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Biochimica Et Biophysica Acta.... Oct 2022Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene...
Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.
Topics: Cytostatic Agents; Ergosterol; Ginsenosides; Saccharomyces cerevisiae; Sterols; Sugars; Triterpenes
PubMed: 35724740
DOI: 10.1016/j.bbamem.2022.183993 -
Synthetic Biology (Oxford, England) 2022Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species....
Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of . Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract.
PubMed: 35774105
DOI: 10.1093/synbio/ysac008 -
Plant Methods Jun 2024PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin...
PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.
PubMed: 38825682
DOI: 10.1186/s13007-024-01182-7 -
Discover Nano Jan 2024In this work, iron oxide (FeO) magnetic nanoparticles (MNPs) and graphene oxide (GO) nanosheets were prepared via the co-precipitation technique and the Modified Hummer...
In this work, iron oxide (FeO) magnetic nanoparticles (MNPs) and graphene oxide (GO) nanosheets were prepared via the co-precipitation technique and the Modified Hummer method. FeO MNPs and GO nanosheets were combined to prepare FeO/GO nanocomposite and subsequently conjugated with Digitonin (DIG) in order to obtain a dual-targeted delivery system based on DIG/FeO/GO nanocomposite. SEM images reveal the presence of FeO MNPs at a scale of 100 nm, exhibiting dispersion between the GO nanosheets. Aggregation of the DIG/FeO/GO nanocomposite was observed at various size scales. The XRD structural analysis confirms the crystal structure of the prepared samples. The FeO MNPs demonstrated the main XRD-diffracted peaks. Also, GO nanosheets exhibit crystalline characteristics on the (001) and (002) planes. The predominant peaks observed in the DIG/GO/FeO nanocomposite are attributed to the crystal phases of FeO MNPs. The FT-IR vibrational modes observed in the GO/DIG/FeO nanocomposite indicate the presence of crosslinking between GO nanosheet layers and the FeO MNPs. The antioxidant activity of the prepared samples was measured and the DIG/GO/FeO nanocomposite demonstrated a significantly high antioxidant activity in both 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) tests.
PubMed: 38253925
DOI: 10.1186/s11671-024-03960-7 -
The Journal of Biological Chemistry Jul 2019Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial...
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-C]glucose to [C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.
Topics: Acetyl Coenzyme A; Acyltransferases; Animals; Carbon; Cardiolipins; Cell Line; Citric Acid Cycle; Energy Metabolism; Enzyme Activation; Lipids; Mice; Mice, Knockout; Mitochondria; Pyruvate Carboxylase; Pyruvate Dehydrogenase Complex; Transcription Factors
PubMed: 31186346
DOI: 10.1074/jbc.RA119.009037 -
Frontiers in Pharmacology 2021The Na/K-ATPase α1 subunit (ATP1A1) is a potential target for hepatic carcinoma (HCC) treatment, which plays a key role in Na/K exchange, metabolism, signal...
The Na/K-ATPase α1 subunit (ATP1A1) is a potential target for hepatic carcinoma (HCC) treatment, which plays a key role in Na/K exchange, metabolism, signal transduction, etc. , we found that saponins (PNS) could inhibit tumor growth and significantly downregulate the expression and phosphorylation of ATP1A1/AKT/ERK in tumor-bearing mice. Our study aims to explore the potential effects of PNS on the regulation of ATP1A1 and the possible mechanisms of antitumor activity. The effects of PNS on HepG2 cell viability, migration, and apoptosis were examined . Fluorescence, Western blot, and RT-PCR analyses were used to examine the protein and gene expression. Further analysis was assessed with a Na/K-ATPase inhibitor (digitonin) and sorafenib . We found that the ATP1A1 expression was markedly higher in HepG2 cells than in L02 cells and PNS exhibited a dose-dependent effect on the expression of ATP1A and the regulation of AKT/ERK signaling pathways. Digitonin did not affect the expression of ATP1A1 but attenuated the effects of PNS on the regulation of ATP1A1/AKT/ERK signaling pathways and enhanced the antitumor effect of PNS by promoting nuclear fragmentation. Taken together, PNS inhibited the proliferation of HepG2 cells downregulation of ATP1A1 and signal transduction. Our findings will aid a data basis for the clinical use of PNS.
PubMed: 34690763
DOI: 10.3389/fphar.2021.720368 -
Scientific Reports Jul 2021In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced...
In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced genome, conferring it the status of a model organism; nonetheless, a high-resolution structure of its Photosystem II is missing. We present the first Cryo-EM high-resolution structure of Arabidopsis PSII supercomplex with average resolution of 2.79 Å, an important model for future PSII studies. The digitonin extracted PSII complexes demonstrate the importance of: the LHG2630-lipid-headgroup in the trimerization of the light-harvesting complex II; the stabilization of the PsbJ subunit and the CP43-loop E by DGD520-lipid; the choice of detergent for the integrity of membrane protein complexes. Furthermore, our data shows at the anticipated MnCaO-site a single metal ion density as a reminiscent early stage of Photosystem II photoactivation.
Topics: Arabidopsis; Cryoelectron Microscopy; Digitonin; Photosystem II Protein Complex
PubMed: 34330992
DOI: 10.1038/s41598-021-94914-x -
Archives of Biochemistry and Biophysics Sep 2020To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the...
