Journal: bioRxiv

Article Title: Unanchored ubiquitin chains promote the non-canonical inflammasome via UBXN1

doi: 10.1101/2024.10.30.621131

Figure Lengend Snippet: ( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and K63 Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.

Article Snippet: Caspase-4 (Cat# 4450S, 1:1000), Gasdermin D (Cat# 39754S, 1:1000), Cleaved Gasdermin D (Asp275) (Cat# 36425S, 1:500), Cleaved Gasdermin D (Asp276) (Cat# 10137S, 1:1000), NLRP3 (Cat# 15101S, 1:1000), Tubulin (Cat# 2144S, 1:2000), Actin (Cat#4967S, 1:2000), UBXN3B (Cat# 34945S, 1:1000), K63-linkage specific polyubiquitin (Clone D7A11, Cat# 5621S,1:500), K48-linkage specific polyubiquitin (Clone D9D5, Cat# 12805S, 1:1000), Phospho-Jak1 (Tyr1034/1035) (Cat# 74129S, 1:1000), Phospho-Stat1 (Tyr701) (Cat# 9167S, 1:1000), Myc-Tag (Cat# 2276S, 1:2000) and HA-Tag (Clone C29F4, Cat# 3724S, 1:1000) antibodies were from Cell Signaling.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Knock-Out, Mutagenesis

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: 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