Functional and structural studies of two enzymes: membrane bound kinase and Z-DNA/Z-RNA-binding protein

Dissertação para obtenção do Grau de Doutor em Sistemas de Bioengenharia

The neocortex is a unique structure designed for higher cognitive and associative functions. Herein, we will refer to the work performed to assess the role of TAOK2α, a specific membrane bound kinase, in the mammalian neocortical development. The results obtained, delineate a pathway whereby Semaphorin3A and Neuropilin1 transduce signals through TAOK2α and c-Jun N-terminal Kinase to regulate basal dendrite development and migration in cortical neurons. This work represents the first approach aimed at understanding the mechanisms responsible for the delineation of basal and apical dendrites during pyramidal neuron development in the embryo, and how such mechanism may evolve to neocortical disconnection disorders. Additional work performed on humanTAOK2α focused at the determination of its three-dimensional structure by X-ray crystallography to elucidate its regulatory mechanism. The 20th century exciting discovery of the DNA left-handed conformation, and the fact it binds to certain classes of proteins with high affinity and specificity, indicated a biological role to it. However, a full function is still to be elucidated. The human double-stranded RNA adenosine deaminase (ADAR1) is the best characterized of all Z-DNA binding proteins, where Zα domain binds and stabilizes Z-DNA/Z-RNA forms upon binding. The second part of this thesis describes the work on the ZαADAR1 domain that binds to Z-RNA/Z-DNA. When a section of a DNA or RNA molecule forms a left-handed Z-DNA/Z-RNA segment, two B-Z/A-Z junctions are formed. Herein, we describe the study carried out on the formation of Z-Z junctions from DNA and, also, the approach on trying to describe the Z-Z junction for RNA when interacting with ZαADAR1. The structure of the Z-Z-DNA junction consists of a single base pair that leads to partial or full disruption of the helical stacking. The junction region allows intercalating agents to insert themselves into the left-handed helix, which is otherwise resistant to intercalation.

Fundação para a Ciência e Tecnologia - PhD grant reference SFRH / BD / 37676 / 2007