CREB3L1/OASIS: cell cycle regulator and tumor suppressor.

Cell cycle checkpoints detect DNA errors, eventually arresting the cell cycle to promote DNA repair. Failure of such cell cycle arrest causes aberrant cell proliferation, promoting the pathogenesis of multiple diseases, including cancer. Endoplasmic reticulum (ER) stress transducers activate the unfolded protein response, which not only deals with unfolded proteins in ER lumen but also orchestrates diverse physiological phenomena such as cell differentiation and lipid metabolism. Among ER stress transducers, cyclic AMP-responsive element-binding protein 3-like protein 1 (CREB3L1) [also known as old astrocyte specifically induced substance (OASIS)] is an ER-resident transmembrane transcription factor. This molecule is cleaved by regulated intramembrane proteolysis, followed by activation as a transcription factor. OASIS is preferentially expressed in specific cells, including astrocytes and osteoblasts, to regulate their differentiation. In accordance with its name, OASIS was originally identified as being upregulated in long-term-cultured astrocytes undergoing cell cycle arrest because of replicative stress. In the context of cell cycle regulation, previously unknown physiological roles of OASIS have been discovered. OASIS is activated as a transcription factor in response to DNA damage to induce p21-mediated cell cycle arrest. Although p21 is directly induced by the master regulator of the cell cycle, p53, no crosstalk occurs between p21 induction by OASIS or p53. Here, we summarize previously unknown cell cycle regulation by ER-resident transcription factor OASIS, particularly focusing on commonalities and differences in cell cycle arrest between OASIS and p53. This review also mentions tumorigenesis caused by OASIS dysfunctions, and OASIS's potential as a tumor suppressor and therapeutic target.

Converting cytochrome c into a DyP-like metalloenzyme.

Dye-decolorizing peroxidase (DyP), which can degrade anthraquinone dyes using H2O2, is an attractive prospect for potential biotechnological applications for environmental purification. We previously designed an artificial DyP with an optimal pH for reactive blue 19 (RB19) degradation shifting from pH 4.5 to 6.5. We then attempted to degrade RB19 using Escherichia coli expressing this mutant, but RB19 was degraded equally compared with bacteria expressing wild-type (WT) DyP because most DyP was expressed in a heme-free form. In this study, we attempted to design an artificial peroxidase based on cytochrome c (cyt c), whose heme is covalently bound to the protein. We found that cyt c can degrade RB19, but its ability at pH 7.0 was ∼60% of that of DyP from Vibrio cholerae at pH 4.5. To enhance this activity we constructed several mutants using three approaches. Initially, to improve reactivity with H2O2, Met80 was replaced with a noncoordinating residue, Ala or Val, but catalytic efficiency (kcat/Km) was increased by only ∼1.5-fold. To enhance the substrate binding affinity we introduced an additional Trp by replacing Pro76 (P76W). The catalytic efficiency of this mutant was ∼3-fold greater than that of WT cyt c. Finally, to form a hydrogen bond to axial histidine Gly29 was replaced with Asp (G29D). This mutant exhibited an ∼80-fold greater dye-decolorizing activity. Escherichia coli expressing the G29D mutant was unable to degrade RB19 in solution due to degradation of heme itself, but this study provides new insights into the design of artificial DyPs.

Radical transfer but not heme distal residues is essential for pH dependence of dye-decolorizing activity of peroxidase from Vibrio cholerae.

Dye-decolorizing peroxidase (DyP) is a heme-containing enzyme that catalyzes the degradation of anthraquinone dyes. A main feature of DyP is the acidic optimal pH for dye-decolorizing activity. In this study, we constructed several mutant DyP enzymes from Vibrio cholerae (VcDyP), with a view to identifying the decisive factor of the low pH preference of DyP. Initially, distal Asp144, a conserved residue, was replaced with His, which led to significant loss of dye-decolorizing activity. Introduction of His into a position slightly distant from heme resulted in restoration of activity but no shift in optimal pH, indicating that distal residues do not contribute to the pH dependence of catalytic activity. His178, an essential residue for dye decolorization, is located near heme and forms hydrogen bonds with Asp138 and Thr278. While Trp and Tyr mutants of His178 were inactive, the Phe mutant displayed ~35% activity of wild-type VcDyP, indicating that this position is a potential radical transfer route from heme to the active site on the protein surface. The Thr278Val mutant displayed similar enzymatic properties as WT VcDyP, whereas the Asp138Val mutant displayed significantly increased activity at pH 6.5. On the basis of these findings, we propose that neither distal amino acid residues, including Asp144, nor hydrogen bonds between His178 and Thr278 are responsible while the hydrogen bond between His178 and Asp138 plays a key role in the pH dependence of activity.