Search Results (4)

Fluorescent proteins (FPs) are widely used as optical probes in molecular and cell biology. tKeima is a tetrameric, large Stokes shift red fluorescent protein and the ancestral protein of mt-Keima, which is widely applied as a pH-sensitive fluorescent probe. While the pH sensitivity of mt-Keima is well characterized, the pH-dependent properties of the ancestral tKeima have not been comprehensively elucidated. To obtain a better understanding of the effects of pH on tKeima, its fluorescent emission intensity at various pH levels was measured, and its crystal structure at pH 4.0 was determined at a resolution of 2.2 Å. The fluorescence emission intensity of tKeima at pH 4.0 decreased by approximately 65% compared with its peak emission at pH 10.0. The crystal structure of tKeima at pH 4.0 revealed both cis and trans conformations of the chromophore, in contrast to previously determined structures at pH 8.0, which showed only the cis conformation. This indicates that pH induces a conformational change of the chromophore in tKeima. Both the cis and trans conformations in tKeima were stabilized by hydrogen bonds with neighboring residues. A comparison of tKeima at pH 4.0 with tKeima at basic pH, as well as with mKeima, highlights its unique structural properties. These results provide a deeper understanding of the structural basis for the pH-induced fluorescence emission changes in the Keima family. Full article

The Keima family comprises large Stokes shift fluorescent proteins that are useful for dual-color fluorescence cross-correlation spectroscopy and multicolor imaging. The tKeima is a tetrameric large Stokes shift fluorescent protein and serves as the ancestor fluorescent protein for both dKeima and mKeima. The spectroscopic properties of tKeima have been previously reported; however, its structural basis and molecular properties have not yet been elucidated. In this study, we present the crystallographic results of the large Stokes shift fluorescent protein tKeima. The purified tKeima protein spontaneously crystallized after purification without further crystallization. The crystal structure of tKeima was determined at 3.0 Å resolution, revealing a β-barrel fold containing the Gln-Tyr-Gly chromophores mainly with cis-conformation. The tetrameric interfaces of tKeima were stabilized by numerous hydrogen bonds and salt–bridge interactions. These key residues distinguish the substituted residues in dKeima and mKeima. The key structure-based residues involved in the tetramer formation of tKeima provide insights into the generation of a new type of monomeric mKeima. This structural analysis expands our knowledge of the Keima family and provides insights into its protein engineering. Full article

Particulate matter (PM) has deleterious consequences not only on the respiratory system but also on essential human organs, such as the heart, blood vessels, kidneys, and liver. However, the effects of PM inhalation on skeletal muscles have yet to be sufficiently elucidated. Female C57BL/6 or mt-Keima transgenic mice were randomly assigned to one of the following four groups: control (CON), PM exposure alone (PM), treadmill exercise (EX), or PM exposure and exercise (PME). Mice in the three-treatment group were subjected to treadmill running (20 m/min, 90 min/day for 1 week) and/or exposure to PM (100 μg/m³). The PM was found to exacerbate oxidative stress and inflammation, both at rest and during exercise, as assessed by the levels of proinflammatory cytokines, manganese-superoxide dismutase activity, and the glutathione/oxidized glutathione ratio. Furthermore, we detected significant increases in the levels of in vivo mitophagy, particularly in the PM group. Compared with the EX group, a significant reduction in the level of mitochondrial DNA was recorded in the PME group. Moreover, PM resulted in a reduction in cytochrome c oxidase activity and an increase in hydrogen peroxide generation. However, exposure to PM had no significant effect on mitochondrial respiration. Collectively, our findings in this study indicate that PM has adverse effects concerning both oxidative stress and inflammatory responses in skeletal muscle and mitochondria, both at rest and during exercise. Full article

Peroxisomes are functionally specialized organelles that harbor multiple hydrogen peroxide (H₂O₂)-producing and -degrading enzymes. Given that this oxidant functions as a major redox signaling agent, peroxisomes have the intrinsic ability to mediate and modulate H₂O₂-driven processes, including autophagy. However, it remains unclear whether changes in peroxisomal H₂O₂ (po-H₂O₂) emission impact the autophagic process and to which extent peroxisomes with a disturbed H₂O₂ metabolism are selectively eliminated through a process called “pexophagy”. To address these issues, we generated and validated HEK-293 and HeLa pexophagy reporter cell lines in which the production of po-H₂O₂ can be modulated. We demonstrate that (i) po-H₂O₂ can oxidatively modify multiple selective autophagy receptors and core autophagy proteins, (ii) neither modest nor robust levels of po-H₂O₂ emission act as a prime determinant of pexophagy, and (iii) high levels of po-H₂O₂ impair autophagic flux by oxidative inhibition of enzymes involved in LC3II formation. Unexpectedly, our analyses also revealed that the autophagy receptor optineurin can be recruited to peroxisomes, thereby triggering pexophagy. In summary, these findings lend support to the idea that, during cellular and organismal aging, peroxisomes with enhanced H₂O₂ release can escape pexophagy and downregulate autophagic activity, thereby perpetuating the accumulation of damaged and toxic cellular debris. Full article