No abstract

A versatile fluorescent probe, PITE, based on alkyl-substituted pyridoindole (PI) and tetraphenylethylene (TE), which exhibits facile pH-induced fluorescence switching in solution, as nanoparticles, and in the solid state, is presented. Strong fluorescence in the solid state, as well as in solution and the aggregated state, allow sensing of toxic acid vapors. Fluorescence "off-on" switching of PITE through exposure to trifluoroacetic acid and triethylamine vapor is visualized by the naked eye. A unified picture of the switchable fluorescence of PITE is obtained by comprehensive spectroscopic investigations coupled with quantum mechanical calculations. Strong fluorescence, a large Stokes shift, high photostability, and biocompatibility of PITE make it a viable probe for subcellular imaging. Extensive fluorescence microscopic studies by employing organisms including lower and higher eukaryotes reveal specific localization of PITE to lipid droplets (LDs). LDs are dynamic subcellular organelles linked to various physiological processes and human diseases. Hence, the specific detection of LDs in diverse organisms is important to biomedical research and healthcare. Isolation of LDs and subsequent colocalization studies ascertain selective targeting of LDs by the easily affordable, lipophilic bioprobe, PITE. Thus, PITE is a promising multifunctional probe for chemosensing and the selective tracking of LDs.

Chromatin regulates gene expression and genome maintenance, and consists of histones and other components. The post‐translational modification of histones plays a key role in maintaining the structure and function of chromatin under different pathophysiological stress conditions. Here, we investigate the functions of previously unexplored amino acid residues in histones H3 and H4. To do so, we screened a library of yeast histone mutants following DNA damage and identified that substitution mutations of histone H3 (H3Q5A/E and H3Q120A) and H4 (H4Y88A/E and H4R78K) render yeast cells sensitive to DNA‐damaging agents. These histone mutants show an activated DNA damage response, Rad53 phosphorylation and Sml1 degradation in the presence of methyl methanesulfonate (MMS). In histone H3Q5A/E mutants, RNR2 and RNR3 genes were induced at low level, as was RNR3 in H4 histone mutants following DNA damage. In H3 mutant cells, the cell cycle was deregulated, leading to inefficient cell cycle arrest in the presence of MMS, and genes involved in aging and DNA damage repair pathways were constitutively upregulated. In H3 mutants (H3Q5A, H3Q5E and H3Q120A), we observed reduced chronological lifespan (CLS), compared with extended CLS in the H4R78K mutant. Histone mutants also showed altered H3K4me and H3K56ac modifications and improper activation of the stress responsive Slt2 and Hog1 kinases. Thus, we have determined the significance of previously uncharacterized residues of H3 and H4 in DNA damage response, cell cycle progression and cellular aging.

Arsenic is an environmental carcinogen that causes many diseases in humans, including cancers and organ failures, affecting millions of people in the world. Arsenic trioxide is a drug used for the treatment of acute promyelocytic leukemia (APL). In the present study, we screened the synthetic histone H3 and H4 library in the presence of arsenite to understand the role of histone residues in arsenic toxicity. We identified residues of histone H3 and H4 crucial for arsenite stress response. The residues H3T3, H3G90, H4K5, H4G13, and H4R95 are required for the activation of Hog1 kinase in response to arsenite exposure. We showed that a reduced level of Hog1 activation increases the intracellular arsenic content in these histone mutants through the Fps1 channel. We have also noticed the reduced expression of ACR3 exporter in the mutants. The growth defect of mutants caused by arsenite exposure was suppressed in hyperosmotic conditions, in a higher concentration of glucose, and upon deletion of the FPS1 gene. The arsenite sensitive histone mutants also showed a lack of H3K4 methylation and reduced H4K16 acetylation. Altogether, we have identified the key residues in histone H3 and H4 proteins important for the regulation of Hog1 signaling, Fps1 activity, and ACR3 expression during arsenite stress.

Histone residues play an essential role in the regulation of various biological processes. In the present study, we have utilized the H3/H4 histone mutant library to probe functional aspects of histone residues in amino acid biosynthesis. We found that histone residue H3R72 plays a crucial role in the regulation of isoleucine biosynthesis. Substitution of arginine residue (H3R72) of histone H3 to alanine (H3R72A) renders yeast cells unable to grow in the minimal media. Histone mutant H3R72A requires the external supplementation of either isoleucine, serine, or threonine for the growth in minimal media. We also observed that H3R72 residue and leucine amino acid in synthetic complete media might play a crucial role in determining the intake of isoleucine and threonine in yeast. Further, gene deletion analysis of ILV1 and CHA1 in H3R72A mutant confirmed that isoleucine is the sole requirement for growth in minimal medium. Altogether, we have identified that histone H3R72 residue may be crucial for yeast growth in the minimal medium by regulating isoleucine biosynthesis through the Ilv1 enzyme in budding yeast Saccharomyces cerevisiae.