Journal: Nature communications

Article Title: Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer.

doi: 10.1038/s41467-017-01974-7

Figure Lengend Snippet: Fig. 5 In vitro assays to test ubiquitination of E6AP substrates identified by OUT. wt UB was transferred through wt Uba1-UbcH7-E6AP cascade to the potential substrate proteins expressed from E. coli cells. E6AP ubiquitination of MAPK1 a, PRMT5 b, CDK1 c, CDK4 d, β-catenin e, and UbxD8 f, were confirmed. E6AP-catalyzed ubiquitination of HHR23A, a previously reported E6AP substrate, was also assayed g. All blots are representative of at least three independent experiments

Article Snippet: The mammalian cell expression vectors for MAPK1 (39230), CDK1 (27652), and UbxD8 (53777) and pGEX-HHR23A (10864) were from Addgene.

Techniques: In Vitro, Ubiquitin Proteomics

Journal: Nature communications

Article Title: Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer.

doi: 10.1038/s41467-017-01974-7

Figure Lengend Snippet: Fig. 6 Cellular assays to test the ubiquitination of E6AP substrates identified by OUT. a Inhibition of E6AP expression in HEK293 cells by shE6AP was confirmed with Western blot probed with an antibody against E6AP. b–g Ubiquitination of MAPK1 b, PRMT5 c, CDK1 d,CDK4 e, β-catenin f and UbxD8 g in HECK293 cells was assayed by immunoprecipitation with antibodies against each substrate proteins and probing the ubiquitination levels of the proteins with an anti-UB antibody on the western blots. After 1.5-h treatment of cells with MG132, ubiquitination of each target protein was compared among the blank HEK293 cell (HEK293), HEK293 expressing shE6AP (shE6AP), HEK293 expressing both shE6AP and recombinant E6AP (shE6AP + OE), and HEK293 expressing recombinant E6AP (OE). h Ubiquitination of HHR23A, a known E6AP substrate, was assayed as a control. Rabbit IgG was used as a control for immunoprecipitation in b and e. Mouse IgG was used as a control for immunoprecipitation in c,d and f–h. All blots are representative of at least three independent experiments

Article Snippet: The mammalian cell expression vectors for MAPK1 (39230), CDK1 (27652), and UbxD8 (53777) and pGEX-HHR23A (10864) were from Addgene.

Techniques: Ubiquitin Proteomics, Inhibition, Expressing, Western Blot, Immunoprecipitation, Recombinant, Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: (A) PCR genotyping. The 187-bp and 245-bp products were obtained from wild-type and Ubxd8 flox/flox mice, respectively. (B) Western blot showing liver-specific depletion of UBXD8 in the UBXD8-LKO mouse. Tissue lysates were prepared from control ( Cre -/-, Ubxd8 flox/flox) and UBXD8-LKO ( Cre +/-, Ubxd8 flox/flox) mice, and an equal amount (30 μg) of protein was loaded for each lane in SDS-PAGE. (C) Western blot showing depletion of UBXD8 in hepatocytes isolated from the UBXD8-LKO mouse.

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Western Blot, Control, SDS Page, Isolation

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: Control and UBXD8-LKO mice (30 wk old) were fed a normal diet for 26 wk. 15 females (A) and 15 males (B) were examined for each group. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles; and outliers are represented by dots. The notches are defined as ± 1.58*IQR/sqrt(n) and represent the 95% confidence interval for each median. Indices marked with an asterisk (*) differed significantly between the two groups (P < 0.05; non-paired Student's t test). Numerical data (means ± SEM) and data on additional indices are shown in . BW: body weight; LW: liver weight; Glu: glucose; FFA: free fatty acid; T-Cho: total cholesterol.

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: Body weight, liver weight, and blood chemistry results of mice fed a normal diet.

