Journal: The Journal of Biological Chemistry

Article Title: Engineering an orthogonal ubiquitin transfer cascade with RING E3 RNF38 by phage display to reveal its regulation of nuclear transport

doi: 10.1016/j.jbc.2026.111199

Figure Lengend Snippet: Identifying the substrates of RNF38 in HEK293 cells by the OUT cascade. A , expression of the OUT-cascade components Flag-xUba1, V5-xUbcH5b, GFP-xRNF38 for the transfer of HBT-xUB in HEK293 cells. The expression of the cascade enzymes in OUT cells with the full xUba1-xUbcH5b-xRNF38 cascade and control cells excluding xRNF38 was verified by western blots of the cell lysates probed with antibodies specific for the tags on each component ( left panels ). The formation of HBT-xUB conjugates with xUba1, xUbcH7, and xRNF38 was confirmed by purifying the xUB-conjugates in the cell lysate by Ni-NTA column and detecting each component with specific tags ( right panels ). B , tandem purification of xUB-conjugated proteins from OUT and the control cells. Lane assignments: 1. cell lysates before Ni-NTA binding; 2. flow-through of the cell lysates after Ni-NTA binding; 3. wash of the Ni-NTA beads; 4. elution from the Ni-NTA beads; 5. flow-through from the streptavidin beads; 6. wash of the streptavidin beads; 7. proteins bound to the streptavidin beads. The western blot was probed with an anti-UB antibody to reveal ubiquitinated species enriched by tandem purification. C , Volcano plot of RNF38 substrates identified by the OUT screen. N = 3 independent biological replicates. Red dots designate proteins with Log 2 [PSM ratio OUT/control] >1 and -Log 10 p > 1. D , heatmap with normalized PSM values for a short list of potential RNF38 substrates from the OUT screen, including RanGAP1, ELAVL1/HuR, Ran, Rack1, VPS35, and KPNA2, that were further characterized in this study (names in red ). P1-P3: 3 replicates from the OUT cells expressing the OUT cascade of RNF38; C1-C3, 3 replicates from the control cells expressing xUba1-xUbcH5b without xRNF38. E , protein–protein interaction network generated by STRING for RNF38 substrates associated with the intracellular and nuclear transport pathways.

Article Snippet: The following plasmids were acquired from Addgene for the expression of RNF38 substrates: pEGFP-C2 RanGAP1 (Plasmid #13378), pET11d-RAN (Plasmid #108919), pCMVTNT-T7-KPNA2 (Plasmid #26678), pFRT_TO_eGFP_ELAVL1 (Plasmid #106105), pEGFP-N1-Rack1 (item #41088), and pLenti6/V5/DEST-VPS35 (item #21691 ).

Techniques: Expressing, Control, Western Blot, Purification, Binding Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: Engineering an orthogonal ubiquitin transfer cascade with RING E3 RNF38 by phage display to reveal its regulation of nuclear transport

doi: 10.1016/j.jbc.2026.111199

Figure Lengend Snippet: In vitro ubiquitination of RNF38 substrates in reconstituted reactions. A–F , reactions were set up with ATP and wt Uba1, UbcH5b, and RNF38 for the transfer of wt UB to RNF38 substrates with various tags for detection - Flag-Ran, Flag-RanGAP1, KPNA2, GFP-HuR, Rack1, and VPS35. In control reactions, each component of the UB transfer cascade of RNF38 was excluded. Lane assignments: 1, reaction of the substrate protein and the Uba1-UbcH5b-RNF38 cascade for the transfer of wt UB to the substrates; 2, reaction missing Uba1 as the E1; 3, reaction missing UbcH5b as the E2; 4, reaction missing RNF38 as the E3; 5, reaction missing wt UB.

Article Snippet: The following plasmids were acquired from Addgene for the expression of RNF38 substrates: pEGFP-C2 RanGAP1 (Plasmid #13378), pET11d-RAN (Plasmid #108919), pCMVTNT-T7-KPNA2 (Plasmid #26678), pFRT_TO_eGFP_ELAVL1 (Plasmid #106105), pEGFP-N1-Rack1 (item #41088), and pLenti6/V5/DEST-VPS35 (item #21691 ).

