Kevin Larade

Accumulation and translation of ferritin heavy chain transcripts following anoxia exposure in a marine invertebrate

SUMMARY Differential screening of a Littorina littorea (the common periwinkle) cDNA library identified ferritin heavy chain as an anoxia-induced gene in hepatopancreas. Northern blots showed that ferritin heavy chain transcript levels were elevated twofold during anoxia exposure, although nuclear run-off assays demonstrated that ferritin heavy chain mRNAs were not transcriptionally upregulated during anoxia. Polysome analysis indicated that existing ferritin transcripts were actively translated during the anoxic period. This result was confirmed via western blotting, which demonstrated a twofold increase in ferritin heavy chain protein levels during anoxia, with a subsequent decrease to control levels during normoxic recovery. Organ culture experiments using hepatopancreas slices demonstrated a >50% increase in ferritin heavy chain transcript levels in vitro under conditions of anoxia and freezing, as well as after incubation with the second messenger cGMP. Taken together, these results suggest that ferritin heavy chain is actively regulated during anoxia exposure in the marine snail, L. littorea.

Reversible suppression of protein synthesis in concert with polysome disaggregation during anoxia exposure in Littorina littorea

Many marine invertebrates can live without oxygen for long periods of time, a capacity that is facilitated by the ability to suppress metabolic rate in anoxia to a value that is typically less than 10% of the normal aerobic rate. The present study demonstrates that a reduction in the rate of protein synthesis is one factor in the overall anoxia-induced metabolic suppression in the marine snail, Littorina littorea. The rate of [3H]leucine incorporation into newly translated protein in hepatopancreas isolated from 48 h anoxic snails was determined to be 49% relative to normoxic controls. However, protein concentration in hepatopancreas did not change during anoxia, suggesting a coordinated suppression of net protein turnover. Analysis of hepatopancreas samples from snails exposed to 24–72 h anoxia showed a gradual disaggregation of polysomes into monosomes. A re-aggregation of monosomes into polysomes was observed after 3 h of aerobic recovery. Analysis of fractions from the ribosome profile using radiolabeled probe to detect α-tubulin transcripts confirmed a general decrease in protein translation during anoxia exposure (transcript association with polysomes decreased) with a reversal during aerobic recovery. Western blotting of hepatopancreas samples from normoxic, 24 h anoxic, and 1 h aerobic recovered snails demonstrated that eIF-2α is substantially phosphorylated during anoxia exposure and dephosphorylated during normoxia and aerobic recovery, suggesting a decrease in translation initiation during anoxia exposure. These results suggest that metabolic suppression during anoxia exposure in L. littorea involves a decrease in protein translation.

Living without Oxygen: Anoxia-Responsive Gene Expression and Regulation

Many species of marine mollusks demonstrate exceptional capacities for long term survival without oxygen. Analysis of gene expression under anoxic conditions, including the subsequent translational responses, allows examination of the functional mechanisms that support and regulate natural anaerobiosis and permit noninjurious transitions between aerobic and anoxic states. Identification of stress-specific gene expression can provide important insights into the metabolic adaptations that are needed for anoxia tolerance, with potential applications to anoxia-intolerant systems. Various methods are available to do this, including high throughput microarray screening and construction and screening of cDNA libraries. Anoxia-responsive genes have been identified in mollusks; some have known functions in other organisms but were not previously linked with anoxia survival. In other cases, completely novel anoxia-responsive genes have been discovered, some that show known motifs or domains that hint at function. Selected genes are expressed at different times over an anoxia-recovery time course with their transcription and translation being actively regulated to ensure protein expression at the optimal time. An examination of transcript status over the course of anoxia exposure and subsequent aerobic recovery identifies genes, and the proteins that they encode, that enhance cell survival under oxygen-limited conditions. Analysis of data generated from non-mainstream model systems allows for insight into the response by cells to anoxia stress.

Characterization of a novel gene up-regulated during anoxia exposure in the marine snail, Littorina littorea.

Gene expression was investigated during anoxia exposure in the marine snail, Littorina littorea. Differential screening of a cDNA library made from hepatopancreas of anoxic L. littorea yielded a 525 bp clone coding for the novel gene kvn. The deduced amino acid sequence of the KVN protein contained 99 amino acid residues with a predicted molecular weight of 12 kDa and showed an N-terminal secretory signal. Analysis of hepatopancreas samples over a time course of anoxia exposure showed a maximum increase in transcript levels of 5.8-fold after 48 h relative to normoxic animals, with a subsequent decrease in transcript levels during normoxic recovery. Nuclear run-off assays confirmed the observed transcriptional up-regulation of kvn during anoxia. Organ culture experiments were performed to determine a possible pathway of up-regulation of kvn, with data indicating a putative role for cGMP in signal transduction. Profiles of ribosome distribution in polysomes versus monosomes revealed a reduction in the polysome peak during anoxia and a shift in the position of kvn transcripts to association with the lower density polysome/higher density monosome region. The data suggest that the kvn transcript is both transcribed and translated during anoxia, indicating a possible significant role for the KVN protein in the survival of anoxia by L. littorea.

