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February 2011

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Intramural papers of the month

By Erin Hopper and Ritu Rana
February 2011

MicroRNA in glucocorticoid-induced lymphocyte apoptosis

A collaborative study involving NIEHS, SRA International, and the University of North Carolina concluded that the expression of small noncoding microRNAs is reduced during glucocorticoid- mediated lymphocyte apoptosis, also known as immune cell death. The results suggest a role for microRNA processors and specific microRNAs in cell life and death decisions.

MicroRNAs are important regulatory elements in a cell because they are involved in development, differentiation, and apoptosis. Glucocorticoids induce apoptosis, which influences lymphocyte development. The authors found that primary microRNA transcripts were not repressed during the lymphocyte apoptosis, but key microRNA processing enzymes - Dicer, Drosha, and DGCR8/Pasha - were reduced. The investigators silenced Dicer expression in two human leukemic cell lines and saw that the depletion of Dicer enhanced glucocorticoid-induced apoptosis in both cell lines.

The overexpression of the microRNA, which is repressed by glucocorticoids, decreased glucocorticoid-induced apoptosis in both human and mouse lymphocytes, suggesting an important role for microRNAs.

Citation: Smith LK, Shah RR, Cidlowski JA. ( Exit NIEHS 2010. Glucocorticoids modulate microRNA expression and processing during lymphocyte apoptosis. J Biol Chem 285(47):36698-36708.

Ubiquitination through lysine 63 mediates adhesion and migration of MDA-MB-435 cells

Scientists at NIEHS recently evaluated the role of ubiquitination in arachidonic acid-induced adhesion and migration of cells from the highly metastatic breast cancer cell line MDA-MB-435. Cellular adhesion and migration are processes that are required for cancer metastasis, and previous studies from this group have established that arachidonic acid, an essential dietary fatty acid, can stimulate cellular adhesion to collagen type IV, a component of the extracellular matrix.

Although ubiquitin is well known for its involvement in protein degradation, it can function in non-proteolytic pathways as well. Ubiquitin chains linked through lysine 48 appear to target the ubiquitinated protein for degradation, but ubiquitin chains linked through lysine 63 are not as well understood and seem to play different roles in the cell, including signal transduction.

The goal of this study was to investigate the role of ubiquitin in arachidonic acid-induced cellular adhesion and migration. To this end, the researchers constructed a series of mutants in which selected lysine residues were changed to arginine, and they examined the effects of these mutations on adhesion and migration of MDA-MB-435 cells exposed to arachidonic acid. These experiments demonstrated that ubiquitination was required for cellular adhesion on collagen type IV and that polyubiquitination through lysine 63 is critical for arachidonic acid-induced cellular adhesion and migration.

Citation: Ray DM, Rogers BA, Sunman JA, Akiyama SK, Olden K, Roberts JD. ( Exit NIEHS 2010. Lysine 63-linked ubiquitination is important for arachidonic acid-induced cellular adhesion and migration. Biochem Cell Biol 88(6):947-956.

The function of BAF155 in the SWI/SNF chromatin remodeling complex

NIEHS scientists determined that Brg-1-associated factor 155 (BAF155) plays a key scaffolding role within the SWI/SNF chromatin remodeling complex and is an important factor in the maintenance of subunit stoichiometry. The SWI/SNF complex acts to disrupt tightly packed chromatin, which allows the transcriptional machinery to access regulatory regions. This action controls the expression of genes involved in various cellular processes. The authors believe that understanding the SWI/SNF complex may lead to the identification of pathways that lead to cancer and other illnesses.

Previous studies found that BAF155 interacts and stabilizes BAF57, and that the proteasome - cellular machinery that degrades protein - regulates BAF57. The research team created BAF155 and BAF57 expression plasmids, including BAF57 mutants that lacked the principal lysine residues required for ubiquitination, and transfected UL3 cells to immunoprecipitate the proteins. They also used a small interfering RNA targeting the E3 ubiquitin ligase TRIP12 to illustrate its role in this cellular mechanism.

The study showed that TRIP12 ubiquitinates free BAF57, which targets it for degradation by the proteasome. The role of BAF155 is to interact with and stabilize BAF57 by blocking TRIP12 from adding ubiquitin molecules. BAF57 mutants incapable of being ubiquitinated could still be stabilized by BAF155, indicating an ubiquitin-independent mechanism of BAF57 degradation that involves direct interaction with the proteasome. 

Citation: Keppler BR, Archer TK. ( Exit NIEHS 2010. Ubiquitin-dependent and ubiquitin-independent control of subunit stoichiometry in the SWI-SNF complex. J Biol Chem 285(46):35665-35674.

Mutation of proline 180 markedly reduces the rate of desensitization of nAChR

NIEHS researchers recently investigated the contribution of a beta-sheet proline residue to the desensitization of rat a7 nicotinic acetylcholine receptor (nAChR), a protein that belongs to the cys-loop superfamily of ligand-gated ion channels. When the neurotransmitter ACh binds nAChR, it leads to a conformational change that allows ions to pass through the channel pore.

Exposure of alpha 7 nAChR to high concentrations of agonist results in rapid desensitization of the receptor, and this desensitization hinders efforts to perform pharmacological assays on this protein. Although the precise structure of alpha 7 nAChR is not known, a number of mutations have been shown to slow the rate of nAChR desensitization. In this study, mutation of proline 180 was found to slow the rate of desensitization, and a series of mutants was created to further investigate the role of this amino acid in the desensitization of nAChR.

Mutation of proline 180 to threonine or serine dramatically decreased the rate of desensitization. The mutants P180Y and P180F exhibited a modest decrease in the rate of desensitization, suggesting that side-chain hydroxyl groups at position 180 slow desensitization but are not its sole determinant. Molecular Dynamics simulations also indicated that an increase in hydrogen bonding at position 180 may play a role in slowing desensitization. 

Citation: McCormack TJ, Melis C, Colón J, Gay EA, Mike A, Karoly R, Lamb PW, Molteni C, Yakel JL. ( Exit NIEHS 2010. Rapid desensitization of the rat alpha7 nAChR is facilitated by the presence of a proline residue in the outer beta-sheet. J Physiol 588(22):4415-4429.

(Erin Hopper, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Structural Biology Mass Spectrometry Group. Ritu Rana, Ph.D., is a visiting fellow in the NIEHS Laboratory of Toxicology and Pharmacology Human Metabolism Group.)

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