plasmid pet28 his mube1  (Addgene inc)

Journal: Advanced Science

Article Title: TBK1 Induces the Formation of Optineurin Filaments That Condensate with Polyubiquitin and LC3 for Cargo Sequestration

doi: 10.1002/advs.202509927

Figure Lengend Snippet: The M1‐ubiquitin‐specific deubiquitinase OTULIN prevents and reverses phase separation of phosphorylated Optineurin in vitro and in cells. A) Deubiquitylation activity of recombinant OTULIN. 4xM1‐ub (20 µM) was incubated with and without OTULIN (2 µM, 1 h at 37 °C) and analyzed by immunoblotting using an anti‐ubiquitin antibody (P4D1). B) OTULIN prevents the formation of pOPTN condensates. pOPTN (5 µM) and M1‐ub x chains (40 µM) were incubated with or without OTULIN (5 µM) for 60 min followed by fluorescence microscopy (3D reconstruction). Scale bar: 5 µm. C) OTULIN reverses the formation of pOPTN condensates. pOPTN and M1‐ub x were incubated for 10 or 60 min. Then OTULIN was added followed by fluorescence microscopy (3D reconstruction) after 60 min incubation in the presence of OTULIN. Scale bar: 5 µm. D) Quantification of the number of pOPTN condensates corresponding to the conditions shown in C. Data represent the mean ± SD of four independent experiments. The statistical analysis was performed using a Mann‐Whitney test (*** p ≤ 0.001). E) OTULIN suppresses the formation of Optineurin condensates in SH‐SY5Y cells. OPTN‐KO SH‐SY5Y cells generated by CRISPR/Cas9 were transiently transfected with Optineurin fused with eGFP (OPTN‐eGFP) and HA‐OTULIN or a control plasmid (‐ HA‐OTULIN) and analyzed by immunocytochemistry and fluorescence microscopy 24 h after transfection using an anti‐HA antibody. The lowest panel represents a zoom of the area marked by a square. Scale bar: 10 µm, zoom‐in scale bar: 5 µm. F) Percentage of transfected cells with pOPTN condensates corresponding to the conditions shown in E. The percentage of transfected cells (expressing OPTN‐eGFP and HA) showing at least one assembly with a diameter ≥ 0.5 µm was quantified. Three biological replicates with two fields of view each were analyzed. The bars indicate the mean ± SD and the data points of each replicate are presented as points. The statistical analysis was performed using a Mann‐Whitney test (*** p ≤ 0.001). G) Immunoblot analysis of wildtype (WT) and OPTN‐KO SH‐SY5Y cells, confirming the defective expression of Optineurin in OPTN‐KO SH‐SY5Y cells. An anti‐Optineurin antibody (HPA003279, Merck) was used for detection. H) Optineurin condensates formed in SH‐SY5Y cells co‐localize with M1‐linked ubiquitin chains. OPTN‐KO SH‐SY5Y cells transiently transfected with OPTN‐eGFP were analyzed by immunocytochemistry and fluorescence microscopy 24 h after transfection using the 1E3 anti‐M1‐ubiquitin and LC3 antibody. Scale bar: 10 µm. I) Inhibition of ubiquitylation or HOIP interferes with the formation of Optineurin condensates in SH‐SY5Y cells. OPTN‐KO SH‐SY5Y cells transiently transfected with OPTN‐eGFP were treated with the E1 ubiquitin‐activating enzyme inhibitor TAK243 (1 µM, 24 h) or the HOIP inhibitor HOIPIN‐8 (30 µM, 24 h) 4 h after transfection and then analyzed by fluorescence microscopy. Scale bar: 10 µm. J) Percentage of transfected cells with Optineurin condensates corresponding to the conditions shown in I. Cells expressing OPTN‐eGFP were classified as positive when they showed at least one assembly ≥ 0.5 µm in diameter. Five areas of view each from three biological samples were analyzed. The statistical analysis was performed using a Mann‐Whitney test (** p ≤ 0.0025).

Article Snippet: The following plasmids were obtained from Addgene: pEGFP‐N1‐OPTN‐WT (Addgene plasmid number: 27052), pEGFP‐N1‐OPTN‐Q398X(Addgene plasmid number: 68849), pEGFP‐N1‐OPTN‐E478G (Addgene number: 68848), pET28b His‐mouse UBE1 (Addgene plasmid number: 32534), pET28a‐LIC UBE2V1 (Addgene Plasmid number: 25619), pOPINF‐OTULIN (Addgene plasmid number: 61464).