Development of Diabetes in Lean Ncb5or-Null Mice is Associated with Manifestations of Endoplasmic Reticulum and Oxidative Stress in Beta Cells

NADH-cytochrome b5 oxidoreductase (Ncb5or) is an endoplasmic reticulum (ER)-associated redox enzyme involved in fatty acid metabolism, and phenotypic abnormalities of Ncb5or(-/-) mice include diabetes and lipoatrophy. These mice are lean and insulin-sensitive but become hyperglycemic at age 7 weeks as a result of β-cell dysfunction and loss. Here we examine early cellular and molecular events associated with manifestations of β-cell defects in Ncb5or(-/-) mice. We observe lower islet β-cell content in pancreata at age 4 weeks and prominent ER distention in β-cells by age 5 weeks. Ultrastructural changes progress rapidly in severity from age 5 to 6 weeks, and their frequency rises from 10% of β-cells at 5 weeks to 33% at 6 weeks. These changes correlate temporally with the onset of diabetes. ER stress responses and lipid load in Ncb5or(-/-) β-cells were assessed with isolated islets from mice at age 5 weeks. Expression levels of the stress marker protein Grp78/BiP and of phosphorylated eIF2α protein were found to be reduced, although their transcript levels did not decline. This pattern stands in contrast to the canonical unfolded protein response. Ncb5or(-/-) β-cells also accumulated higher intracellular levels of palmitate and other free fatty acids and exhibited greater reactive oxygen species production than wild-type cells. An alloxan-susceptible genetic background was found to confer accelerated onset of diabetes in Ncb5or(-/-) mice. These findings provide the first direct evidence that manifestations of diabetes in lean Ncb5or(-/-) mice involve saturated free fatty acid overload of β-cells and ER and oxidative stress responses.

The reductase NCB5OR is responsive to the redox status in β-cells and is not involved in the ER stress response

The novel reductase NCB5OR (NADPH cytochrome b5 oxidoreductase) resides in the ER (endoplasmic reticulum) and may protect cells against ER stress. Levels of BiP (immunoglobulin heavy-chain-binding protein), CHOP (CCAAT/enhancer-binding protein homologous protein) and XBP-1 (X-box-binding protein-1) did not differ in WT (wild-type) and KO (Ncb5or-null) tissues or MEFs (mouse embryonic fibroblasts), and XBP-1 remained unspliced. MEFs treated with inducers of ER stress demonstrated no change in Ncb5or expression and expression of ER-stress-induced genes was not enhanced. Induction of ER stress in beta-cell lines did not change Ncb5or expression or promoter activity. Transfection with Ncb5or-specific siRNA (small interfering RNA) yielded similar results. Microarray analysis of mRNA from islets and liver of WT and KO animals revealed no significant changes in ER-stress-response genes. Induction of oxidative stress in betaTC3 cells did not alter Ncb5or mRNA levels or promoter activity. However, KO islets were more sensitive to streptozotocin when compared with WT islets. MEFs incubated with nitric oxide donors showed no difference in cell viability or levels of nitrite produced. No significant differences in mRNA expression of antioxidant enzymes were observed when comparing WT and KO tissues; however, microarray analysis of islets indicated slightly enhanced expression of some antioxidant enzymes in the KO islets. Short-term tBHQ (t-butylhydroquinone) treatment increased Ncb5or promoter activity, although longer incubation times yielded a dose-dependent decrease in activity. This response appears to be due to a consensus ARE (antioxidant-response element) present in the Ncb5or promoter. In summary, NCB5OR does not appear to be involved in ER stress, although it may be involved in maintaining or regulating the redox status in beta-cells.

Constructing and Screening a cDNA Library

Many organisms provide excellent models for studying disease states or for exploring the molecular adaptations that allow cells and organisms to cope with or survive different stresses. The construction of a cDNA library and subsequent screening for genes of interest allows researchers to select for genes that are likely to play key roles in the regulation or response to the condition or stress of interest, those that may not be expressed (or exist) in other systems. Determination of the open reading frame(s) of novel genes, and extensive analysis of the proteins they encode, can open up new avenues of research and promote intelligent design of downstream projects.

Identification of a granulin-like transcript expressed during anoxic exposure and translated during aerobic recovery in a marine gastropod.

A novel transcript encoding a cysteine-rich granulin-like peptide (l-grn) was identified in the hepatopancreas of the marine intertidal gastropod, Littorina littorea, an anoxia-tolerant species. Experimental exposure of snails to anoxia induced a gradual accumulation of l-grn transcripts over time, with expression regulated in vitro through elements responsive to second messengers of protein kinases A, C and G. Translation of this transcript was analyzed by examining l-grn association with ribosomes during normoxia, anoxia, and aerobic recovery. Transcripts of l-grn were associated with polysomes during normoxia, moved into the monosome fractions under anoxia, but shifted back to the polysomal fractions during aerobic recovery. Western blotting confirmed this with a granulin-like protein detected under normoxic conditions, but not during anoxia exposure. A significant increase in the precursor protein and peptide (L-GRN) was observed during the aerobic recovery period. The accumulation of l-grn transcripts during anoxic exposure and subsequent translation following the return to aerobic conditions may be a response to oxidant damage that occurs during re-oxygenation. Overall, the data show that the l-grn gene is anoxia-responsive in this species and may have pro-survival functions during the recovery period.