Techniques: In Vitro, Ubiquitin Proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: Engineering an orthogonal ubiquitin transfer cascade with RING E3 RNF38 by phage display to reveal its regulation of nuclear transport

doi: 10.1016/j.jbc.2026.111199

Figure Lengend Snippet: V alidation of RNF38-catalyzed ubiquitination of substrate proteins identified by the OUT cascade in cells. RNF38 was overexpressed in HEK293 cells with an increasing transfection of the RNF38 expression plasmid. The cells were treated with proteasome inhibitor MG132 to accumulate ubiquitinated proteins, and the designated substrates were immunoprecipitated from the cell with specific antibodies for probing their ubiquitination levels on the western blots with an anti-UB antibody. Bands designated with a star on the western blots correspond to the size of the IgG heavy chain. A , increasing expression of RNF38 in transfected HEK293 cells. B and C , increasing expression of RNF38 led to enhanced ubiquitination of known RNF38 substrates ACTN4 and p53. D–I , verifying the enhanced ubiquitination of OUT-identified RNF38 substrates, including Ran, RanGAP1, KPNA2, HuR, Rack1, and VPS35, with increasing levels of RNF38 expression.

Article Snippet: The following plasmids were acquired from Addgene for the expression of RNF38 substrates: pEGFP-C2 RanGAP1 (Plasmid #13378), pET11d-RAN (Plasmid #108919), pCMVTNT-T7-KPNA2 (Plasmid #26678), pFRT_TO_eGFP_ELAVL1 (Plasmid #106105), pEGFP-N1-Rack1 (item #41088), and pLenti6/V5/DEST-VPS35 (item #21691 ).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Engineering an orthogonal ubiquitin transfer cascade with RING E3 RNF38 by phage display to reveal its regulation of nuclear transport

doi: 10.1016/j.jbc.2026.111199

Figure Lengend Snippet: Regulation of the substrate stability in the cell by RNF38. A , enhanced expression of RNF38 in the cell suppressed the level of the substrate proteins identified by OUT. HEK293 cells were transfected with increasing amounts of RNF38 expression plasmid, and the levels of the substrate proteins in the cell were assayed by substrate-specific antibodies. The correlation of decreased substrate levels with increased RNF38 expression was shown in the companion plot. B , RNF38 expression in HEK293 cells accelerated the degradation of the substrate proteins in the cell. RNF38 expression plasmid was transfected into the HEK293 cells, and cells were treated with cycloheximide (CHX) to inhibit protein expression. The degradation of the RNF38 substrates was followed by substrate-specific antibodies in cells with enhanced expression of RNF38 and compared with background HEK293 cells. The degradation of Ran, KPNA2, HuR, and Ran at different chasing times after the addition of CHX was plotted in the panels on the right. The plots for the degradation of ACTN4, RanGAP1, and VPS35 were shown in .

Article Snippet: The following plasmids were acquired from Addgene for the expression of RNF38 substrates: pEGFP-C2 RanGAP1 (Plasmid #13378), pET11d-RAN (Plasmid #108919), pCMVTNT-T7-KPNA2 (Plasmid #26678), pFRT_TO_eGFP_ELAVL1 (Plasmid #106105), pEGFP-N1-Rack1 (item #41088), and pLenti6/V5/DEST-VPS35 (item #21691 ).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Engineering an orthogonal ubiquitin transfer cascade with RING E3 RNF38 by phage display to reveal its regulation of nuclear transport

doi: 10.1016/j.jbc.2026.111199

Figure Lengend Snippet: A model for the regulation of nuclear transport pathways by RNF38. Cargo proteins, including HuR, E2F1, and p-STAT3, are assembled with the importin complex composed of the KPNA2-KPNB1 pair in the cytoplasm and transported across the nuclear pore complex (NPC) to enter the nucleus. The association of Ran-GTP with the cargo-importin complex results in the disassembly of the complex and the release of cargo proteins into the nucleus. Cargo proteins in the nucleus are also bound to exportin for their transport to the cytoplasm. RanGTP hydrolysis to RanGDP in the cytoplasm is activated by RanGAP1, triggering the disassociation of the cargo proteins from exportin. The OUT screen identified RNF38-catalyzed ubiquitination of nuclear transport components, including Ran, RanGAP1, KPNA2, and KPNB1, as well as the cargo protein HuR. We also verified the role of RNF38 in regulating the import of KPNA2 cargos E2F1 and p-STAT3 into the nucleus.

Article Snippet: The following plasmids were acquired from Addgene for the expression of RNF38 substrates: pEGFP-C2 RanGAP1 (Plasmid #13378), pET11d-RAN (Plasmid #108919), pCMVTNT-T7-KPNA2 (Plasmid #26678), pFRT_TO_eGFP_ELAVL1 (Plasmid #106105), pEGFP-N1-Rack1 (item #41088), and pLenti6/V5/DEST-VPS35 (item #21691 ).

Techniques: Ubiquitin Proteomics

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Mutagenesis, In Vivo, Plasmid Preparation, Software