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: Control and UBXD8-LKO mice (30 wk old) fed a high-fat diet for 26 wk. 15 females (A) and 15 males (B) were examined for each group. Indices marked with an asterisk (*) differed significantly between the two groups (P < 0.05; non-paired Student's t test). Box plots were prepared in the same manner as . Numerical data (means ± SEM) and data on additional indices are shown in . BW: body weight; LW: liver weight; Glu: glucose; FFA: free fatty acid; T-Cho: total cholesterol.

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: Body weight, liver weight, and blood chemistry results of mice fed a high-fat diet.

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: (A) Histology of liver sections obtained from control and UBXD8-LKO mice (30 wk old) fed a high-fat diet for 26 wk. The perivenular area (zone 3) of both groups showed steatosis of the predominantly microvesicular type, whereas the periportal area (zone 1) showed macrovesicular steatosis only in UBXD8-LKO mice. CV: central vein, PV: portal vein. (B) Analysis of lipids extracted from livers of control and UBXD8-LKO mice fed a high-fat diet for 26 wk. TG, total cholesterol, and free cholesterol did not exhibit significant differences between the two groups (n = 5; means ± SEM).

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: Histological evaluation of liver at 30 wk of age.

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: (A) Lipoprotein profile obtained by gel filtration–HPLC of mouse serum at 30 wk old. A representative result on TG (blue) and total cholesterol (red) is shown for sera of normal and UBXD8-LKO male mice fed a normal (A) or a high-fat diet (B). Sera from female mice gave similar results. Sera for the UBXD8-LKO group fed a high-fat diet were taken from mice showing periportal steatosis. Lower panels show a magnified view of the VLDL portion. FG: free glycerol. (B) VLDL-TG was lower in UBXD8-LKO mice than in normal mice, even when they were fed a normal diet. The difference between the control and UBXD8-LKO mouse became significantly larger when mice were fed a high-fat diet. P values were obtained by non-paired Student's t test (n = 3; means ± SEM). (C) The TG-to-cholesterol ratio in VLDL tended to be lower in the UBXD8-LKO than in the control mouse. P values were obtained by non-paired Student's t test (n = 3; means ± SEM).

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Filtration, Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: TG secretion estimated by the Triton WR-1339 injection method. Control and UBXD8-LKO mice, both male and female, were fed a normal diet until 30–33 wk of age. Serum TG levels were measured before and 1 and 2 h after the Triton WR-1339 injection. P values were obtained by non-paired Student's t test (n = 4; means ± SEM).

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Injection, Control

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: (A) Cultured primary hepatocytes obtained from the UBXD8-LKO mice secreted lower levels of ApoB than cells obtained from the control mice. Hepatocytes obtained from female mice fed a normal diet until 12 wk of age were cultured, and ApoB and albumin secreted into the culture medium were examined by densitometry of Western-blot signals. The albumin signal was used for normalization. The amount of secreted ApoB was lower in hepatocytes obtained from UBXD8-LKO mice than in those obtained from control mice, whether the cells were cultured in the normal medium (DMEM + 10% FBS) (left) or in the medium supplemented with 0.4 mmol/l OA (right). P values were obtained by non-paired Student's t test (n = 3; means ± SEM). (B) HepG2 cells transfected with control or UBXD8 siRNA were pulse-labeled with 35 S-methionine and treated with 0.4 mmol/l oleic acid. ApoB immunoprecipitated from the medium at 1 and 3 h was subjected to SDS-PAGE and quantitated by radiography. ApoB secreted in the medium is shown as the ratio relative to total cellular ApoB immediately after pulse-labeling. (C) Primary hepatocytes obtained from UBXD8-LKO mice showed ApoB-crescent structures. The ApoB-crescent was observed as a crescent-shaped ApoB labeling (red) adjacent to LDs (green). Nuclei were stained with Hoechst dye (blue). The ApoB-crescent was present only in cells derived from UBXD8-LKO mice, and not in those obtained from control mice. Cells were cultured in the presence of 1.2 mmol/l OA.