Techniques: Ubiquitin Proteomics, In Vitro, Activity Assay, Recombinant, Incubation, Western Blot, Fluorescence, Microscopy, MANN-WHITNEY, Generated, CRISPR, Transfection, Control, Plasmid Preparation, Immunocytochemistry, Expressing, Inhibition

Journal: Cell reports

Article Title: The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome.

doi: 10.1016/j.celrep.2025.115736

Figure Lengend Snippet: Figure 2. Disrupting interactions between Rpt5 and Rpn10 stabilizes the s1 state (A and B) Atomic models based on the cryo-EM structures of the engagement-competent, s1-like E A2 state (A, PDB ID: 6MSB) and the processing-competent, non-s1-like E C1 state (B, PDB ID: 6MSG) of the human 26S proteasome, with the ATPase ring shown in blue, Rpn11 in green, Rpn10’s von Willebrand factor type A (VWA) domain in orange, and ubiquitin in pink. Insets depict the details of Rpn11-bound ubiquitin contacting the same region between K51 and H64 (dark blue) of Rpt5’s coiled coil (A) as Rpn10’s VWA domain in the non-s1 state (B), leading to a competition where ubiquitin stabilizes the s1 relative to non-s1 states. (C) Atomic model based on the cryo-EM structure of the S. cerevisiae 26S proteasome in the non-s1 state (s2 state, PDB ID: 4CR3), with Rpn10’s VWA domain contacting Rpt5’s coiled coil in a homologous region (R42-H53 shown in dark blue) as in the human proteasome (B). The critical residues R23, D31, and E68 of Rpn10 are shown in stick representation and colored red. (D) Effect of the R23E, D31K, and E68K triple mutations in Rpn10’s VWA domain (Rpn10 EKK ) on SspB-mediated substrate degradation. Shown are the averages of three technical replicates with error bars indicating the standard errors of the mean. Statistical significance was calculated using an ordinary one-way ANOVA test. **p = 0.0012, ***p = 0.0003. (E) Effect of triple mutations in Rpn10’s VWA domain (Rpn10 EKK ) on the s1-to-non-s1 transition rates as derived from the single-molecule FRET-based conformational dynamics assay. Shown are the transition rates calculated by fitting the s1-state dwell time distribution of >2,900 transition events observed for at least 200 FRET-efficiency traces from two technical replicates of the proteasome conformational dynamics assay, with error bars representing the standard errors of the fit. (F) Effect of triple mutations in Rpn10’s VWA domain (Rpn10 EKK ) on the non-s1-to-s1 transition rates. Shown are the transition rates calculated by fitting the non- s1-state dwell time distribution of >2,900 transition events observed for at least 200 FRET-efficiency traces from two technical replicates of the proteasome conformational dynamics assay, with error bars representing the standard errors of the fit.