CBP/p300 is a cell type-specific modulator of CLOCK/BMAL1-mediated transcription

<p>Abstract</p> <p>Background</p> <p>Previous studies have demonstrated tissue-specific regulation of the rhythm of circadian transcription, suggesting that transcription factor complex CLOCK/BMAL1, essential for maintaining circadian rhythm, regulates transcription in a tissue-specific manner. To further elucidate the mechanism of the cell type-specific regulation of transcription by CLOCK/BMAL1 at the molecular level, we investigated roles of CBP/p300 and tissue-specific cofactors in CLOCK/BMAL1-mediated transcription.</p> <p>Results</p> <p>As shown previously, CBP/p300 stimulates CLOCK/BMAL1-mediated transcription in COS-1 cells. However, CBP/p300 repressed CLOCK/BMAL1-mediated transcription in NIH3T3 cells and knockdown of CBP or p300 expression by siRNA enhanced this transcription. Studies using GAL4-fusion proteins suggested that CBP represses CLOCK/BMAL1-mediated transcription by targeting CLOCK. We further investigated mechanisms of this cell type-specific modulation of CLOCK/BMAL1-mediated transcription by CBP by examining roles of co-repressor HDAC3 and co-activator pCAF, which are highly expressed in NIH3T3 and COS cells, respectively. CBP repressed CLOCK/BMAL1-mediated transcription in COS-1 cells when HDAC3 was overexpressed, but activated it in NIH3T3 cells when pCAF was overexpressed. CBP forms a complex with CLOCK by interacting with HDAC3 or pCAF; however, direct interaction of CBP with CLOCK was not observed.</p> <p>Conclusion</p> <p>Our findings indicate possible mechanisms by which CBP/p300 tissue-specifically acts cooperatively with pCAF and HDAC3 either as a co-activator or co-repressor, respectively, for CLOCK/BMAL1.</p

The acute effects of antidepressants on the human AEP (Auditory Evoked Potential) and EEG

The acute effect of clomipramine hydrochloride (CMI) was studied by auditory evoked potential (AEP) and compared with those of mianserin hydrochloride (MSR), with each 12 and 16 healthy male subjects, respectively. In the two experimental session on different days, CMI (0.5mg/kg) or MSR (0.3mg/kg) were orally administered for each subjects. EEGs containing AEPs evoked by click stimuli once every 5 sec were derived from the two derivations ( 3ch : Cz→A1+2 , 6ch : Cz→T5) and recorded into magnetic tape. Reproducing the tape, AEPs before and 120 min after the administration of these drugs, with 1024 msec of analysis time were obtained by averaging 100 responses, and EEGs were subjected to the frequency analysis. The changes of the waveform of group mean AEP were studied. Individual AEPs were subjected to the component analysis, and to the statistical assessment together with EEG. The following, statistically significant results were obtained. 1. After the administration of CMI, only P8 and N8 latencies of long latency components significantly increased (P<0.05), while the peak-to-peak amplitudes of middle latency components significantly increased (P<0.01, P<0.05). In EEG, the α1 power% significantly increased (P<0.01). In conclusion, stimulatory effect of CMI besides inhibitory effect was verified by AEP. 2. After the administration of MSR, P2 and P3 latencies of the middle latency components and those of long latency components (P7~) significantly increased (P<0.01, P<0 05). All of significant changes were decrease for the peak-to-peak amplitudes (P<0.01, P<0.05). In EEG, the power% were significantly increased for δ and θ, but significantly decreased for α2 and β2 (P<0.01, P<0.05). In conclusion, sedative effect of MSR was verified by AEP

Ca2+ Regulates ERp57-Calnexin Complex Formation

ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+

Functional Interplay between P5 and PDI/ERp72 to Drive Protein Folding

The physiological functions of proteins are destined by their unique three-dimensional structures. Almost all biological kingdoms share conserved disulfide-catalysts and chaperone networks that assist in correct protein folding and prevent aggregation. Disruption of these networks is implicated in pathogenesis, including neurodegenerative disease. In the mammalian endoplasmic reticulum (ER), more than 20 members of the protein disulfide isomerase family (PDIs) are believed to cooperate in the client folding pathway, but it remains unclear whether complex formation among PDIs via non-covalent interaction is involved in regulating their enzymatic and chaperone functions. Herein, we report novel functional hetero complexes between PDIs that promote oxidative folding and inhibit aggregation along client folding. The findings provide insight into the physiological significance of disulfide-catalyst and chaperone networks and clues for understanding pathogenesis associated with disruption of the networks.P5 is one of protein disulfide isomerase family proteins (PDIs) involved in endoplasmic reticulum (ER) protein quality control that assists oxidative folding, inhibits protein aggregation, and regulates the unfolded protein response. P5 reportedly interacts with other PDIs via intermolecular disulfide bonds in cultured cells, but it remains unclear whether complex formation between P5 and other PDIs is involved in regulating enzymatic and chaperone functions. Herein, we established the far-western blot method to detect non-covalent interactions between P5 and other PDIs and found that PDI and ERp72 are partner proteins of P5. The enzymatic activity of P5-mediated oxidative folding is up-regulated by PDI, while the chaperone activity of P5 is stimulated by ERp72. These findings shed light on the mechanism by which the complex formations among PDIs drive to synergistically accelerate protein folding and prevents aggregation. This knowledge has implications for understanding misfolding-related pathology