The 24-hour periodicity of biological rhythms relies on the circadian clock circuitry, made of peripheral oscillators operated by molecular clockworks and synchronized through humoral and neural outputs by central oscillators located in the hypothalamic suprachiasmatic nuclei. Circadian variation of gene expression is driven by the biological clock, operated by a transcriptional-translational negative feed-back loop hardwired by circadian proteins encoded by the core clock genes ARNTL1-2 or BMAL1, CLOCK, CRY1-2, PER1-3, NR1D1-2, RORA. An important paralogue of CLOCK in neural tissues is represented by the Neuronal PAS domain protein 2, NPAS2, a mammalian transcription factor that binds DNA as an obligate dimeric partner of BMAL1 and is implicated in the regulation of circadian rhythm. NPAS2, whose structure and biochemical facets are presently not well known, appears to reversibly bind heme as a prosthetic group to form a gas-regulated sensor, in which a heme-binding domain controls the DNA-binding activity of an additional domain in the protein triggering its transcriptional function. It is currently not completely clear how heme ligands of the ferrous state (such as the natural ligands CO, NO and O2) or the ferric state (cyanide, azide) may regulate the function of NPAS2, and consequently circadian gene expression. In this project we wish to: i) clone, express and purify recombinant NPAS2, ii) characterize the NPAS2 protein from a biochemical point of view, with particular emphasis on its thermodynamic and kinetic properties by spectroscopic techniques, and its structure features by x-ray crystallography and cryo-EM, and iii) explore its role in neural differentiation of human induced pluripotent stem cells (iPSCs).
Circadian rhythms are fundamental mechanisms observed in almost all living organisms including cyanobacteria, drosophila, arabidopsis and humans. These rhythms control behavioral and physiological phenomena, which are entrained by the daily light-dark cycle in a 24-hour period. At the molecular level, it is known that the intracellular positive and negative transcriptional/translational feedback loops of clock genes and proteins generate the oscillations of circadian clocks. NPAS2 is a transcription factor belonging to the bHLH-PAS superfamily and each of two PAS domain binds heme as a prosthetic group, and the PASA domain acts as a gas sensor to regulate the DNA-binding activity of the NPAS2/ BMAL1 heterodimer by CO and NO binding to the ferrous heme. NADH and NADPH enhance the DNA-binding activity of the NPAS2/ BMAL1 heterodimer, whereas NAD+ and NADP+ inhibit its activity, suggesting regulation by the redox state of the NAD cofactors.
Actually, the biochemical properties of NPAS2 protein are only partially known, while the secondary, tertiary (and quaternary) structural features are still undefined. A better characterization of NPAS2 protein from the point of view of its biochemical aspects and structural features would expand our knowledge about its functional aspects and physiological roles. A more accurate characterization of NPAS2 is even more relevant considering its interplay with NAD cofactors, which are well known as essential electron carriers used in a huge number of metabolic pathways including glycolysis, TCA cycle, ß-oxidation, and biosynthesis of fatty acids, sterols and nucleotides. . This evidence pinpoints a key role for NPAS2 in metabolism regulation and suggests a crucial involvement of NPAS2 signaling deregulation, through ¿metaflammation¿ and ¿inflammaging¿, in metabolic and neurodegenerative diseases, which represent an ever-increasing socio-economic burden to contemporary society.