The lipid peroxidation product and environmental pollutant acrolein participates in many diseases. Because of its formation during tobacco combustion, its role in various smoking-related respiratory conditions including lung cancer has received increasing attention. As a reactive electrophile, acrolein seems likely to disrupt many biochemical pathways, but these are poorly characterized on a genome-wide basis. This study used microarrays to study short-term transcriptional responses of A549 human lung cells to acrolein, with cells exposed to 100 microM acrolein for 1, 2, or 4 h prior to RNA extraction and transcription profiling. Major pathways dysregulated by acrolein included those involved in apoptosis, cell cycle control, transcription, cell signaling, and protein biosynthesis. Although HMOX1 is a widely used marker of transcriptional responses to acrolein, this gene was the sole upregulated member of the Nrf2-driven family of antioxidant response genes. Transcript levels of several members of the metallothionein class of cytoprotective metal-chelating proteins decreased strongly in response to acrolein. Other novel findings included strong and persistent upregulation of several members of the early growth response (EGR) class of zinc finger transcription factors. Real-time PCR and Western blotting confirmed strong upregulation of a key member of this family (EGR-2), the DNA damage response gene GADD45beta, the heat shock response participant Hsp70, and also HMOX1. Consistent with changes in Nur77 mRNA levels during the microarray study, Western blotting confirmed strong Nur77 induction at the protein level, raising the possibility that this death-inducing protein contributes to the loss of cell viability during acrolein exposure. Collectively, the transcriptional response to acrolein is complex and dynamic, with future work needed to determine whether acrolein-responsive genes identified in this study contribute to cell and tissue injury in the smoke-exposed lung.
The smoke-borne electrophile acrolein reacts extensively with proteins, forming carbonyl-retaining Michael adducts that may be attacked by adjacent protein nucleophiles to form cross-links. Because little information is available concerning the extent of intermolecular protein cross-linking during acrolein toxicity in cells, we used an antibody against a known target for toxic carbonyls, the chaperone protein Hsp90, to detect the formation of high-mass protein complexes in acrolein-exposed A549 cells. A 3 h exposure to acrolein (0 to 200 microM) resulted in concentration-dependent formation of a single high-mass band (approx. 180 kDa). This species was detected in cells exposed to just 50 microM acrolein, a concentration that did not elicit acute cell death as assessed by measurements of cell ATP levels. The formation of cross-linked Hsp90 coincided with a rapid loss of carbonyl adducts within cells that had been subjected to a brief "pulse" exposure to a subtoxic concentration of acrolein, suggesting Michael adducts are short-lived within cells due in part to consumption during reactions with protein nucleophiles. Cross-linked Hsp90 persisted following an overnight recovery incubation, suggesting the cellular ability to repair or degrade these species is limited. Two known carbonyl scavengers, hydralazine and bisulfite, strongly protected against the ATP depletion accompanying acrolein exposure, but only the latter suppressed protein adduction and Hsp90 cross-linking. As previously shown for hydralazine, mass spectrometry studies using a model peptide indicated that bisulfite traps carbonyl groups possessed by Michael addition adducts, and such adduct-trapping reactivity appeared to contribute to the blockade of Hsp90 cross-linking in acrolein-preloaded cells. Collectively, these findings establish that formation of stable intermolecular protein cross-links accompanies exposure to acrolein. Future clarification of the chemistry underlying this damage may provide novel biomarkers of acrolein exposure.
Enzymes such as family 11 xylanases are increasingly being used for industrial applications. Here, the cloning, structure determination and temperature-stability data of a family 11 xylanase, Xyn11X, from the alkali-tolerant Bacillus subtilis subspecies B230 are reported. This enzyme, which degrades xylan polymers, is being produced on an industrial scale for use in the paper-bleaching industry. Xyn11X adopts the canonical family 11 xylanase fold. It has a greater abundance of side chain to side chain hydrogen bonds compared with all other family 11 xylanase crystal structures. Means by which the thermostability of Xyn11X might be improved are suggested.
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