Protocol for determination of phosphatidylinositol 3-phosphate levels and localization in Candida glabrata by confocal microscopy.

Phosphatidylinositol 3-phosphate (PI3P) levels govern membrane trafficking in Candida glabrata. Here, we present a confocal imaging-based protocol for PI3P localization analysis using the GFP-FYVE (found in Fab1, YOTB, Vac1, and EEA1) fusion protein. We describe steps for cloning the FYVE domain into the GFP-containing vector backbone, transforming FYVE-GFP into C. glabrata, and preparing slides with FYVE-GFP-expressing C. glabrata cells. We then detail procedures for acquiring and analyzing images and quantifying signal data. This protocol is adaptable to subcellular localization analysis of other low-abundant lipid and protein molecules. For complete details on the use and execution of this protocol, please refer to Askari et al. (2023).1.

Phosphatidylinositol 3-phosphate regulates iron transport via PI3P-binding CgPil1 protein.

Iron homeostasis, which is pivotal to virulence, is regulated by the phosphatidylinositol 3-kinase CgVps34 in the human fungal pathogen Candida glabrata. Here, we identify CgPil1 as a phosphatidylinositol 3-phosphate (PI3P)-binding protein and unveil its role in retaining the high-affinity iron transporter CgFtr1 at the plasma membrane (PM), with PI3P negatively regulating CgFtr1-CgPil1 interaction. PI3P production and its PM localization are elevated in the high-iron environment. Surplus iron also leads to intracellular distribution and vacuolar delivery of CgPil1 and CgFtr1, respectively, from the PM. Loss of CgPil1 or CgFtr1 ubiquitination at lysines 391 and 401 results in CgFtr1 trafficking to the endoplasmic reticulum and a decrease in vacuole-localized CgFtr1. The E3-ubiquitin ligase CgRsp5 interacts with CgFtr1 and forms distinct CgRsp5-CgFtr1 puncta at the PM, with high iron resulting in their internalization. Finally, PI3P controls retrograde transport of many PM proteins. Altogether, we establish PI3P as a key regulator of membrane transport in C. glabrata.

Thymoquinone binds and activates human salivary aldehyde dehydrogenase: Potential therapy for the mitigation of aldehyde toxicity and maintenance of oral health.

Human salivary aldehyde dehydrogenase (hsALDH) is a very important anti-oxidant enzyme present in the saliva. It is involved in the detoxification of toxic aldehydes and maintenance of oral health. Reduced level of hsALDH activity is a risk factor for oral cancer development. Thymoquinone (TQ) has many pharmacological activities and health benefits. This study aimed to examine the activation of hsALDH by TQ. The effect of TQ on the activity and kinetics of hsALDH was studied. The binding of TQ with the enzyme was examined by different biophysical methods and molecular docking analysis. TQ enhanced the dehydrogenase activity of crude and purified hsALDH by 3.2 and 2.9 fold, respectively. The Km of the purified enzyme decreased and the Vmax increased. The esterase activity also increased by 1.2 fold. No significant change in the nucleophilicity of the catalytic cysteine residue was observed. TQ forms a strong complex with hsALDH without altering the secondary structures of the enzyme. It fits in the active site of ALDH3A1 close to Cys 243 and the other highly conserved amino acid residues which lead to enhancement of substrate binding affinity and catalytic efficiency of the enzyme. TQ is expected to give better protection from toxic aldehydes in the oral cavity and to reduce the risk of oral cancer development through the activation of hsALDH. Therefore, the addition of TQ in the diet and other oral formulations is expected to be beneficial for health.