Serio, Laura (2011) Human aldolase C: gene transcriptional regulation and protein functional role. [Tesi di dottorato] (Inedito)

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Abstract

Aldolase C is the brain-specific aldolase isoenzyme. In the human brain, aldolase C messenger and protein are expressed in a stripe-like pattern in the Purkinje cells of the cerebellum, in the inferior olives and in the Goll and Burdach nuclei of the posterior horn in the spinal cord. Notwithstanding numerous studies have been conducted on aldolase C transcriptional regulation, promoter regions governing brain- and cell-specific expression in human are not yet precisely known. We analysed transcriptional regulation of human aldolase C gene through in vitro and in vivo systems. We previously demonstrated that cAMP increased human aldolase C gene expression in PC12 cells through NGFI-B binding to element D in the distal promoter region of the gene. Here we demonstrate that NGF up-regulates human aldolase C gene expression in PC12 cells. We identified the element E in the distal promoter region as responsive to NGF treatment and demonstrated that USF1 binds to this region thus mediating transcriptional up-regulation. We also analyzed the transcriptional regulation of the human aldolase C gene using transgenic mice as in vivo model. We found that the construct pAldC2500-LacZ, containing the cis-elements A, B, C, D and E in the promoter region, was able to direct specific, high levels AldC/LacZ hybrid messenger expression in the brain of transgenic mice, as well as stripe-like expression in the Purkinje cells of the cerebellum. Construct pAldC1500-LacZ, containing elements A, B and C in the proximal promoter region, plus element D of the distal region, was able to direct low levels brain-specific AldC/LacZ and also the stripe-like expression in the Purkinje cell layer. Here we analysed three new transgenic mice lines carrying constructs containing different deleted promoter regions of human aldolase C gene fused to LacZ reporter gene, in order to further point out the role of distal and proximal regions in governing brain-specific and stripe-like expression in vivo. Our results indicate that proximal region contains all the elements required for low-levels, stripe-like expression of aldolase C in the cerebellar Purkinje cell layer; the distal region is responsible for high-levels, stripe-like expression in the cerebellum and in distinct brain areas. The second part of this thesis focuses on the functional role(s) of aldolase C protein. To improve our knowledge on the canonical glycolytic role of aldolase C, linking isozyme structure with function, we produced recombinant aldolases by swapping Isozymes Specific Residues (ISRs) from aldolase A to C and viceversa and we analysed their kinetic properties. Finally, to shed light on the putative additional functions we have long intended aldolase C exert, we started a project aimed to the production of a conditional aldolase C knockout mouse.

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