RESUMEN
CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.
Asunto(s)
Criptocromos/química , Isoformas de Proteínas/química , Animales , Sitios de Unión , Relojes Circadianos , Criptocromos/genética , Fibroblastos/metabolismo , Células HEK293 , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones Noqueados , Modelos Moleculares , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/genética , TermodinámicaRESUMEN
Temporal changes of mRNA expression of three clock genes, qPer2, qPer3 and qClock, were studied in the suprachiasmatic nucleus (SCN) of Japanese quail under different light conditions, as well as under the condition of continuous melatonin. In addition, the expression of melatonin receptor genes, Mel1a and Mel1c, in the SCN were also examined. The expression of qPer2 mRNA showed robust oscillation during both light and dark (LD) 12:12 cycles and under constant dark conditions (DD), but did not exhibit circadian rhythmicity in constant light conditions (LL), instead being expressed at a consistently high level. Expression of qPer3 also showed robust oscillation under both LD and DD conditions. Unlike qPer2 however, qPer3 mRNA expression remained rhythmic under LL conditions. Contrary to the findings on the other clock genes, no remarkable rhythmicity was detectable in either light condition. Both Mel1a and Mel1c mRNAs were detected in the SCN, however, Mel1a mRNA levels were higher than Mel1c and showed daily rhythmicity. Although implantation of melatonin tubes caused constant high levels of plasma melatonin and consequently masked the endogenous daily melatonin rhythm, no significant differences in the expression pattern of any of the three clock genes were observed between birds with and without constant melatonin. In addition, a single injection of melatonin did not affect mRNA expression of these clock genes. These results suggest that melatonin does not affect transcription of clock genes, but may act on the mechanism of synchronization among SCN oscillatory cells.