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Intramural Papers of the Month

By Robin Arnette
December 2008

Crystal Structure of RACK1 in Arabidopsis thaliana

Researchers at NIEHS and Howard University have crystallized an isoform of the activated C-kinase 1 (RACK1) protein from the plant Arabidopsis thaliana. The crystal structure of RACK1, a scaffolding protein implicated in signaling pathways, revealed that the protein contained highly conserved surface residues that could be involved in vital protein-protein interactions. These data provide a future framework for studying RACK1's role in cellular stress, protein translation and developmental processes in both plants and animals.

Work from other labs determined that RACK1 was a member of the WD40 repeat family of β-propeller proteins, and early biochemical efforts used the β subunit of mammalian G proteins (Gβ) as a model. However, the model lacked precise structural detail with regard to diverse loop regions and surface side chain conformations that make RACK1 and Gβ functionally distinct. As mammal and plant forms of RACK1 have failed to crystallize on their own, the NIEHS team fused the c-terminal of a mutant maltose binding protein (MBP) - specifically designed to improve the chances of crystallization - to the n-terminus of the A. thaliana isoform RACK1A and solved the structure at 2.4 Å.

RACK1A is a canonical seven-bladed β-propeller with each propeller blade consisting of a four-stranded antiparallel β-sheet. The blades are arranged around an axis that creates a water accessible channel in the middle of the propeller. Earlier mutational studies have implicated blades 5 and 6 as important docking stations for a host of signaling proteins.

Ullah H, Scappini EL, Moon AF, Williams LV, Armstrong DL, Pedersen LC. (http://www.ncbi.nlm.nih.gov/pubmed/18715992?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2008. Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana. Protein Sci 17(10):1771-1780.

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SNPs in Human Ion Channel Genes Increase Susceptibility to Disease by Creating New Phosphorylation Sites in the Channel Proteins

NIEHS scientists have determined that a common single nucleotide polymorphism (SNP) in a cardiac potassium channel gene, hERG1, creates a phosphorylation site within the human Kv11.1 channel protein that inhibits channel activity. Kv11.1 channels are necessary for rhythmic excitability of cardiac muscle and endocrine cells, and they are regulated by hormonal signaling through the phosphatidylinositol 3-kinase (PI3K), which normally increases channel activity by stimulating a protein phosphatase, PP5, which dephosphorylates the channel protein.

However, in human channels with the K897T polymorphism, in which a single nucleotide change alters the translation of codon 897 from a lysine to a threonine, hormonal signaling through PI3K has the opposite effect on phosphorylation of the channel and on its activity. With a threonine (Thr) instead of a lysine at residue 897, hormonal signaling through PI3K leads to channel phosphorylation on Thr 897 by the Akt protein kinase, and to inhibition of channel activity. Human cardiac myocytes with less Kv11.1 channel activity have longer action potentials, as indicated by longer QT intervals in the electrocardiogram, and an increased risk of developing an arrhythmia, which can be fatal.

The finding suggests that human genetic differences can alter susceptibility to disease by changing phosphorylation sites in proteins and altering their regulation, a phenomenon the authors have named "phosphorylopathy." The paper also reports the results of a bioinformatics search for other SNPs in human ion channel genes that are known to be associated with disease. The authors identify 15 other SNPs in nine genes that are predicted to create or destroy putative phosphorylation sites. They are currently investigating these genes in collaboration with clinical investigators at NIEHS.

Gentile S, Martin N, Scappini E, Williams J, Erxleben C, Armstrong DL. (http://www.ncbi.nlm.nih.gov/pubmed/18791070?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2008. The human ERG1 channel polymorphism, K897T, creates a phosphorylation site that inhibits channel activity. Proc Natl Acad Sci USA 105(38):14704-14708.

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Cumene Exposure Leads to K-ras and p53 Mutations That Are Linked to Lung Tumors in Mice

B6C3F1 mice treated with the solvent cumene had significantly greater incidences of alveolar/bronchiolar adenomas and carcinomas than control mice according to a team of researchers from NIEHS. The team evaluated the cause of these lung neoplasms and determined that the mutations occurred in the K-ras oncogene and p53 tumor suppressor gene, the same genes that are often mutated in human lung cancer.

Cumene, also known as isopropylbenzene, is a component of crude oil and is mainly used to produce acetone and phenol. Humans are probably exposed to the chemical by breathing air-borne cumene molecules during petroleum refining or the combustion of petroleum products. Using PCR-amplified DNA isolated from paraffin-embedded neoplasms, the researchers detected K-ras mutations in 87 percent of cumene-induced lung neoplasms. The most common mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions.

p53 protein expression was detected by immunohistochemistry in 56 percent of cumene-induced lung neoplasms, and mutations were detected in 52 percent of lung neoplasms. In addition, cumene-induced lung carcinomas exhibited loss of heterozygosity (LOH) on chromosome four and six, while no LOH occurred in spontaneous carcinomas or normal lung tissue.

The K-ras and p53 pattern of mutations suggested that DNA damage and genomic instability contribute to the development of lung cancer in mice that may be of relevance to humans.

Hong HH, Ton TV, Kim Y, Wakamatsu N, Clayton NP, Chan PC, Sills RC, Lahousse SA. (http://www.ncbi.nlm.nih.gov/pubmed/18648094?ordinalpos=&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.SmartSearch&log$=citationsensor) Exit NIEHS 2008. Genetic alteration in K-ras and p53 cancer genes in lung neoplasms from B6C3F1 mice exposed to cumene. Toxicol Pathol 36(5):720-726.

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Inducible Nitric Oxide Synthase Is Involved With Streptozotocin-induced Diabetes

Recent studies from researchers at NIEHS and the Universidad de la Republica in Uruguay indicate that inducible nitric oxide synthase (iNOS) is a significant source of the free radical intermediates that are formed in streptozotocin (STZ)-induced diabetic rats. The finding sheds new light on the mechanisms involved in diabetes.

Diabetes mellitus leads to a myriad of medical complications in humans, with cardiovascular disease and atherosclerosis being significant causes of death. Earlier published reports, using STZ-induced diabetic rats as a model, suggested that oxidative stress and free radicals are contributing to diabetes and its complications through various mechanisms. To determine the source of the free radicals, the research team employed electron paramagnetic resonance (EPR) spectroscopy, in vivo spin-trapping, isotope labeling experiments and immunological techniques.

The results indicated that iNOS was the main source of radical generation, and isotope labeling determined that the lipid-derived radicals detected by EPR spectra were induced by hydroxyl radicals. L-arginine pretreatment and 1400W, a specific iNOS inhibitor, reduced EPR signals to baseline levels, which indicated that peroxynitrite was the source of the hydroxyl radicals. Immunohistochemistry of the liver and kidney of the diabetic rats determined the correlation and co-localization between iNOS, nitrotyrosine and 4-hydroxynonenal as a lipid peroxidation end product in the tissues.

Stadler K, Bonini MG, Dallas S, Jiang J, Radi R, Mason RP, Kadiiska MB. (http://www.ncbi.nlm.nih.gov/pubmed/18620046?ordinalpos=19&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2008. Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes. Free Radic Biol Med 45(6):866-874.



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