Biochemical and structural characterization of two novel flavoenzymes implicated in cytoskeleton plasticity and systemic blood pressure control.
Progetto Cancer, cardiovascular and neurodegenerative diseases are leading causes of death and severe disability in the developed countries. The mechanisms underlying these processes are complex and far from being understood at the molecular level, thus hampering targeted therapy. In all these processes, a prominent role is played by the redox state of the cell. Not surprisingly novel flavoenzymes are being identified as key components in processes governing cell growth, differentiation and mobility, apoptosis, degeneration and communication. Indeed, the enzymes that use the FAD and FMN cofactors typically catalyze redox reactions using a variety of substrates, including protein sidechains and nucleic acids, and they contribute to the generation of and protection from oxidative stress.
In this context, we have recently focused our attention on two novel flavoenzymes that in vivo and ex vivo studies have demonstrated to be important in human health and disease.
1. MICAL indicates a family of multidomain proteins expressed in animals, which participate in the control of cytoskeletal rearrangements associated with cell migration during differentiation or metastatization, intracellular vesicle trafficking, cell adhesion and axon growth (Kolk & Pasterkamp Adv Exp Med Biol 600, 38, 2007). The N-terminal domain of MICAL is structurally related to the FAD-containing p-hydroxybenzate hydroxylase (PHBH) suggesting an oxidase or monoxygenase (MO) activity for this domain. It has been shown that the integrity of the MO domain is essential for transduction of the signals mediating cytoskeletal rearrangements. However, no information is available yet on the reaction catalyzed by MICAL and on its regulation as a function of its localization and interacting proteins.
2. Recently, another flavoprotein, also showing sequence similarity with PHBH, has been implicated as a key component for the control of systemic blood pressure and heart contractility. The protein was named RENALASE being produced in the kidney and secreted in the blood stream. The physiological role of renalase is unknown; it is even unknown if its effect on the cardiovascular system is linked to its catalytic activity or if the protein moonlights between an oxidoreductase (in the kidney cell) and a scaffold/modulatory protein (when secreted into the blood stream; Xu & Desir, Curr Opin Nephrol Hypertens 16, 373,2007).
We have produced and purified, for the first time, the recombinant human MO domain of MICAL and the full-length form of renalase. Here, we propose to extend their characterization through the identification of their substrates by combining kinetic and equilibrium studies on wild-type and engineered protein forms with the determination of their three-dimensional structures through X-ray crystallography.
This knowledge will shed light on the functional roles of the enzymes, thus contributing to the understanding of the fundamental processes in which they are involved.