22 research outputs found
Identification of novel small molecule inhibitors of proteins required for genomic maintenance and stability
Indiana University-Purdue University Indianapolis (IUPUI)Targeting uncontrolled cell proliferation and resistance to DNA damaging chemotherapeutics using small molecule inhibitors of proteins involved in these pathways has significant potential in cancer treatment. Several proteins involved in genomic maintenance and stability have been implicated both in the development of cancer and the response to chemotherapeutic treatment. Replication Protein A, RPA, the eukaryotic single-strand DNA binding protein, is essential for genomic maintenance and stability via roles in both DNA replication and repair. Xeroderma Pigmentosum Group A, XPA, is required for nucleotide excision repair, the main pathway cells employ to repair bulky DNA adducts. Both of these proteins have been implicated in tumor progression and chemotherapeutic response. We have identified a novel small molecule that inhibits the in vitro and cellular ssDNA binding activity of RPA, prevents cell cycle progression, induces cytotoxicity and increases the efficacy of chemotherapeutic DNA damaging agents. These results provide new insight into the mechanism of RPA-ssDNA interactions in chromosome maintenance and stability. We have also identified small molecules that prevent the XPA-DNA interaction, which are being investigated for cellular and tumor activity. These results demonstrate the first molecularly targeted eukaryotic DNA binding inhibitors and reveal the utility of targeting a protein-DNA interaction as a therapeutic strategy for cancer treatment
DJ-1 is not a deglycase and makes a modest contribution to cellular defense against methylglyoxal damage in neurons
Human DJ-1 is a cytoprotective protein whose absence causes Parkinson\u27s disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity
Targeting the OB-Folds of Replication Protein A with Small Molecules
Replication protein A (RPA) is the main eukaryotic single-strand (ss) DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs) that show in vitro inhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B) of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold) containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment
Metal-Assisted Protein Quantitation (MAPq): Multiplex Analysis of Protein Expression Using Lanthanide-Modified Antibodies with Detection by Inductively Coupled Plasma Mass Spectrometry.
Targeted Inhibition of Replication Protein A Reveals Cytotoxic Activity, Synergy with Chemotherapeutic DNA-Damaging Agents, and Insight into Cellular Function
Mass Spectrometric Methods for the Analysis of Nucleoside–Protein Cross-Links: Application to Oxopropenyl-deoxyadenosine
Electrophilic DNA adducts produced
following oxidative stress can
form DNA–protein cross-links (DPCs), dramatically altering
genomic maintenance pathways. Complete characterization of DPCs has
been hindered, in part, because of a lack of comprehensive techniques
for their analysis. We have, therefore, established a proteomics approach
to investigate sites of cross-link formation using <i>N</i><sup><i>6</i></sup>-(3-oxo-1-propenyl)-2′-deoxyadenosine
(OPdA), an electrophilic DNA adduct produced from oxidative stress.
OPdA was reacted with albumin and reduced with NaBH<sub>4</sub> to
stabilize DPCs. Using LC-MS/MS proteomics techniques, high-resolution
peptide sequence data were obtained; however, using a database searching
strategy, adducted peptides were only identified in samples subjected
to chemical depurination. This strategy revealed multiple oxopropenyl
adenine-lysine adducts and oxopropenyl-lysine adducts with the most
reactive lysines identified to be Lys256 and Lys548. Manual interrogation
of the mass spectral data provided evidence of OPdA deoxynucleoside
conjugates to lysines and cross-links that underwent facile collision-induced
dissociation to release an unmodified peptide without subsequent fragmentation.
These fragmentations precluded adduct detection and peptide sequencing
using database searching methods. Thus, comprehensive analysis of
DPCs requires chemical depurination of DNA–protein reaction
mixtures followed by a combination of database-dependent and manual
interrogation of LC-MS/MS data using higher-energy collision-induced
dissociation. In the present case, this approach revealed that OPdA
selectively modifies surface lysine residues and produces nucleoside–protein
cross-links and oxopropenyl lysine
Metal-Assisted Protein Quantitation (MAPq): Multiplex Analysis of Protein Expression Using Lanthanide-Modified Antibodies with Detection by Inductively Coupled Plasma Mass Spectrometry.
