Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Structure of TAM predicted using AlphaFold2 and depicted embedded into both the inner membrane (IM) and outer membrane (OM). POTRA and β-barrel domains of TamA (yellow), and the location of an N-terminal His-tag, are indicated. The conserved TamB domain of unknown function 490 (DUF490) is colored light-green and the remaining N-terminal region of TamB is grey. The location of a Twin-StrepII (TS)-tag introduced into TamB is indicated. ( b ) E. coli BL21 cultures expressing His TamAB, His TamAB TS and BamABCDE, His TamAB490 TS and BamABCDE, or possessing an empty pTrc99a plasmid were induced with 0.4 mM IPTG at 25 ℃ for 1h. Total cell protein was probed by western immunoblotting using αTamA, αTamB, or αStrepII and culture density (OD 600 ) was recorded that this time point (n = 3). TAM subunit expression in uninduced samples was also probed by immunoblot (Fig. S1). Asterisk, putative TamA prior to signal peptide cleavage. Statistical tests are in Table S2. ( c ) His TamAB490 TS purified in LMNG detergent (left) or reconstituted into E. coli phospholipid membrane nanodiscs with membrane scaffold protein 1D1 (MSP1D1) (right) was resolved by SDS-PAGE. ( d ) Cryo-EM map of His TamAB490 TS in LMNG detergent (3.5 Å average resolution). His TamA and TamB490 TS are colored orange and dark-green respectively. ( e ) Cryo-EM map of His TamAB490 TS in phospholipid nanodiscs (left, 3.7 Å average resolution) and model (right). ( f ) Comparison of His TamAB490 TS -nanodisc (orange, dark-green) to the crystal structure of TamA alone (white, PDB ID: 4C00 ). Models were aligned on TamA β-barrel α-carbons Y440-L577. The difference in the position of α-carbon Y274 (red sphere, TamA β1) between each model was measured. ( g ) As in f except His TamAB490 TS -nanodisc and TamAB His -amphipol (TamA, cream; TamB, teal-green; PDB ID: 9XDC ). Additional comparisons in Fig. S8 and S9.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Membrane, Expressing, Plasmid Preparation, Western Blot, Purification, SDS Page, Cryo-EM Sample Prep, Comparison, Cream

Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Magnified His TamAB490 TS -nanodisc structure showing hybridization interface between TamA β-strand 1 (β1) and TamB DUF490 C-terminal β-signal (βS) strand. Register-paired lumen-facing residues that are substituted for cysteine in experiments in b and f are indicated by matching colors, except for T270 (grey) which is membrane-facing. ( b ) E. coli expressing wild-type (WT) His TamAB TS , or derivatives with cysteine-pair substitutions at the indicated residues, were mock treated (Ox -) or treated with 200 µM 4-DPS (Ox +), n = 2. Empty pTrc99a as vector control. Intermolecular disulfide-bonds (•) in total cell protein extracts were detected by double-immunoblotting with antibodies against TamA (αTamA) and the TS-tag in TamB (αStrepII). See Fig. S10 and S11 for reduced sample and single-cysteine substitution controls, respectively. ( c ) Efficiency of plating assay. Serial dilutions of WT or mutant derivatives of E. coli K-12 W3110 were spotted onto plain LB agar plates or plates containing 5 µg/mL vancomycin, n = 3. See Fig. S12 for results with 10 µg/mL vancomycin or with 0.2% deoxycholate. ( d ) Phospholipidomic analysis of W3110 or W3110Δ tamAB Δ yhdP outer membranes, (n = 4). d i , Abundance of phospholipid classes phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), and lysophosphatidylethanolamine (LPE) possessing species with significant differences as in d ii . d ii , Volcano plot showing significant fold-changes in abundance of specific phospholipid species between strains (above the red dashed line is significant with false discovery rate correction). Letters correspond to the same species in d ii and Fig 5c. d ii , Abundances of all significantly different phospholipid species. See Table S3 for all identified lipid species ( e ) Experiment as in c except with W3110 Δ tamAB Δ yhdP or Δ tamAB Δ yhdP Δ rcsF strains spotted onto plain LB agar plates or plates containing 10 µg/mL vancomycin, n = 3. ( f ) Experiment as in c except with W3110 Δ tamAB Δ yhdP complemented with empty pTrc99a or harboring genes for expression of His TamAB TS , or derivatives with cysteine-pair substitutions at positions as in a , and spotted onto plates containing 50 µM 4-DPS and the absence or presence of 20 µg/mL vancomycin, n = 3. See Fig. S16 for different treatment concentrations of vancomycin, or 0.2% deoxycholate, and no-4-DPS controls.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Hybridization, Membrane, Expressing, Plasmid Preparation, Control, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Far left, His TamAB490 TS -nanodisc structure with views aligned to the TamB lipophilic β-taco. Left, His TamAB490 TS -nanodisc cryo-EM map with vertical slice across the TamB lipophilic β-taco revealing potential lipid densities (grey). Additional obstructing densities are transparent. Map was sharpened with LocScale2. See Fig. S21 and Videos S16-18 for cryo-EM map comparisons of sharpening methods and 3DVA analysis of rivulet of additional dynamic densities in the β-taco. Middle, model showing TamB β -taco surface hydrophobicity (blue, hydrophilic; brown, hydrophobic). TamA is transparent. Right, model showing TamB β -taco surface electrostatics (red, negative; blue, positive; white, neutral). Dashed box indicates channel terminus and the position of TamB I1102 (purple outline). Far right, magnified view of channel terminus, αH1-3 locations (2 and 3 transparent), and orientation of I1102. ( b ) Efficiency of plating experiment as in except Δ tamAB Δ yhdP strain complemented with empty pTrc99a or harboring genes for expression of His TamAB TS , or His TamAB I1102R TS . See Fig. S22 for 0.2% deoxycholate treatment condition. ( c ) Phospholipidomic experiment as in except that outer membrane phospholipids from plasmid complemented strains in b were analyzed, (n = 4). c i , Volcano plot showing significant fold-changes in abundance of specific phospholipid species in the absence of TamAB (vector) relative to the presence of WT His TamAB TS . Letters correspond to the same species in c ii and . c ii , as in c i except comparing His TamAB I1102R TS to His TamAB TS . c iii , Abundances of all significantly different phospholipid species. See Table S4 for all identified lipid species. c vi , Sum of abundances of all significantly different phospholipid species into classes. ( d ) Molecular mechanism of TAM-mediated phospholipid transport to the bacterial OM. Phospholipids enter the N-terminus of the TamB β-taco, move towards the OM through DUF490, and are released into the outer membrane through a reaction that requires the dynamicity of the conserved amphipathic α-helices.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Cryo-EM Sample Prep, Expressing, Membrane, Plasmid Preparation