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains Escherichia coli Rosetta2 (DE3) pLysS Novagen 71403–3 Escherichia coli Bl21-star (DE3) Thermofisher C601003 Chemicals, peptides, and recombinant proteins 5-FAM-HHHHHHLPETGG Genscript N/A LD555-maleimide Lumidyne Technologies N/A Dibenzocyclooctyne (DBCO)–conjugated LD555 Lumidyne Technologies N/A DBCO–conjugated LD655 Lumidyne Technologies N/A 4-azido-L-phenylalanine Acrotein ChemBio Inc. Cat.# A-7137 Experimental models: Organisms/strains Saccharomyces cerevisiae yAM54 with Pre1-3xFLAG Beckwith et al. 23 N/A Saccharomyces cerevisiae yAM80 expressing Pre1-Avi-HRV-3xFLAG Jonsson et al. 36 N/A Recombinant DNA Mouse E1 Jorge Eduardo Azevedo, Carvalho et al. 52 Addgene 32534 Rpn10 Lander et al. 53 N/A Ubiquitin Worden et al. 45 N/A Rsp5 Worden et al. 24 N/A Ubc1 Lander et al. 53 N/A pAM80 pACYCDuet-1_Sem1-Hsp90 Bard et al. 12 N/A pAM81 pETDuet-1_Rpn1_Rpn2_Rpn13 Bard et al. 12 N/A pAM83 pACYCDuet-1_Nas2_Nas6_Hsm3_Rpt14_RILrare-tRNAs Bard et al. 12 N/A pAM85 pETDuet-1_Rpn9_Rpn11_Rpn8_MBP-HRV-Rpn6_Rpn5 Bard et al. 12 N/A pAM86 pCOLADuet-1_His6-HRV-Rpn12_Rpn7_Rpn3 Bard et al. 12 N/A pAM87 pUltra_AzFRS.2.t1_UAG-tRNA Bard et al. 12 N/A pAM88 pCOLADuet-1_FLAG-Rpt1[I191TAG]_ Rpt2_His6-Rpt3_Rpt4_Rpt5_Rpt6 Bard et al. 12 N/A pAM89 pCOLADuet-1_FLAG-Rpt1_Rpt2_ His6-Rpt3_Rpt4_Rpt5[Q49TAG]_Rpt6 Bard et al. 12 N/A pAM210 pCOLADuet-1_FLAG-Rpt1_sspB-Rpt2_ His6-Rpt3_Rpt4_Rpt5_Rpt6 Jonsson et al. 36 N/A pAM239 pACYC-His6-Rpn10 Beckwith et al. 23 N/A pAM242 pET28a-His6-TEV-Sortase Theile et al. 54 N/A pAM314 pETDuet-1_Rpn9[F2TAG]_Rpn11_Rpn8_MBP-HRV-Rpn6_Rpn5 Jonsson et al. 36 N/A pAM315 pACYC-His6-Rpn10[ΔUIM] This paper N/A pAM321 pETDuet-1_Rpn1[D541A, D548R, E552R]_Rpn2_Rpn13 This paper N/A pAM322 pETDuet-1_Rpn1_Rpn2_Rpn13[E41K, E42K, L43A, F45A, S93D] This paper N/A pAM323 pETDuet-1_Rpn1[D541A, D548R, E552R]_Rpn2 _Rpn13[E41K, E42K, L43A, F45A, S93D] This paper N/A pAM326 pACYC-His6-Rpn10[R23E, D31K, E68K] This paper N/A pAM341 pETDuet-1_Rpn9_Rpn11[A89F]_ Rpn8_MBP-HRV-Rpn6_Rpn5 This paper N/A pAM342 pETDuet-1_Rpn9[F2TAG]_Rpn11[A89F]_ Rpn8_MBP-HRV-Rpn6_Rpn5 This paper N/A pAM345 pETDuet-1_Rpn9_Rpn11[A89I]_Rpn8_ MBP-HRV-Rpn6_Rpn5 This paper N/A (Continued on next page) Cell Reports 44, 115736, June 24, 2025 13