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Cell Culture, Control, Western Blot, Transfection, Labeling, Immunoprecipitation, SDS Page, Staining, Derivative Assay

Journal: PLoS ONE

Article Title: Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0127114

Figure Lengend Snippet: In normal hepatocytes, lipidated ApoB destined for proteasomal degradation is translocated to the cytoplasmic side and subjected to ubiquitination by a UBXD8-dependent mechanism. In the absence of UBXD8, lipidated ApoB accumulates at the ER–LD interface and disturbs the normal ApoB lipidation process. This probably results in a decrease in VLDL-TG secretion in UBXD8-null hepatocytes.

Article Snippet: Rabbit anti-UBXD8 antibody (GeneTex, Irvine, CA), goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), goat anti–apolipoprotein B antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti–β-actin antibody (Sigma–Aldrich, St. Louis, MO), and secondary antibodies conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) or fluorochromes (Thermo Fisher Scientific; Jackson ImmunoResearch Lab, West Grove, PA) were obtained from the indicated suppliers.

Techniques: Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a Schematic depicting LD biogenesis from the ER membrane. ERTOLD hairpin proteins are considered to integrate first into the cytosolic leaflet of the ER membrane in a monotopic topology, which presumably enables them to partition from the ER bilayer to the LD monolayer membrane during LD biogenesis. NL neutral lipids. b Dual localization of opUBXD8 53-153 mCherry to the ER and LDs. Top: Schematic outline of the opsin (op) and mCherry-tagged opUBXD8 53-153 mCherry construct. HR hydrophobic region. Bottom: Fluorescence micrographs of oleate-treated cells transfected with opUBXD8 53-153 mCherry representative for 3 independent experiments. LipidTox marks LDs. Scale bar: 10 µm. c Isolation of opUBXD8 53-153 mCherry-containing LDs from cells. Left: Schematic outline for isolation of UBXD8-containing LDs. Right: immunoblot of post-nuclear supernatant (PNS), membranes (M), cytosol (C), and LD fractions derived from oleate-treated cells expressing OpUBXD8 53-153 mCherry using anti-calnexin (ER-resident protein), anti-tubulin (cytosolic protein) and anti-mCherry antibodies. Non-transfected cells (NT) serve as specificity control for the antibody. Data are representative for 3 independent experiments. d Integration of opUBXD8 53-153 mCherry into rough microsomes (RMs). Left: Schematic outline of co-translational protein insertion into RMs employing in vitro translation of UBXD8 mRNAs in rabbit reticulocyte lysate (RRL) with subsequent fractionation into soluble and membrane-inserted proteins by centrifugation. Right: Immunoblot of soluble (S) and membrane-inserted (M) fractions derived from in vitro translations reactions using anti-mCherry antibodies (representative for n = 3 independent experiments). mRNA encoding either opUBXD8 53-153 mCherry or UBXD8 53-153 mCherryOP and RMs were added to the reaction as indicated. Arrows indicate glycosylated forms of the respective proteins. e – g opUBXD8 53-153 mCherry single cysteine mutants can be PEGylated in ER bilayer and LD monolayer membranes when the cysteine is solvent-exposed. Top: Principle of solvent-accessibility probing of opUBXD8 53-153 mCherry single cysteine mutants by PEGylation in ER bilayer and LD monolayer membranes, respectively. Only solvent-exposed cysteines are accessible to mPEG forming covalent adducts, while bilayer-embedded cysteines are not reactive with mPEG. Bottom: Proof-of-concept immunoblots probed with anti-mCherry antibodies after PEGylation reaction on RM-inserted and LD-inserted opUBXD8 53-153 mCherry single cysteine mutants as indicated. Non-PEGylated proteins are indicated by (0 PEG) and PEGylated proteins by (1 PEG). TX-100: Triton X-100. Quantifications for multiple replicates of these experiments are shown in Fig. .

