Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Dynein-mediated nephrin degradation and recycling in HG treatment podocytes. (A) Tracking of nephrin and the coexpressed GFP-Rab7 or GFP-Rab11. Endocytosed nephrin proteins in HG-treated cells were enriched in Rab7-positive lysosomes with reduced Rab11 coating compared with NG-treated cells. These changes were much less pronounced in HG-treated cells in which dynein activity was inhibited with Ciliobrevin D (versus HG+0.3% DMSO) or siRNA-mediated Dynll1 knockdown (versus cells transfected with control siRNA). (B) Cells were pretreated with cycloheximide (to reduce the background of newly synthesized nephrin) and underwent nephrin cross-linking and internalization. The extent of postendocytic degradation of nephrin in these cells was reflected by the reduction in nephrin protein levels as measured by Western blotting. Nephrin cross-linking in HG-treated cells resulted in reduced nephrin, which was rescued by incubation with Ciliobrevin D (50 µM), Leupeptin (200 µg/ml), or bortezomib (100 nm). 0.3% DMSO was added to control cells without exposure to specific chemical compounds. Compared with cells transfected with control siRNA, Dynll1 knockdown reduced the cross-linking–induced depletion of nephrin protein in HG-grown cells. n=3, *P<0.05 versus HG+DMSO after nephrin cross-linking. ^P<0.05 versus HG+Control siRNA after nephrin cross-linking. (C) Nephrin recycling was examined by using a surface biotinylation-based recycling assay as outlined in the schematics. Cells went through the following steps: (1) biotinylation of all surface nephrin; (2) antibody-mediated internalization of nephrin; (3) first surface stripping with Mesna to remove biotin from uninternalized nephrin, i.e., to leave only the internalized nephrin biotinylated; (4) recycling of the biotinylated nephrin; and (5) second surface stripping with Mesna to remove biotin from the recycled nephrin. Cell lysates collected at steps (4) and (5) were subjected to streptavidin IP to pull-down biotinylated nephrin. The biotinylated nephrin measured in specimen 4 and specimen 5 reflects initially internalized nephrin and residual nonrecycled nephrin, respectively. The percentages of recycled nephrin were determined with the following formula: % recycled=[(initially internalized nephrin)−(nonrecycled nephrin)]/(initially internalized nephrin)]×100%. n=3, *P<0.05 versus HG+DMSO; ^P<0.05 versus HG+control siRNA. IP, immunoprecipitation.

Article Snippet: Cells were then returned to 37°C and underwent internalization, followed by surface stripping by incubating with 100 mM Mesna (Santa Cruz, CAS 19767-45-4) at 4°C to remove biotin from uninternalized nephrin.

Techniques: Activity Assay, Knockdown, Transfection, Control, Synthesized, Western Blot, Incubation, Stripping Membranes, Immunoprecipitation

Journal: Nature

Article Title: Ubiquitin ligation to F-box protein targets by SCF–RBR E3–E3 super-assembly

doi: 10.1038/s41586-021-03197-9

Figure Lengend Snippet: a , Strategy to generate an ABP to visualize TS1 (TS1 ABP). The goal was to generate an ABP with a warhead between the catalytic cysteine of UBE2L3 and the C terminus of ubiquitin that would react with ARIH1 only when assembled with a neddylated CRL. An intein-based semisynthesis route was used to couple Ub(1–75)–MESNa and (E)-3-[2-(bromomethyl)-1,3-dioxolan-2-yl]prop-2-en-1-amine (BmDPA) to yield a cyclic ketal-protected ubiquitin species. Acidic deprotection of the cyclic ketal yields a reactive ubiquitin species , which when conjugated to a single-cysteine-containing version of UBE2L3 produces an ABP with a Michael acceptor between the C terminus of ubiquitin and the active site of UBE2L3. b , Quality controls comparing predicted masses for Ub–MESNa, ABP precursor and TS1 ABP entities with measurements obtained by electrospray ionization–time-of-flight mass spectrometry. c , SDS–PAGE gel confirming TS1 ABP reaction depends on the catalytic cysteine of ARIH1 (C>S refers to serine replacement). Gel image is representative of independent technical replicates ( n = 2). d , SDS–PAGE gel demonstrating TS1 ABP reaction with ARIH1 depends on neddylated CUL1–RBX1 and ARIH1 residues required for ubiquitylating client substrates bound to an F-box protein . Gel image is representative of independent technical replicates ( n = 2). e , Because structural biology is an empirical endeavour, various TS2 ABP approaches were tested to identify a strategy yielding high-quality electron microscopy data visualizing TS2. The concept was to place a warhead between a substrate and the C terminus of ubiquitin, to generate an ABP that would react only with ARIH1 super-assembled with the SCF containing the cognate F-box protein of the substrate. The fully synthetic TS2 ABP alternative (left) displayed reactivity and specificity matching the native reaction when assembled with cyclin E or p27 phosphopeptide substrate mimics, as shown by SDS–PAGE gel (right). f , In parallel, we tested a semisynthetic strategy, which led to high-resolution cryo-EM structures, and thus complexes generated with this strategy are referred to as TS2 throughout the article. Ub–MESNa and a substrate phosphopeptide with an N-terminal cysteine placed to mimic the acceptor site were fused via native chemical ligation and the free cysteine was converted to dehydroalanine. g , SDS–PAGE showing TS2 p27 ABP reaction with ARIH1 requires all elements needed for native TS2, or use of a mutant version of ARIH1 (ARIH1(F430A/E431A/E503A)) bypassing the need for NEDD8 for this reaction. Gel image is representative of independent technical replicates ( n = 2). h , SDS–PAGE testing specificity of TS2 ABPs for cognate F-box proteins. Phosphorylated cyclin E and p27 are substrates of SCF FBXW7 and SCF SKP2 , respectively. All experiments with SCF SKP2 also contained the essential protein partner CKSHS1 unless otherwise indicated. Gel image is representative of independent technical replicates ( n = 2).

Article Snippet: The basis for the formation of the TS1 ABP–UBE2L3~Ub was coupling His–Ub(1–75)–MESNa to (E)-3-[2-(bromomethyl)-1,3-dioxolan-2-yl]prop-2-en-1-amine (BmDPA) (>95% purity, ChiroBlock) to yield a reactive species .

Techniques: Mass Spectrometry, SDS Page, Electron Microscopy, Cryo-EM Sample Prep, Generated, Ligation, Mutagenesis