Signaling mechanisms for gene regulation by metals and metal mixtures

Numerous epidemiological studies associate chronic inhalation of metal mixtures with increased risk of pulmonary diseases. Although exposure to metal mixtures is of great public health relevance, the integration of cellular responses to metals within these mixtures that promote disease is poorly understood. This dissertation investigated the hypothesis that chromium (VI) (Cr(VI)) stimulates signaling that alters transcriptional complexes to silence protective gene induction by nickel (Ni). In airway epithelial (BEAS-2B) cells, Cr(VI) activated signal transducer and activator of transcription 1 (STAT1)-dependent signaling within 1 h of exposure. This activation was dependent on Src family kinases (SFKs) since inhibiting SFKs prevented Cr(VI)-stimulated STAT1 signaling. Moreover, Cr(VI) activated STAT1 in wild-type mouse embryonic fibroblast (MEF) cells, but no response was observed in MEF cells null for the SFKs, Src, Yes, and Fyn. However, reconstituting human Fyn in the deficient MEF cells restored the Cr(VI) response. These data indicate that Cr(VI)-activated STAT1 is mediated by Fyn. This signaling may be detrimental as STAT1 has been implicated as an inflammatory mediator in asthma patients that is specifically activated in bronchial epithelial cells (1). Metallothionein (MT) and vascular endothelial growth factor A (VEGFA) are involved in protecting the lung from injury by sequestering metals and promoting wound repair, respectively. Ni-induced MT2A, the most abundant human isoform, required zinc (Zn) redistribution which directly activated metal transcription factor-1 (MTF-1). A prolonged induction was mediated by secondary signaling pathways. Cr(VI) negatively regulated the secondary pathway and had no effect on Zn mobilization. For VEGFA induction, Ni activated a complex signaling cascade involving ERK. Ni-stimulated ERK was upstream of hypoxia-inducible factor-1á (HIF-1á) and Src-mediated Sp1 transactivation. Cr(VI) inhibited Ni-activated ERK, HIF-1á stabilization, Src phosphorylation, and VEGFA induction. The current study demonstrated that Cr(VI)-activated STAT1 is responsible for the silencing of inducible genes. In BEAS-2B cells stably expressing STAT1 shRNA, Cr(VI) no longer had an inhibitory effect on Ni-induced MT or VEGFA mRNA expression and positively interacted with Ni to induce both genes. These data indicate that Cr(VI)-activated STAT1 may play a role in the pathogenesis of Cr(VI)-induced pulmonary diseases by silencing the protective gene transcription in the airway epithelium

A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

<div><p>Several germline single nucleotide polymorphisms (SNPs) have been identified in the <em>POLB</em> gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the <em>POLB</em> gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the <em>POLB</em> germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility.</p> </div

Chromosomal aberrations in P242R-expressing cells.

<p>Representative metaphase spread of MCF10A expressing (A.) WT or (B.) P242R Pol β. Chromosomal fusions are shown with the gray arrow and fragments are shown with black arrows. C. Number of aberrations per metaphase. A total of at least 50 metaphases were scored for each cell line.</p

An Allosteric Interaction Network Promotes Conformation State-Dependent Eviction of the Nas6 Assembly Chaperone from Nascent 26S Proteasomes

Summary: The 26S proteasome is the central ATP-dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation-specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP binding by the ATPase subunit Rpt3 promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly-coupled allosteric signals promote chaperone eviction and provides a framework for understanding the eviction of other chaperones from this biomedically important molecular machine. : Nemec et al. report how the evolutionarily conserved Nas6 assembly chaperone is evicted from nascent 26S proteasomes. Nucleotide binding events within the nascent proteasome trigger formation of conformation-specific intersubunit contacts that expel Nas6. This mechanism may serve a quality control function by blocking formation of 26S proteasomes from defective components. Keywords: proteasome, macromolecular complex, ubiquitin, proteolysis, gankyrin, Nas6, assembly, oncogen

P242R Pol β confers slight sensitivity to MMS compared to WT.

<p>Clonogenic survival assays were conducted with (A.) Pol β^−/− MEFs, (B.) Pol β^+/+ MEFs, or (C.) MCF10A pools expressing WT or P242R Pol β. Filled circles represent results from pools expressing empty vector, filled squares represent pools expressing WT Pol β, and filled triangles represent pools expressing P242R Pol β. Data are plotted as the mean ± SEM (n = 3).</p

Accumulation of BER intermediates in MCF10A cells expressing P242R Pol β.

<p>A. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 30 minutes and allowed to recover for 0, 30, or 60 min and single-strand breaks (SSBs) were analyzed by comet assay. The percentage of tail DNA is plotted on the Y-axis. B. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 30 min and allowed to recover for 0, 30, or 60 min, stained with γH2AX antibody, and analyzed by flow cytometry. Cells were treated for 120 min as a positive control. C–D. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 2 h and allowed to recover for 0 or 2 hours. Cells were stained with γH2AX antibody and propidium iodide to assess the levels of double-strand breaks (DSBs) and the cell cycle phase, respectively, and analyzed by flow cytometry. Data are plotted as the mean ± SEM. Data are plotted as the mean ± SEM (n = 3). A and C. ** and *** denote <i>p</i><0.01 and 0.001, respectively. ∧ denotes <i>p</i><0.05 comparing 0 vs 2 h recovery within each cell line. ∧∧∧ denotes <i>p</i><0.001 comparing 30 or 60 min recovery to 0 recovery. D. *** denotes <i>p</i><0.001 comparing WT+MMS to P242R+MMS in each phase of the cell cycle. ∧ and ∧∧∧ denote <i>p</i><0.05 and 0.001, respectively, comparing WT+MMS+recovery to P242R+MMS+recovery in each phase of the cell cycle.</p

The P242R germline variant of Pol β is slow and binds DNA tightly.

<p>A. Representative results from a presteady-state burst assay. Results for the WT are shown as filled squares fit with a solid curve. Results for the P242R are shown as open triangles fit with a dashed curve. The assay was repeated four times for each protein. B. Representative results from a gel electrophoretic mobility shift assay. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003052#s2" target="_blank">Results</a> for the different proteins are shown as in A.</p