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Membrane, Construct, Fluorescence, Transfection, Isolation, Western Blot, Derivative Assay, Expressing, Control, In Vitro, Fractionation, Centrifugation, Solvent

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a Atomistic MD simulations of UBXD8 80-128 in a POPC bilayer membrane with a deeply inserted starting structure. Left: Starting structure Right: average structure after 2 µs. b Atomistic MD simulations of UBXD8 80-128 in a POPC bilayer membrane with a partially inserted starting structure. Left: Starting structure Right: average structure after 2 µs. c Atomistic MD simulations of UBXD8 80-128 in a POPC-triolein/cholesteryl-oleate-POPC trilayer system mimicking the LD monolayer membrane. Left: Starting structure; Middle: side view of average structure after 2 µs; Right: top view of average structure after 2 µs; d Center of mass distances of the Cα atom in P102 of UBXD8 80-128 and the P atom in the phospholipid headgroup (dotted line) during the simulation time of 2 µs, and upon UBXD8 80-128 insertion into POPC bilayers or into trilayer systems as indicated. PI partially inserted, DI deeply inserted, TRIO Triolein, CLOL (cholesteryl-oleate). e Center of mass distances of amino acid Cα atoms in UBXD8 80-128 and the P atom in the phospholipid headgroup (dotted line) in the average structures obtained after 2 µs simulations. UBXD8 80-128 was inserted into POPC bilayers or into trilayer systems as indicated. Max. penetration into bilayer is ~2 nm and into monolayer is ~1 nm. Five independent simulations with the CHARMM36m force field over 2 μs were performed. For ( d ) and ( e ): Lines and shaded areas show mean and ±SEM, respectively ( n = 5 simulations). Source data are provided as a source data file.

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Membrane

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a Positioning of the amino acids L91 and L118 within the atomistic starting structure of UBXD8 80-128 that were mutated to a cysteine pair in opUBXD8 53-153 L91C_L118C_mCherry. Cα atoms are highlighted as purple spheres. b Violin plots of distances between the Cα atoms of amino acids L91 and L118 in MD simulations in different membrane systems as shown in Figs. , respectively. DI deeply inserted, TRIO Triolein, CLOL (cholesteryl-oleate). Vertical bars indicate median and maximum/minimum values of the distributions ( n = 5 simulations). c Schematic representation to illustrate the principle of the combined intramolecular crosslinking - PEGylation assay. For clarity, a double-cysteine-containing peptide is only schematically depicted in a bilayer membrane and does not particularly reflect closed or open conformations of UBXD8 in different types of membranes. d , e Immunoblots using anti-mCherry antibodies showing intramolecular crosslinking/PEGylation experiments of opUBXD8 53-153 L91C_L118C_mCherry in either RMs ( d ) or in isolated LDs from cells ( e ). Non-PEGylated protein species (0 PEG) as well as species with one mPEG (1 PEG) or two mPEG (2PEG) molecules attached, are indicated. High molecular weight adducts derived from inter -molecular crosslinking of opUBXD8 53-153 L91C_L118C_mCherry on LDs are indicated (inter x-link). f Quantification of relative opUBXD8 53-153 L91C_L118C_mCherry intramolecular crosslinking efficiencies in RMs versus LDs. From experiments as shown in ( d , e ), bands corresponding to non-PEGylated/intramolecularly crosslinked OpUBXD8 53-153 L91C_L118CmCherry (0 PEG) were quantified. The relative increase in these bands upon addition of crosslinker (lane 2 versus negative DMSO control in lane 1) was calculated from three independent experiments and the values were normalized to the highest value, which was set to 100%. Scatter plots show the mean values with SEM from three independent experiments as well as the individual values for each replicate. Source data are provided as a source data file.