Techniques: Cryo-EM Sample Prep, Ubiquitin Proteomics, Derivative Assay

Journal: Cell reports

Article Title: The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome.

doi: 10.1016/j.celrep.2025.115736

Figure Lengend Snippet: Figure 5. Rpn11 functions as the allosteric ubiquitin sensor (A) Rates for the SspB-mediated degradation of the slowly engaging GS substrate in its unmodified or mono-ubiquitinated form by triple-receptor- deficient 26S proteasome. Shown are the aver- ages of three technical replicates with error bars indicating the standard deviation. Statistical sig- nificance was calculated using a standard un- paired t test ***p = 0.0003. (B) Deubiquitinase activity of the isolated Rpn11/ Rpn8 heterodimer containing the Rpn11 A89F or L132R mutation at the ubiquitin-binding interface. Shown are the averages of three technical repli- cates with error bars representing standard devi- ation. Statistical significance was calculated using an ordinary one-way ANOVA test: *p = 0.0151, ***p = 0.001. (C) Rates for the SspB-mediated degradation of the slowly engaging GS substrate in its unmodified or mono-ubiquitinated form by triple-receptor-defi- cient 26S proteasome with wild-type Rpn11, Rpn11 A89F, or Rpn11 L132R. Shown are the av- erages of three technical replicates with error bars indicating the standard deviation. Statistical sig- nificance was calculated using an ordinary one- way ANOVA test: ****p < 0.0001; ns, not significant with p = 0.8005. (D) Model for the allosteric regulation of proteaso- mal substrate engagement through early ubiquitin binding to Rpn11. The substrate-free proteasome spontaneously switches between the engage- ment-competent s1 state (left) and the processing- competent non-s1 states (right). Binding of a ubiquitinated substrate (purple with pink ubiquitin chain) to the Rpn10 receptor (orange) allows ubiq- uitin binding to Rpn11 (green) and a simultaneous contact with Rpt5’s coiled coil (red ellipse), which stabilizes the s1 state by competing with the non- s1-state-specific interaction between Rpn10’s VWA domain and Rpt5’s coiled coil (yellow ellipse). This shifts the conformational equilibrium toward the s1 state with an accessible central channel and thus facilitates the insertion of a substrate’s flexible initiation region into the ATPase motor. Upon suc- cessful substrate engagement, the proteasome switches to non-s1 processing-competent states for processive substrate translocation, unfolding, and co-translocational deubiquitination.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains Escherichia coli Rosetta2 (DE3) pLysS Novagen 71403–3 Escherichia coli Bl21-star (DE3) Thermofisher C601003 Chemicals, peptides, and recombinant proteins 5-FAM-HHHHHHLPETGG Genscript N/A LD555-maleimide Lumidyne Technologies N/A Dibenzocyclooctyne (DBCO)–conjugated LD555 Lumidyne Technologies N/A DBCO–conjugated LD655 Lumidyne Technologies N/A 4-azido-L-phenylalanine Acrotein ChemBio Inc. Cat.# A-7137 Experimental models: Organisms/strains Saccharomyces cerevisiae yAM54 with Pre1-3xFLAG Beckwith et al. 23 N/A Saccharomyces cerevisiae yAM80 expressing Pre1-Avi-HRV-3xFLAG Jonsson et al. 36 N/A Recombinant DNA Mouse E1 Jorge Eduardo Azevedo, Carvalho et al. 52 Addgene 32534 Rpn10 Lander et al. 53 N/A Ubiquitin Worden et al. 45 N/A Rsp5 Worden et al. 24 N/A Ubc1 Lander et al. 53 N/A pAM80 pACYCDuet-1_Sem1-Hsp90 Bard et al. 12 N/A pAM81 pETDuet-1_Rpn1_Rpn2_Rpn13 Bard et al. 12 N/A pAM83 pACYCDuet-1_Nas2_Nas6_Hsm3_Rpt14_RILrare-tRNAs Bard et al. 12 N/A pAM85 pETDuet-1_Rpn9_Rpn11_Rpn8_MBP-HRV-Rpn6_Rpn5 Bard et al. 12 N/A pAM86 pCOLADuet-1_His6-HRV-Rpn12_Rpn7_Rpn3 Bard et al. 12 N/A pAM87 pUltra_AzFRS.2.t1_UAG-tRNA Bard et al. 12 N/A pAM88 pCOLADuet-1_FLAG-Rpt1[I191TAG]_ Rpt2_His6-Rpt3_Rpt4_Rpt5_Rpt6 Bard et al. 12 N/A pAM89 pCOLADuet-1_FLAG-Rpt1_Rpt2_ His6-Rpt3_Rpt4_Rpt5[Q49TAG]_Rpt6 Bard et al. 12 N/A pAM210 pCOLADuet-1_FLAG-Rpt1_sspB-Rpt2_ His6-Rpt3_Rpt4_Rpt5_Rpt6 Jonsson et al. 36 N/A pAM239 pACYC-His6-Rpn10 Beckwith et al. 23 N/A pAM242 pET28a-His6-TEV-Sortase Theile et al. 54 N/A pAM314 pETDuet-1_Rpn9[F2TAG]_Rpn11_Rpn8_MBP-HRV-Rpn6_Rpn5 Jonsson et al. 36 N/A pAM315 pACYC-His6-Rpn10[ΔUIM] This paper N/A pAM321 pETDuet-1_Rpn1[D541A, D548R, E552R]_Rpn2_Rpn13 This paper N/A pAM322 pETDuet-1_Rpn1_Rpn2_Rpn13[E41K, E42K, L43A, F45A, S93D] This paper N/A pAM323 pETDuet-1_Rpn1[D541A, D548R, E552R]_Rpn2 _Rpn13[E41K, E42K, L43A, F45A, S93D] This paper N/A pAM326 pACYC-His6-Rpn10[R23E, D31K, E68K] This paper N/A pAM341 pETDuet-1_Rpn9_Rpn11[A89F]_ Rpn8_MBP-HRV-Rpn6_Rpn5 This paper N/A pAM342 pETDuet-1_Rpn9[F2TAG]_Rpn11[A89F]_ Rpn8_MBP-HRV-Rpn6_Rpn5 This paper N/A pAM345 pETDuet-1_Rpn9_Rpn11[A89I]_Rpn8_ MBP-HRV-Rpn6_Rpn5 This paper N/A (Continued on next page) Cell Reports 44, 115736, June 24, 2025 13

Techniques: Ubiquitin Proteomics, Standard Deviation, Activity Assay, Isolation, Mutagenesis, Binding Assay, Translocation Assay