Overexpression and analysis on posttranslational modification of the retinoic acid related orphan receptor alpha4

The retinoic acid related orphan receptor alpha (RORalpha) regulates the expression of various target genes by binding to specific response elements in their promoter region. RORalpha is an interesting pharmaceutical target since it positively affects several pathophysiological processes of clinical relevance. RORalpha enhances the expression of Apo-AI protein, the major constituent of HDL, which is responsible for the cholesterol transportation. RORalpha notably contributes to the bone mineralization and generation of the extracellular bone matrix, demonstrating its involvement in osteoporosis, and by up-regulating the gene for IKBalpha, RORalpha has anti-inflammatory effects. Moreover, RORalpha is necessary for cerebellar development and the maintenance of the mammalian day-night periodicity governed by the core-clock within the suprachiasmatic nuclei. RORalpha receptors have been reported to bind cholesterol, melatonin, or to function ligand-independent. By monomeric binding to the recognition motif AGGTCA preceded by an A/T-rich sequence (ROR response element, RORE), RORalpha constitutively activates gene transcription. However, RORalpha activity is passively suppressed by its opponents RevErbalpha and RevErbbeta, which both bind to the same target sequence. ...Der Retinoic Acid Related Orphan Recepor alpha (RORalpha) ist ein nukleärer Rezeptor, der die Expression verschiedener Targetgene reguliert, indem er an spezifische Erkennungssequenzen in deren Promotorbereiche bindet. Pharmazeutisches Interesse erweckt RORalpha, weil es einige klinisch relevante pathophysiologische Prozesse positiv beeinflusst. Anti-atherosklerotische Eigenschaften werden RORalpha nachgesagt, da es die Expression von Apo-AI aktiviert, welches den Hauptbestandteil des Cholesterol-transportierenden HDLs darstellt. Durch die Verstärkung der Knochenmineralisierung und dem Aufbau der extrazellulären Knochenmatrix kann RORalpha einer Osteoporose entgegenwirken. Außerdem wirkt RORalpha anti-inflammatorisch, da es die Expression von IKBalpha verstärkt und damit den NF-alphaB-Signalweg hemmt. Des weiteren ist RORalpha an der Gehirnentwicklung und an der Aufrechterhaltung des Tag-Nacht- Rhythmus, der bei Säugetieren in den suprachiasmatischen Nuclei erzeugt wird, beteiligt. Bei Kristallisationsexperimenten wurde Cholesterin in der Ligandenbindungstasche von RORalpha gefunden, und es wird diskutiert, ob auch Melatonin mit dem nukleären Rezeptor interagiert. Allerdings gibt es ebenso Studien die beweisen, dass RORalpha auch ohne Ligand in der aktivierten Konformation vorliegt. Durch monomere Bindung an das DNA-Erkennungsmotiv AGGTCA (ROR response element, RORE), welchem eine A/T-reiche Sequenz vorangeht, kann RORalpha konstitutiv aktivierend wirken. Allerdings kann diese Aktivität durch die Gegenspieler RevErbalpha und RevErbbeta passiv reprimiert werden, da jene die gleichen Motive erkennen und RORalpha aus seiner Bindung verdrängen können. ..

Overexpression, refolding, and purification of polyhistidine-tagged human retinoic acid related orphan receptor RORα4

RORα4 is a nuclear receptor activating the transcription of genes that are important for a variety of physiological processes like muscle differentiation, lipid and bone metabolism, cerebellar development, and inflammation. Furthermore, it plays an essential role in maintaining circadian rhythmicity of the core clock in the suprachiasmatic nuclei (SCN). Here, we describe the successful overexpression and purification of human full-length RORα4 in Escherichia coli using a T7 expression system. The expressed protein formed inclusion bodies which were solubilized in the presence of 6 M guanidinium–HCl and renatured by gradual removal of guanidinium–HCl and addition of l-arginine. The refolded protein was purified by nickel affinity chromatography due to an N-terminal polyhistidine tag which can be cleaved with thrombin subsequently. This method permitted us to obtain up to 20 mg of pure and native RORα4 protein per liter of E. coli culture. The DNA binding activity of the refolded protein was demonstrated by electrophoretic mobility shift assay (EMSA) using an oligonucleotide comprising the ROR-response element (RORE) motif (A/G)GGTCA. In addition, we developed a new monoclonal antibody to human RORα in mice with high sensitivity and specificity

Extracellular signal-regulated kinase-2 phosphorylates RORα4 in vitro

The retinoic acid related orphan receptor RORα activates transcription of genes that play an important role in cerebellar development, the protection against age-related degenerative processes, the regulation of inflammatory responses, and is one of the pivotal participants that control the circadian rhythmicity in the core-clock of mammals. We identified the extracellular signal-regulated kinase 2 (ERK-2) as RORα4 phosphorylating kinase in vitro. The primary sequence of RORα4 contains an ERK-2 recognition motif (P-L-T128-P) within the hinge domain, and mutation of Thr-128 to Ala prevents RORα4 phosphorylation by ERK. The RORα4-T128A mutant exhibits an increased DNA-binding affinity, an increased transcriptional activity and, in the interplay with the opponent RevErbα, acts as a stronger competitor at ROR response elements than RORα4-WT