Langellotti, Simona (2013) Unravelling aldolase C moonlighting functions: a multi-task approach. [Tesi di dottorato]

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Abstract

The aldolase C protein is the brain-specific isoform of the glycolytic enzyme fructose 1-6 bisphosphate aldolase. It catalyzes the reversible cleavage of fructose-1,6-bisphosphate (Fru 1,6-P2) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P). Vertebrates express three tissue-specific isoforms of aldolase namely aldolase A, B and C. Aldolase A is ubiquitous and highly expressed in muscle tissues. Aldolase B is mainly expressed in the liver, where it is involved in the utilization of exogenous fructose. Aldolase C is selectively expressed in the central nervous system (CNS) but its physiological role is still unclear. Indeed, although aldolase C is undoubtedly involved in glycolysis within the brain and other tissues of neuronal origin, its glycolytic function is not believed sufficient to account for the selective expression of this isozyme in nervous tissues, for two main reasons: 1) In the CNS aldolase C is invariably expressed together with aldolase A, which could be explained as a redundancy function since aldolase A and C catalyze the same reaction in glycolysis. 2) Aldolase C is distributed in a peculiar stripe-like pattern in such areas of the human, mouse and rat brain, as the Purkinje cell layer of the cerebellum. These observations prompted the hypothesis that aldolase C, beside its role in glycolysis, exerts other specific physiological functions (which may be called “moonlighting”) within the CNS. To date, only a few observations have been reported about the possible association of the aldolase C protein with other functions. These include: an involvement in a sensory pattern of transmission and in cerebellar development; a protective function in Purkinje cells after cerebral trauma and AMPA-mediated excitoxicity; and the ability to interact with and regulate the stability of light neurofilaments (NF-L) mRNA, whose proper expression is essential since alterations have been associated to neurodegeneration. Moreover, aldolase C is the only isozyme for which no gene variants have been found so far in animals or humans. Hence, the physiological role of this enzyme isoform is basically unknown and multidisciplinary studies are required to unravel this complex biological question. The purpose of my PhD project was to gain insight into the physiology of the aldolase C protein in the CNS, to propose new hypotheses, to investigate further previously reported hypotheses, and to provide novel tools to be applied in research on aldolase C. To this aim I carried out three main tasks, which were devoted to the: 1) Identification of novel aldolase C protein interactors using a functional proteomics approach. 2) Study of the putative RNA-binding ability of the aldolase C protein to the NF-L mRNA. 3) Antigenic characterization of two new, non-commercial anti-human aldolase C antibodies. The results of each task are summarized below: 1) The functional proteomic approach led to the identification of the 14-3-3 γ protein as a novel molecular interactor of aldolase C; the 14-3-3 γ protein is involved in a broad spectrum of cellular functions. Although the functional role of this interaction is still unclear, our results might imply a role for this binding in NF-L mRNA physiology, and preliminary data also suggest a potential involvement in the NGF-mediated differentiation process. 2) The interaction of aldolase C with the NF-L mRNA was proposed by Canete-Soler and colleagues based on their finding that aldolase C interacts with this transcript through direct binding. We verified the presence of the aldolase C protein in a multimeric complex that interacts with this target molecule, thereby confirming the hypothesis that aldolase C is involved in NF-L mRNA physiology. Importantly, however, our experiments also demonstrated that no direct interaction occurs between aldolase C and the NF-L mRNA, thus not supporting the direct binding previously proposed. 3) The complementary methodologies we used to characterize two novel, non-commercial anti-human aldolase C antibodies resulted in their successful epitope mapping: both antibodies were found to target an epitope localized within a short peptidic region of the aldolase C protein, spanning from residue 85 to residue 102. Moreover, we also demonstrated that these antibodies perform well in multiple applications including immunobloting, ELISA and immunoprecipitation. Consequently, we have obtained new, effective tools for routine experiments involving aldolase C and that can also be used to design novel approaches for the investigation of the still unclear physiological functions of this brain-specific isozyme.

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