Understanding the complex relationships between genomics, transcriptomics, and proteomics requires the development of more sensitive and rapid methods of multiplexed protein analysis. This is necessary to understand the relationship between cellular responses to environmental stresses, disease progression, and/or drug treatment; however, most methods are limited by low sensitivity, nonspecificity, and minimal multiplexing capacity. To more fully explore the relationship between multiple cellular pathways, we have developed a novel antibody-based multiplex assay using inductively coupled plasma mass spectrometry (ICP-MS), which we term metal-assisted protein quantitation (MAPq). MAPq utilizes lanthanide-conjugated antibodies to simultaneously quantify up to 35 proteins with low pg/mL sensitivity. This method is especially advantageous for low-abundance proteins, a significant limitation of many multiplex MS methods. We observed a limit of detection of 0.5 pg/mL and a limit of quantitation of 5 pg/mL with virtually no background signal. We applied this method to both cultured cells and mouse tissues to investigate changes in low-abundance nuclear and cytoplasmic proteins following drug or environmental stresses. MAPq was found to be at least 10 times more sensitive than Western blots and could detect quantitative changes in protein expression not readily observed using conventional approaches
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DNA Advanced Glycation End Products (DNA-AGEs) Are Elevated in Urine and Tissue in an Animal Model of Type 2 Diabetes.
More precise identification and treatment monitoring of prediabetic/diabetic individuals will require additional biomarkers to complement existing diagnostic tests. Candidates include hyperglycemia-induced adducts such as advanced glycation end products (AGEs) of proteins, lipids, and DNA. The potential for DNA-AGEs as diabetic biomarkers was examined in a longitudinal study using the Leprdb/db animal model of metabolic syndrome. The DNA-AGE, N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) was quantified by mass spectrometry using isotope dilution from the urine and tissue of hyperglycemic and normoglycemic mice. Hyperglycemic mice (fasting plasma glucose, FPG, ≥ 200 mg/dL) displayed a higher median urinary CEdG value (238.4 ± 112.8 pmol/24 h) than normoglycemic mice (16.1 ± 11.8 pmol/24 h). Logistic regression analysis revealed urinary CEdG to be an independent predictor of hyperglycemia. Urinary CEdG was positively correlated with FPG in hyperglycemic animals and with HbA1c for all mice. Average tissue-derived CEdG was also higher in hyperglycemic mice (18.4 CEdG/106 dG) than normoglycemic mice (4.4 CEdG/106 dG). Urinary CEdG was significantly elevated in Leprdb/db mice relative to Leprwt/wt, and tissue CEdG values increased in the order Leprwt/wt < Leprwt/db < Leprdb/db. These data suggest that urinary CEdG measurement may provide a noninvasive quantitative index of glycemic status and augment existing biomarkers for the diagnosis and monitoring of diabetes
Selection of Monoclonal Antibodies Against 6-oxo-M<sub>1</sub>dG and Their Use in an LC-MS/MS Assay for the Presence of 6-oxo-M<sub>1</sub>dG in Vivo
Oxidative stress triggers DNA and lipid peroxidation,
leading to
the formation of electrophiles that react with DNA to form adducts.
A product of this pathway, (3-(2′-deoxy-β-d-<i>erythro</i>-pentofuranosyl)-pyrimido[1,2-α]purine-10(3<i>H</i>)-one), or M<sub>1</sub>dG, is mutagenic in bacterial and
mammalian cells and is repaired by the nucleotide excision repair
pathway. In vivo, M<sub>1</sub>dG is oxidized to a primary metabolite,
(3-(2-deoxy-β-d-<i>erythro</i>-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3<i>H</i>,5<i>H</i>)-dione, or 6-oxo-M<sub>1</sub>dG,
which is excreted in urine, bile, and feces. We have developed a specific
monoclonal antibody against 6-oxo-M<sub>1</sub>dG and have incorporated
this antibody into a procedure for the immunoaffinity isolation of
6-oxo-M<sub>1</sub>dG from biological matrices. The purified analyte
is quantified by LC-MS/MS using a stable isotope-labeled analogue
([<sup>15</sup>N<sub>5</sub>]-6-oxo-M<sub>1</sub>dG) as an internal
standard. Healthy male Sprague–Dawley rats excreted 6-oxo-M<sub>1</sub>dG at a rate of 350–1893 fmol/kg·d in feces. This
is the first report of the presence of the major metabolite of M<sub>1</sub>dG in rodents without exogenous introduction of M<sub>1</sub>dG