Journal: Molecular Biology Reports

Article Title: Gypenosides mitigate acrylamide-induced oxidative stress, inflammation and lipid metabolic dysregulation in retinal pigment epithelial cells and in zebrafish embryos

doi: 10.1007/s11033-026-11676-3

Figure Lengend Snippet: ( A ) Effects of gypenosides (GYP) on lipid levels in in ACR-treated RPE cells. GYP treatment significantly reduced the levels of total cholesterol, triglycerides, and phospholipids, compared to that of cells treated with ACR alone. ( B ) Effects of GYP on cholesterol metabolism associated gene expression in RPE cells. The relative mRNA levels of CYP27A1 and NR1H3 were quantified by qRT-PCR, calculated using the 2 –ΔΔCT method and normalised to the housekeeping gene, Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). Data are expressed as mean ± SEM. NS, no significance; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Article Snippet: ARPE-19 cells were cultured and treated without or with 2 mM ACR, 5 μg/mL GYP, or 5 μg/mL GYP+ 2mM ACR for 24 h. Following this, the total cholesterol, triglycerides and phospholipids were assessed by, respectively, Amplex Red Cholesterol Assay kit (Thermo Fisher, UK), EnzyChrom Triglyceride assay kit, (BioAssay System, UK) and Phospholipids assay kit (Merck Life Sciences UK Limited, UK), according to the manufacturers’ protocols.

Techniques: Gene Expression, Quantitative RT-PCR

Journal: Molecular Biology Reports

Article Title: Gypenosides mitigate acrylamide-induced oxidative stress, inflammation and lipid metabolic dysregulation in retinal pigment epithelial cells and in zebrafish embryos

doi: 10.1007/s11033-026-11676-3

Figure Lengend Snippet: Effects of gypenosides (GYP) on lipid levels in acrylamide (ACR)-treated zebrafish embryos. ( A ) GYP treatment significantly reduced the levels of total cholesterol, triglycerides, and phospholipids, compared to embryos treated with ACR. ( B ) Expression of lipid synthesis associated genes was detected by qRT-PCR. The relative mRNA levels of acaca , hmgcra , and srebf2 were calculated using the 2 –ΔΔCT method and normalised to s the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase ( gapdh ). Data is expressed as mean ± SEM ( n = 6). NS, no significance; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Article Snippet: ARPE-19 cells were cultured and treated without or with 2 mM ACR, 5 μg/mL GYP, or 5 μg/mL GYP+ 2mM ACR for 24 h. Following this, the total cholesterol, triglycerides and phospholipids were assessed by, respectively, Amplex Red Cholesterol Assay kit (Thermo Fisher, UK), EnzyChrom Triglyceride assay kit, (BioAssay System, UK) and Phospholipids assay kit (Merck Life Sciences UK Limited, UK), according to the manufacturers’ protocols.

Techniques: Expressing, Quantitative RT-PCR