Coppola, Daniela (2011) Structural and functional studies of hemoproteins from polar marine organisms. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||Antarctica, fish, bacteria, hemoglobin, blood substitutes|
|Date Deposited:||06 Dec 2011 11:14|
|Last Modified:||30 Apr 2014 19:48|
Antarctica, more than any other habitat on Earth, represents a unique natural laboratory for fundamental research on the processes that have produced biological diversity in extreme environments and, at the same time, offers potential biotechnological opportunities. One of the most interesting models, within vertebrates, to study the biological responses to cold is provided by Notothenioidei, a group of related species constituting the dominant suborder of teleosts living in Antarctica. To preserve biological activity, five high-Antarctic families of this suborder, living in a stable, extremely cold, and well-oxygenated marine environment, have evolved unique specialisations, including modification of hematological features, having reduced hemoglobin concentration and multiplicity. On the contrary, the three remaining small basal sub-Antarctic families exhibit high hemoglobin multiplicity, probably as a response to temperature differences and fluctuations of temperate waters, and provide an excellent opportunity for understanding the processes involved in cold adaptation. In this thesis the structure and function of the oxygen-transport system of two sub-Antarctic notothenioids, Eleginops maclovinus (family Eleginopidae) and Dissostichus eleginoides (family Nototheniidae), were described and compared with respect to high-Antarctic species, and in particular to the hemoglobin of Trematomus bernacchii (family Nototheniidae). In contrast to high-Antarctic notothenioids, the hemoglobins of E. maclovinus and D. eleginoides show high oxygen affinity and cooperativity, and marked Root effect. In addition, in the major component of E. maclovinus, a strong stabilization of the low affinity T quaternary state and some peculiar features at the level of the tertiary and quaternary structures have been identified. The study of these fish and their adaptations is also interesting in the production of new treatments for blood-related diseases and syndromes, including anaemia, hemoglobinopathies and thalassemias or in the development of hemoglobin-based oxygen carriers, a novel pharmaceutical class used in surgery or emergency medicine. Conjugation of human and animal hemoglobins with polyethylene glycol (PEG) has been widely explored as one the most promising strategies to develop blood substitutes. In fact, PEGylation of human hemoglobin led to products with significantly different oxygen-binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity. In this context, hemoglobins from Notothenioidei are particularly interesting as they show peculiar features, namely exceptionally low oxygen affinity, little or no dissociation of the tetramer into dimers, absence of cysteine β93, that make them potentially less sensitive to the undesirable effects of PEGylation. The action of PEGylation on properties of the oxygen-transport system of Antarctic fish, and in particular of T. bernacchii hemoglobin, was investigated and compared with that of PEGylated human hemoglobin, confirming that these PEGylated Antarctic hemoglobins potentially meet the functional requirements of blood substitutes. A remarkable restriction to the research on Antarctic fish is the current lack of genomic sequence data. In contrast, this is not a problem in microorganisms with their small genomes, and which represent a remarkable source for the discovery of new potential biotechnological products. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) displays many features that make it an organism of choice for fundamental, environmental and biotechnological studies. The genome sequence shows that PhTAC125 copes with the high oxygen solubility, due to cold temperature, by increasing production of oxygen-scavenging enzymes and deleting entire metabolic pathways which generate reactive oxygen species as side products. The presence of multiple genes encoding 2/2 hemoglobins (annotated as PSHAa0030, PSHAa0458, PSHAa2217) and a flavohemoglobin gene (PSHAa2880), that seem be involved in the protection of the cells from nitrosative and oxidative stress, can also be seen in this perspective. While the flavohemoglobin is widely recognised to be involved in the NO detoxification, the role of the 2/2 hemoglobins is still not clear. Recent in vivo results demonstrated that the inactivation of the gene PSHAa0030 encoding the globin Ph-2/2HbO makes the mutant bacterial strain sensitive to high oxygen levels, hydrogen peroxide, and nitrosating agents. On this basis, the physiological role of the globin Ph-2/2HbO was investigated by in vivo and in vitro experiments. The results confirm its involvement in the protection of cells against the toxic effects of NO and related reactive nitrogen species.
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