Concerted primary proton transfer reactions in a thermophilic rhodopsin studied by time-resolved infrared spectroscopy at high temperature.

The primary proton transfer reactions of thermophilic rhodopsin, which was first discovered in an extreme thermophile, Thermus thermophilus JL-18, were investigated using time-resolved Fourier transform infrared spectroscopy at various temperatures ranging from 298 to 343 K (25 to 70 °C) and proton transport activity analysis. The analyses were performed using counterion (D95E, D95N, D229E, and D229N) and proton donor mutants (E106D and E106Q) as well. First, the initial proton transfer from the protonated retinal Schiff base (PRSB) to D95 was identified. The temperature dependency showed that the proton transfer reaction in the intermediate states dramatically changed above 318 K (45 °C). In addition, the proton transfer reaction correlated well with the structural change from turn to ß-strand in the protein moiety, suggesting that this step may be regulated by the rigidity of the loop region. We also elucidated that the proton transfer reaction from proton donor E106 to the retinal Schiff base occurred synchronously with the primary proton transfer from the PRSB to D95. Surprisingly, we discovered that the direction of proton transfer was regulated by the secondary counterion, D229. Comparative analysis of Gloeobacter rhodopsin from the mesophile, Gloeobacter violaceus, highlighted that the primary proton transfer reactions in thermophilic rhodopsin were optimized at high temperatures partly due to the specific turn to ß-strand structural change. This was not observed in Gloeobacter rhodopsin and other related proteins such as bacteriorhodopsin.

Expression Dynamics of Heme Oxygenase-1 in Tumor Cells and the Host Contributes to the Progression of Tumors.

Heme oxygenase (HO-1) plays an important role in cellular protection against various stresses. The induction of HO-1 is an effective strategy for reactive oxygen species-related diseases, inflammatory diseases, as well as suppressing carcinogenesis. On the other hand, the high expression of HO-1 is now well known in many tumors. In this study, we investigated the dynamics of HO-1 expression in the host and the tumor. In the mouse sarcoma S180 solid tumor model and the rat hepatoma AH136B ascitic tumor model, HO-1 expression in the tumor, as indicated by the end product of HO-1 activation, i.e., carbon monoxide, gradually increased along with tumor growth. Over-expression of HO-1 expression in mouse colon cancer C26 tumor cells significantly promoted tumor growth as well as lung metastasis, whereas opposite results were found when the HO-1 expression was reduced in the cells. On the other hand, upregulating HO-1 levels in the host by using an HO-1 inducer protected the progression of the xenograft tumor in mice, whereas lowering HO-1 expression in the host with an HO-1 inhibitor showed accelerated tumor growth and lung metastasis after subcutaneous tumor xenograft inoculation. These findings strongly suggest that the balance of HO-1 levels in the host and the tumor cells is essential for the occurrence, progression, and prognosis of cancer. Maintenance of appropriately high HO-1 levels in the host is favorable for cancer prevention, whereas suppression of HO-1 in the tumor cells may thus become a therapeutic strategy for cancer.

X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION.

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.