To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the biological activity and/or perturb oligomerization of individual MPs. Nanodisc scaffold peptide (NSP), an amphipathic peptide analog of ApoA1, was recently shown to reconstitute detergent solubilized MPs into peptidiscs in vitro. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also known as sterol O-acyltransferase 1 (SOAT1), plays a key role in cellular cholesterol storage in various cell types and is a drug target to treat multiple human diseases. ACAT1 contains nine transmembrane domains (TMDs) and primarily forms a homotetramer in vitro and in intact cells; deletion of the N-terminal dimerization domain produces a homodimer with full retention in catalytic activity. ACAT1 is prone to inactivation by numerous detergents. Here we pursued the use of NSP to overcome the detergent-induced inactivation of ACAT1 by generating near detergent-free ACAT1 peptidiscs. Based on native-PAGE analysis, we showed that NSP reconstitutes ACAT1 into soluble peptidiscs, in which ACAT1 exists predominantly in oligomeric states greater than a homotetramer. The formation of these higher-order oligomeric states was independent of the N-terminal dimerization domain, suggesting that the oligomerization is mediated through hydrophobic interactions of multiple ACAT1 subunits. ACAT1 peptidiscs were still susceptible to heat-mediated inactivation, presumably due to the residual detergent (CHAPS) bound to ACAT1. We then conditioned ACAT1 with phosphatidylcholine (PC) to replace CHAPS prior to the formation of ACAT1 peptidiscs. The results showed, when PC was included, ACAT1 was present mainly in higher-order oligomeric states with greater enzymatic activity. With PC present, the enzymatic activity of ACAT1 peptidiscs was protected from heat-mediated inactivation. These results support the use of NSP to create a near detergent-free solution of ACAT1 in peptidiscs for various in vitro studies. Our current results also raise the possibility that, under certain conditions, ACAT1 may form higher-order oligomeric states in vivo.
Topics: Amino Acid Sequence; Animals; CHO Cells; Cholic Acids; Cricetulus; Detergents; Digitonin; Humans; Peptides; Protein Domains; Protein Multimerization; Sterol O-Acyltransferase; Surface-Active Agents
PubMed: 32735863
DOI: 10.1016/j.abb.2020.108518 -
RSC Advances Jun 2022There is a great demand for the rapid and non-invasive atherosclerosis screening method. Cholesterol content in the epidermis of the skin is an early biomarker for...
There is a great demand for the rapid and non-invasive atherosclerosis screening method. Cholesterol content in the epidermis of the skin is an early biomarker for atherosclerosis. Risk assessment of atherosclerosis can be achieved by measuring cholesterol in the epidermis. Here, we synthesised a new fluorescent digitonin derivative (FDD) for the non-invasive detection of skin cholesterol. The results of fluorescence spectroscopy studies indicated that the probe exhibited desirable selectivity for cholesterol. The proof-of-concept preclinical study confirmed that FDD can detect different concentrations of skin cholesterol; patients diagnosed with atherosclerotic cardiovascular disease and the at-risk atherosclerosis group exhibited higher skin cholesterol content than the normal group. The area under the ROC curve for distinguishing the normal/disease group was 0.9228 (95% confidence interval, 0.8938 to 0.9518), and the area under the ROC curve for distinguishing the normal/risk group was 0.9422 (95% confidence interval, 0.9178 to 0.9665). We anticipate that this non-invasive skin cholesterol test may be used as a risk assessment tool for atherosclerosis screening in a large population for further examination and intervention in high-risk populations.
PubMed: 35799936
DOI: 10.1039/d2ra01982e -
Drug Metabolism and Disposition: the... Nov 2023Our recent study revealed that SLC49A4, known as disrupted in renal carcinoma 2, is a H-coupled lysosomal exporter for pyridoxine (vitamin B6), a cationic compound, and...
Our recent study revealed that SLC49A4, known as disrupted in renal carcinoma 2, is a H-coupled lysosomal exporter for pyridoxine (vitamin B6), a cationic compound, and involved in the regulation of its lysosomal and cellular levels. We here examined a possibility that this transporter might also transport cationic amphiphilic drugs (CADs) that are known to undergo lysosomal trapping, using pyrilamine, an H-antagonist, as a model CAD and the COS-7 cell line as a model cell system for transient introduction of human SLC49A4 and a recombinant SLC49A4 protein (SLC49A4-AA), in which the N-terminal dileucine motif involved in lysosomal localization was removed by replacing with dialanine for redirected localization to the plasma membrane. The introduction of SLC49A4 into COS-7 cells induced a significant decrease in the accumulation of pyrilamine in the intracellular compartments in the cells treated with digitonin for permeabilization of plasma membranes, suggesting its operation for lysosomal pyrilamine export. Accordingly, functional analysis using the SLC49A4-AA mutant, which operates for cellular uptake at the plasma membrane, in transiently transfected COS-7 cells demonstrated its H-coupled operation for pyrilamine transport, which was saturable with a Michaelis constant of 132 μM at pH 5.5. In addition, many CADs that may potentially undergo lysosomal trapping, which include imipramine, propranolol, verapamil, and some others, were found to inhibit SLC49A4-AA-mediated pyrilamine transport, suggesting their affinity for SLC49A4. These results suggest that SLC49A4 is involved in the lysosomal trapping of pyrilamine, operating for its exit. The CADs that inhibited SLC49A4-AA-mediated pyrilamine transport could also be SLC49A4 substrate candidates. SLC49A4 mediates the transport of pyrilamine in a H-coupled manner at the lysosomal membrane. This could be a newly identified mechanism for lysosomal export involved in its lysosomal trapping.
PubMed: 37963658
DOI: 10.1124/dmd.123.001354