is lost or excluded from the nucleus in 85% of ovarian tumors and GATA4 expression is absent in majority of ovarian cancer cell lines. Here, we evaluated their DNA and histone epigenetic modifications in five ovarian epithelial and carcinoma cell lines (human ‘immortalized’ ovarian surface epithelium (HIO)-117, HIO-114, A2780, SKOV3 and ES2). GATA4 and GATA6 gene silencing was found to correlate with hypoacetylation of histones H3 and H4 and loss of histone H3/lysine K4 tri-methylation at their promoters in all lines. Conversely, histone H3/lysine K9 di-methylation and HP1γ association were not observed, excluding reorganization of GATA genes into heterochromatic structures. The histone deacetylase inhibitor trichostatin A, but not the DNA methylation inhibitor 5′-aza-2′-deoxycytidine, re-established the expression of GATA4 and/or GATA6 in A2780 and HIO-114 cells, correlating with increased histone H3 and H4 acetylation, histone H3 lysine K4 methylation and DNase I sensitivity at the promoters. Therefore, altered histone modification of the promoter loci is one mechanism responsible for the silencing of GATA transcription factors and the subsequent loss of a target gene, the tumor suppressor Disabled-2, in ovarian carcinogenesis.

Enzymatic mutation detection technologies.

Mutation is as necessary for life as fidelity is in DNA replication. The study of mutations reveals the normal functions of genes, messages, proteins, the causes of many diseases, and the variability of responses among individuals. Indeed, recent mutations that have not yet become polymorphisms are often deleterious and pertinent to the disease history of afflicted individuals. This review discusses the principles behind a variety of methods for the detection of mutations and factors that should be considered in future methods design. One enzymatic approach in particular using orthologs of the CEL I nuclease that show high specificity for all mismatches, appears to be easy and robust. Further developments of this and other methods will allow mutation detection to become an integral component of individualized medicine.

Proteomic Analyses of Pancreatic Cyst Fluids

Objectives: There are currently no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. Proteomic analyses were performed to explore pancreatic cyst fluids to yield effective diagnostic biomarkers. Methods: We have prospectively recruited 20 research participants and prepared their pancreatic cyst fluids specifically for proteomic analyses. Proteomic approaches applied were as follows: (1) matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry peptidomics with LC/MS/MS (HPLC-tandem mass spectrometry) protein identification; (2) 2-dimensional gel electrophoresis; (3) GeLC/MS/MS (tryptic digestion of proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by LC/MS/MS). Results: Sequencing of more than 350 free peptides showed that exopeptidase activities rendered peptidomics of cyst fluids unreliable; protein nicking by proteases in the cyst fluids produced hundreds of protein spots from the major proteins, making 2-dimensional gel proteomics unmanageable; GeLC/MS/MS revealed a panel of potential biomarker proteins that correlated with carcinoembryonic antigen (CEA). Conclusions: Two homologs of amylase, solubilized molecules of 4 mucins, 4 solubilized CEA-related cell adhesion molecules (CEACAMs), and 4 S100 homologs may be candidate biomarkers to facilitate future pancreatic cyst diagnosis and risk-stratification. This approach required less than 40 &mgr;L of cyst fluid per sample, offering the possibility to analyze cysts smaller than 1 cm in diameter.

An Artificial Gene for Human Porphobilinogen Synthase Allows Comparison of an Allelic Variation Implicated in Susceptibility to Lead Poisoning

Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B12). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference.

The UvrABC endonuclease system of Escherichia coli — A view from Baltimore

Nucleotide excision is initiated by the UvrABC endonuclease system in which the initial DNA interaction is with UvrA which was dimerized in the presence of ATP. Nucleoprotein formation most likely takes place on undamaged regions of DNA by (UvrA)2 which has been dimerized in the presence of ATP. Topological unwinding of DNA, driven by ATP binding, is increased by the presence of UvrB to approximately a single helical turn. The Uvr(A)2B complex translocates to a damaged site by the combined Uvr(A)2B helicase in which the driving force is provided by the UvrB-associated ATPase. The dual incision reaction is initiated by the binding of the UvrC protein to the Uvr(A)2B-nucleoprotein complex. The proteins in this post-incision nucleoprotein complex do not turn over and require the presence of the UvrD protein and DNA polymerase I under polymerizing conditions. The final integrity of the DNA strands is restored with polynucleotide ligase.

Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.

Altered Gene Expression in Morphologically Normal Epithelial Cells from Heterozygous Carriers of BRCA1 or BRCA2 Mutations

We hypothesized that cells bearing a single inherited “hit” in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even prevent progression to carcinoma. We report here on the transcriptomes of primary breast and ovarian epithelial cells cultured from BRCA1 and BRCA2 mutation carriers and controls. Our comparison analyses identified multiple changes in gene expression, in both tissues for both mutations, which were validated independently by real-time reverse transcription-PCR analysis. Several of the differentially expressed genes had been previously proposed as cancer markers, including mammaglobin in breast cancer and serum amyloid in ovarian cancer. These findings show that heterozygosity for a mutant tumor suppressor gene can alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner; these detectable effects of “one hit” represent early molecular changes in tumorigenesis that may serve as novel biomarkers of cancer risk and as targets for chemoprevention. Cancer Prev Res; 3(1); 48–61

Assessment of Two Immunodepletion Methods: Off-Target Effects and Variations in Immunodepletion Efficiency May Confound Plasma Proteomics

Immunodepletion of abundant plasma proteins increases the depth of proteome penetration by mass spectrometry. However, the nature and extent of immunodepletion and the effect of off-target depletion on the quantitative comparison of the residual proteins have not been critically addressed. We performed mass spectrometry label-free quantitation to determine which proteins were immunodepleted and by how much. Two immunodepletion resins were compared: Qproteome (Qiagen) which removes albumin+immunoglobulins and Seppro IgY14+SuperMix (Sigma-Aldrich) which removes 14 target proteins plus a number of unidentified proteins. Plasma collected by P100 proteomic plasma collection tubes (BD) from 20 human subjects was individually immunodepleted to minimize potential variability, prior to pooling. The abundant proteins were quantified better when using only albumin+immunoglobulins removal (Qproteome), while lower abundance proteins were evaluated better using exhaustive immunodepletion (Seppro IgY14+SuperMix). The latter resin removed at least 155 proteins, 38% of the plasma proteome in protein number and 94% of plasma protein in mass. The depth of immunodepletion likely accounts for the effectiveness of this resin in revealing low abundance proteins. However, the more profound immunodepletion achieved with the IgY14+SuperMix may lead to false-positive fold-changes between comparison groups if the reproducibility and efficiency of the depletion of a given protein are not considered.