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Iron binding proteins
- Reptile freeze tolerance: Metabolism and gene expression

Heart and liver of hatchling C. p. marginata showed enhanced expression during freezing and anoxia of the mRNA coding for iron binding proteins including ferritin heavy (H) and light (L) chains, transferrin receptor 2 (TfR2), and hemoglobin α and β chains. Iron is a vital component of many functional proteins in cells but free iron in the ferrous state (Fe2+) participates in the Fenton reaction with hydrogen peroxide and lipid peroxides to generate highly reactive hydroxyl radicals and lipid radicals [45] and [47]. Hence, intracellular free iron levels are kept low by storing the metal in ferritin, a huge protein consisting of 24 H and L subunits that surround a core of up to 4500 iron atoms locked in a low reactivity ferrihydrite state [45]. In plasma, iron is bound tightly to another protein, transferrin (Tf), and iron uptake into cells involves endocytosis of Tf after docking with the cell surface transferrin receptor (TfR) [45]. Acidification of the endosomes releases the iron and Tf and TfR are recycled. The TfR1 isoform is ubiquitous but TfR2 in mammals is apparently restricted to liver and erythroid cells. Ferritin, Tf, TfR and hemoglobin are induced by hypoxia in mammals and the latter three genes are under the control of HIF-1 [72], [86] and [90].

HIF-1 up-regulates genes whose protein products contribute to two main responses to low oxygen: (a) enhancing capacity for anaerobic ATP production (e.g., up-regulation of glycolytic enzymes and glucose transporters), and (b) enhancing oxygen delivery to tissues (e.g., increased synthesis of red blood cells, stimulation of capillary growth). These gene responses to freezing or anoxia in the hatchlings suggest that an activation of HIF-1 occurs under both stresses. This is also supported by our early studies of the hatchlings that found a freeze-responsive increase in the activity of two glycolytic enzymes known to be HIF-1 responsive (pyruvate kinase, lactate dehydrogenase) in liver and skeletal muscle [85]. Thus, it appears that HIF-1 mediated responses to oxygen limitation are being activated by freezing and anoxia exposures in the hatchlings. This differs from the response by adult turtles which do not show up-regulation of genes under HIF-1 control during anoxic submergence; indeed, a gene coding for an inhibitor of vascular growth is one of the prominently up-regulated genes in adult turtles under anoxia [77]. However, our array screening of freeze-responsive genes in wood frogs showed prominent up-regulation of the HIF-1 α subunit and ferritin light chain [75] indicating consistency in the response to freezing. The differences between the hatchling and adult turtle responses to anoxia probably reflect the constitutive enzyme/protein levels at the two life stages. Metabolic adaptations supporting anoxia tolerance are fully developed in adults that experience frequent hypoxia/anoxic excursions while diving or hibernating underwater whereas anoxia tolerance may be less fully developed in hatchlings that have not yet experienced hypoxic/anoxic excursions. Hence, the hatchlings respond to anoxia or freezing with a rapid up-regulation of genes that will enhance their survival at low oxygen.

Ferritin and TfR2 up-regulation under freezing or anoxia (or during recovery) in hatchling turtles would result in increased sequestering of iron. This could help to supply the iron needs for hemoglobin synthesis but may also have another function. In recent studies with marine snails (Littorina littorea) we also found anoxia-responsive up-regulation of ferritin H chain mRNA and protein as well as enhanced levels of ferritin mRNA in the polysome fraction during anoxia. Hence, ferritin is one of just a few actively translated proteins in anoxia [57]. However, snails do not have hemoglobin and so enhanced iron sequestering in anoxia must have another purpose. Two seem possible: (a) to sequester free Fe2+ to minimize iron-mediated generation of ROS, particularly when oxygen is rapidly reintroduced into tissues when the anoxic excursion ends, or (b) to store excess iron during hypometabolism when the net rate of biosynthesis of iron-containing proteins is low. For hatchling turtles, the first possibility seems quite likely especially when put together with the simultaneous up-regulation of antioxidant genes in turtle organs, as discussed next.

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