A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins

Rev-erb␣ and Rev-erb␤ are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erb␣ recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erb␤ (FLRev-erb␤) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo-and heme-replete FLRev-erb␤⅐DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erb␤⅐NCoR1 complex. The interaction between FLRev-erb␤ and NCoR1 as well as Reverb␤ repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erb␤ in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10 ؊6 s ؊1 ) of heme dissociation from Rev-erb␤ rules out a prior proposal that Reverb␤ acts as an intracellular heme sensor.Nuclear receptors (NRs) 2 are eukaryotic transcription factors that activate or repress gene expression in response to binding small signaling molecules such as steroid hormones, lipophilic vitamins, and fatty acids (1). Genes regulated by NRs are involved in a myriad of cellular processes ranging from metabolic homeostasis to growth and development. The subjects of this article, Rev-erb␣ and Rev-erb␤, are NRs that have overlapping functions and tissue expression patterns (2-9) and are under circadian control (10 -12). Rev-erb NRs repress the transcription of key genes, e.g. Bmal1, CLOCK, and Rev-erb␣ itself, involved in regulating the molecular clock (13)(14)(15). This is accomplished in part through the formation of a heterodimeric Bmal1-CLOCK complex that activates the transcription of clock-controlled genes including Rev-erb␣/␤, completing a critical feedback loop required for circadian rhythm maintenance (6, 7). Rev-erb␣ and Rev-erb␤ also regulate the expression of genes involved in gluconeogenesis, lipid homeostasis, and immune responses, ultimately synchronizing these processes to the diurnal cycle (16 -21).The multiple functions of NRs (DNA, ligand, and coregulator binding) are accomplished through their modular structure (22, 23). The N-terminal A/B domain is hypervariable, is involved in ligand-independent transcriptional regulation, and is subject to ...

Section: Methodsmentioning

confidence: 99%

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor

Carter

Gupta

Ragsdale

2016

“…QT1-6CC1 was used as the template in polymerase chain reactions to generate amplified DNA fragments corresponding to residues 1075-1115 (QT1), 1075-1128 (QT1-2), 1075-1140 (QT1-3), 1075-1153 (QT1-4), 1075-1165 (QT1-5), and 1075-1178 (QT1-6), which were subsequently cloned into a pMCSG9 vector (27) using the ligation-independent cloning protocol of Eschenfeldt et al (28). QT1-6CC1 was subcloned in a pET Sumo vector (Invitrogen), and QT5-6 and QT6 were subcloned in pET 15b vectors (Novagen).…”

Section: Methodsmentioning

confidence: 99%

Multiple Recognition Motifs in Nucleoporin Nup159 Provide a Stable and Rigid Nup159-Dyn2 Assembly

Nyarko

Song

Nováček

et al. 2013

Background: Nucleoporin Nup159 has multiple recognition motifs for Dyn2, the yeast ortholog of LC8. Results: Nup159 is intrinsically disordered and binds Dyn2 cooperatively at five of the six recognition motifs. Conclusion: Initial binding aligns two Nup chains in a bivalent scaffold; motifs 5 and 6 underlie rigidity. Significance: Multiple recognition sites provide entropy/enthalpy balance to a stable yet entropically unfavorable rigid complex.

“…The PCR product was introduced into pMCSG7 by ligation-independent cloning (LIC) to generate the plasmid pMCSG7-His-cymR (25). To make pMCSG7-His-cymRC25S plasmid, the QuikChange site-directed mutagenesis (Stratagene) strategy was used as described above.…”

Section: Methodsmentioning

confidence: 99%

Staphylococcus aureus CymR Is a New Thiol-based Oxidation-sensing Regulator of Stress Resistance and Oxidative Response

Zhang

Sun

et al. 2012

Background:The master regulator of cysteine metabolism-CymR influences S. aureus stress resistance and virulence. Results: Mutation of the sole cysteine residue Cys-25 to Ser eradicates the redox sensing ability of the protein. Conclusion:CymR is a new thiol-based oxidation-sensing regulator. Significance: Elucidating the oxidation-sensing mechanism of CymR is important for understanding oxidation sensing by S. aureus.