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Membrane, Western Blot, Isolation, High Molecular Weight, Derivative Assay, Control

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a Average atomistic MD simulation structure of UBXD8 80-128 in a POPC bilayer (as in Fig. ) indicating amino acids that were substituted for single cysteines for cwEPR analyses. b Left: Schematic outline of proteo-aLDs generation from proteo-SUVs and isolation by density gradient centrifugation. Right: photograph showing density gradients after centrifugation with floating aLDs when triolein was present during the reconstitution (+). (−): negative control without triolein. c Immunoblot analysis of proteo-aLDs isolation by density gradient fractionation as indicated in ( b ) using anti-S-tag antibodies. Top: negative control without triolein. Arrowhead indicates MTSL-labeled sUBXD8 71-132 His S127C in the top floating aLD fraction when triolein was present during the reconstitution. Representative for n = 3 independent experiments. d Fluorescence micrograph of the top floating aLDs fraction as shown in ( b ) upon reconstitution of Atto488-labeled sUBXD8 71-132 His T130C (green). LipidTox Red marks the neutral lipid core (red). Scale bar: 10 µm. Representative for n = 3 independent experiments. e First derivative absorption cwEPR spectra of MTSL spin-labeled sUBXD8 71-132 -His single cysteine mutants in POPC/DOPS SUVs (left) and aLDs (right). Spectra were normalized by the height of the central EPR line. Asterisks mark spectra with additional shoulders in the low-field region indicating immobile (i) and mobile (m) motional components. f Schematic illustration of how the spin-label positioning in a membrane protein affects the line shape of cwEPR spectra and the membrane depth parameter (Φ). Solvent-exposed: orange; membrane-associated: red; membrane-embedded: blue. In the bilayer midplane, the O 2 concentration is the highest (green), while NiEDDA is gradually excluded from the membrane (purple). g Exemplary EPR power saturation plots of MTSL spin-labeled sUBXD8 71-132 His Y81C and L118C single cysteine mutants reconstituted into SUVs. The peak-to-peak amplitude of the central EPR line was plotted against the square root of the applied microwave power. Power saturation curves were measured under three conditions: nitrogen gas as control (red circles), molecular O 2 (black squares), and NiEDDA (blue diamonds). P 1/2 values were obtained after curve fitting and are indicated with SEM. h Membrane depth parameter (Φ) analysis of MTSL spin-labeled sUBXD8 71-132 His single cysteine mutants in SUVs using cwEPR power saturation analyses ( n = 2 independent experiments). Positive Φ: membrane-embedding; negative Φ: solvent exposure. i Membrane depth parameter (Φ) analysis of MTSL spin-labeled sUBXD8 71-132 His Y96C and I113C reconstituted into either SUVs or aLDs using cwEPR power saturation analyses ( n = 2 independent experiments). Φ close to 0: proximity to solvent-membrane interface. Results for SUVs are duplicates from ( h ) for direct comparison with aLDs. Source data are provided as a source data file.

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Isolation, Gradient Centrifugation, Centrifugation, Negative Control, Western Blot, Fractionation, Labeling, Fluorescence, Membrane, Solvent, Concentration Assay, Control, Comparison

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a MARTINI-based coarse-grained simulation of a bilayer-embedded LD lens consisting of triolein/cholesteryl oleate. Upper panel: UBXD8 80-128 was integrated into the bilayer in its shallow conformation as assessed by atomistic simulations. After 5 μs, the peptides have partitioned to the LD monolayer surface where they accumulate. Lower panel: When inserted into the bilayer in the deep-V state as suggested by atomistic simulations, UBXD8 80-128 accumulates at the bilayer-LD rim but does not transition to the LD surface within 5 µs. b , c All-atom pulling simulations reveal high free-energy costs involved in pulling the shallow-open state of UBXD8 80-128 downwards ( b ) or the deep-V state upwards ( c ) within a planar POPC bilayer membrane. d All-atom simulation system of a minimal LD-bilayer system used for pulling the deep-V-inserted UBXD8 80-128 along the LD rim towards the open-shallow conformation. e Graphs comparing free-energy profiles during MD pulling experiments of UBXD8 80-128 in planar bilayers versus at the LD rim as derived from ( b – d ). Shaded areas show SEM derived by bootstrapping from the set of 33 umbrella histograms. f Revised model for the intramembrane positioning of UBXD8 in ER bilayer versus LD monolayer membranes indicating that structural rearrangements are required for enabling the partitioning. Positive charges are indicated by “+”. Source data are provided as a source data file.

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Membrane, Derivative Assay