The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition.

BACKGROUND: The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1. RESULTS: To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo. CONCLUSIONS: Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.

Toxicity of smoke extracts towards A549 lung cells: role of acrolein and suppression by carbonyl scavengers.

The noxious 3-carbon electrophile acrolein forms on combustion of diverse organic matter including synthetic polymers such as polyethylene. While known to play a key role in smoke inhalation injury (SII), the molecular basis for the pulmonary toxicity of high dose acrolein-containing smoke is unclear. As a result, drug interventions in SII are poorly directed against pathogenetic smoke toxicants such as acrolein. The first aim of this study was to confirm a role for acrolein in the acute toxicity of smoke extracts towards A549 lung cells by monitoring adduction of known acrolein targets and the expression of acrolein-inducible genes. A second aim was to evaluate carbonyl scavengers for their abilities to protect cell targets and block smoke extract toxicity. Extracts were prepared by bubbling smoke released by smouldering polyethylene through a buffered saline-trap. Acrolein levels in the extracts were estimated via HPLC after derivatisation with 2,4-dinitrophenylhydrazine. Extracts were highly toxic towards A549 cells, eliciting greater ATP depletion than an equivalent concentration of acrolein alone. The toxicity was accompanied by pronounced carbonylation of several cytoskeletal targets, namely vimentin and keratins-7, -8 and -18. Western blotting revealed that polyethylene combustion products also upregulated several acrolein-responsive protein markers, including GADD45beta, NQO1, HMOX, Hsp70, Nur77 and Egr1. Several carbonyl scavengers (bisulfite, d-penicillamine, hydralazine and 1-hydrazinoisoquinoline) strongly attenuated smoke extract toxicity, with bisulfite suppressing both the adduction and cross-linking of intermediate filament targets. Bisulfite also suppressed the cytotoxicity of smoke extracts when detected using real-time monitoring of cellular impedance. These findings confirm a key role for acrolein in smoke cytotoxicity and suggest drugs that block acrolein toxicity deserve further investigation as possible interventions against SII.

Intermediate filament carbonylation during acute acrolein toxicity in A549 lung cells: functional consequences, chaperone redistribution, and protection by bisulfite.

Extensive protein carbonylation accompanies cellular exposure to acrolein, a ubiquitous smoke constituent implicated in life-threatening pulmonary edema in fire victims, a condition involving rapid erosion of the "watertight" properties of respiratory epithelium. Since the identities of lung epithelial proteins that sustain carbonylation by acrolein are unknown, we sought to identify significant targets in subcellular fractions from A549 cells after 30 min exposure to either subtoxic or acutely toxic acrolein concentrations (60 or 360 fmol acrolein/cell). The lower concentration mainly modified cytosolic proteins while the higher concentration also damaged nuclear, membrane, and cytoskeletal proteins. The multifunctional intermediate filament proteins vimentin, keratin-18, keratin-7 and keratin-8, were conspicuous targets. Consistent with their mechanical functions, a loss of cellular adhesive strength accompanied adduction of the two most abundant intermediate filaments in A549 cells, keratins-8 and -18. Acrolein also elicited redistribution of several chaperones (Hsp40, -70, -90, and -110) to intermediate filament fractions, suggesting chaperone-mediated autophagy contributes to the triage of acrolein-adducted proteins. The carbonyl scavenger bisulfite suppressed acrolein toxicity, intermediate filament adduction, vimentin cross-linking, Hsp90 redistribution, and loss of cellular adhesive strength, while also suppressing vimentin hyperphosphorylation. These novel observations identify intermediate filaments as key targets for the reactive smoke constituent acrolein.

Protein alkylation, transcriptional responses and cytochrome c release during acrolein toxicity in A549 cells: influence of nucleophilic culture media constituents.

Acrolein is a toxic combustion product that elicits apoptotic and/or necrotic cell death depending on the conditions under which exposure occurs. As a strong electrophile, side-reactions with nucleophilic media constituents seem likely to accompany study of its toxicity in vitro, but these reactions are poorly characterized. We have thus examined the effect of media composition on the toxicity of acrolein in A549 cells. Cells were exposed to acrolein in either Dulbecco's buffered saline (DBS) or F12 supplemented with various concentrations of fetal bovine serum. Cell viability was assessed using the MTT assay, while heme oxygenase-1 (HO-1) and cytoplasmic cytochrome c were measured as respective markers of transcriptional response and apoptosis. Protein damage was evaluated using the protein carbonyl assay. Compared to F12 media (with or without serum), maximal cell death as evaluated using the MTT assay, as well as adduction of intracellular proteins, occurred when cells were exposed to acrolein in DBS. In contrast, cytochrome c release was maximal in cells exposed to acrolein in serum-containing F12, conditions which inhibited protein modification and overt cell death. These findings highlight the need for careful attention to experimental conditions when conducting in vitro toxicological studies of reactive substances.

Genome-wide transcriptional responses to acrolein.

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.

Characterization of a family 11 xylanase from Bacillus subtillis B230 used for paper bleaching.

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.