Multiplexed Delivery of Synthetic (Un)Conjugatable Ubiquitin and SUMO2 Enables Simultaneous Monitoring of Their Localization and Function in Live Cells

Abstract Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post‐translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.


Fmoc-SPPS general procedure for 2-CTC resin:
2-Chlorotritylchloride (CTC) resin (0.7 mmol/g) was loaded with 0.6 eq. of Fmoc-Gly-OH and 2 eq. of DIEA in dry DCM for 1h. Following resin esterification, the Fmoc-Gly-resin was washed with DCM (X5), methanol (X5), and DMF (X5). The final loading was verified by Fmoc group absorbance after the DBU treatment to be 0.42 mmol/g. After initial resin loading, Fmoc-SPPS 6 was carried on automated peptide synthesizer (CS336X, CSBIO) in presence of 4 eq. of amino acid, 4 eq. of HCTU, and 8 eq. of DIEA to the initial loading of the resin for 50 min. Dipeptides were coupled manually using 2.5 eq. of amino acid, 2.5 eq. of HATU, and 5 eq. of DIEA to the initial loading of the resin for 1 h 30 min. To cleave the peptides from the solid support, the resin was washed with DMF (X5), MeOH (X5), DCM (X5), and dried under high vacuum. A cocktail of TFA:triisopropylsilane (TIS):H2O (95:2.5:2.5) was added to resin and the reaction mixture was shaken for 2h at RT. The resin was filtered, and the combined filtrate was added dropwise to a 10-fold volume of cold ether and centrifuged. The precipitate was purged with nitrogen, dissolved in acetonitrile-water and freeze-drying in the lyophilizer to give the crude peptide.

Fmoc-SPPS general procedure on a Rink amide resin:
Fmoc-SPPS was carried on an automated peptide synthesizer (CS336X, CSBIO) in presence of 4 eq. of amino acid, 4 eq. of HCTU, and 8 eq. of DIEA to the initial loading of the resin for 50 min. The dipeptides were coupled manually using 2.5 eq. of amino acid, 2.5 eq. of HATU, and 5 eq. of DIEA to the initial loading of the resin for 1 h 30 min. To cleave the peptides from the solid support the resin was washed with DMF (X5), MeOH (X5), DCM (X5) and dried under high vacuum. A cocktail of TFA: triisopropyl silane (TIS): H2O (95:2.5:2.5) was added to the resin and the reaction mixture was shaken for 2h at RT. The resin was filtered and the combined filtrate was added dropwise to a 10-fold volume of cold ether and centrifuged. The precipitate was purged with nitrogen, dissolved in acetonitrile-water for freeze-drying in the lyophilizer to give the crude peptide.

HPLC for peptide analysis and purification:
Analytical HPLC was performed on a thermo instrument (dionex ultimate 3000) using analytical column XSelect (thermo scientific hypersil gold, C18, 3 μm, 4.6 × 150 mm) at a flow rate of 1.2 mL/min and preparative HPLC was performed on a thermo instrument (dionex ultimate 3000) using preparative column XSelect (phenomenex jupiter, C18, 5 μm, 250 × 21.2 mm) at a flow rate of 15 mL/min. Semi-preparative HPLC was performed on a thermo instrument (dionex ultimate 3000) using chromatographie multohigh bio C18 10 μm, 250 × 10 mm column, at a flow rate of 4 mL/min. All synthetic products were purified by HPLC and characterized by mass spectrometry using the LCQ Fleet Ion Trap (thermo Scientific). All calculated masses have been reported as an average isotope composition. All HPLC steps were performed using a gradient To detach cells from culture flasks, the media was aspirated and the flask was washed with sterile calcium and magnesium free PBS before cells were treated with 0.25% Trypsin + 0.02% EDTA solution and returned to incubation chamber for 5 min. Trypsin was quenched by adding the FBS supplemented media. The cell suspension was collected and the cells were pelleted (2 min at 1,000xg). Media was then aspirated and the cell pellet was re-suspended in fresh media.
The cell density was determined using automated cells counter (Countess II, Invitrogen). For confocal microscopy, cells were seeded on poly-L-lysine (PLL) treated 8 well chamber slides (Ibidi) with removable silicone chamber in 3.4 × 10 4 per and were allowed to reach ~90% confluence (24 h). 8 9. General procedure for establishing stable parkin over expression: The retroviral plasmid pBMN-Parkin for overexpression of untagged human parkin was a gift from Dr. michael lazarou (Addgene plasmid repository #89299). U2OS cells stably expressing parkin were produced as previously described. [1] 10. General procedure for protein loading via multiplexed bead loading (MBL): Cells were cultured in 8 well ibdi chamber slides to 90% confluence. The protein constructs were diluted from concentrated 1000X DMSO stocks into sterile PBS with 0.1% pluronic® F-68. The and incubated for 30-60 min at 37 °C under 5% CO2 with opti-DMEM to recover from the loading process for a duration of 30 min. [2] 11. General procedure for CCCP and OA/OG mitophagy induction: The peptide delivery was performed according to general procedure and after the medium was Single representative images were analyzed using ZEN 3.0 (ZEN-lite) software. All channels were normalized to uniform intensity by using the range indicator tool (using a representative image) and all adjustments were identically applied to all the images of the different treatments. SRM images were processed in ZEN black 3.0 by SIM 2 algorithm using default parameters (Leap, Low SNR Input, 16 iterations). All channels were normalized to uniform intensity by using the range indicator tool, using a representative image, and all adjustments were identically applied to images of different conditions to avoid data manipulation. Non-linear signal enhancement was applied to SIM 2 images to compensate for low intensity; any modification was identically applied to all images from the different treatments.

General procedure for cell fixation and immunostaining
After the indicated treatments, the cells in ibdi 8 well chamber slides were gently washed twice The slides were stored at -20 °C until imaging.

Dye labeling with maleimide fluorophores
The sulfhydryl-containing protein was dissolved in 6M guanidine hydrochloride (Gn.HCl)/200 mM phosphate buffer pH = 7.3 (2 mM) and cooled to 0 °C. A solution of maleimide conjugated fluorophore (1.2 equiv, dissolved to 10 mM in DMF) was added to the sulfhydryl-containing protein solution and allow the reaction to proceed for 2-4 hours at 0 °C. [3] The reaction was monitored by analytical HPLC (C18 column), using a gradient of 0-60% B over 30 min. Finally, the crude peptide was purified using a C18 semi-preparative column with the gradient flow of 0-12 60% B over 30 min and purified labeled protein was isolated and lyophilized. Dry purified proteins were dissolved in DMSO to X1000 stock and kept at -20 °C. FITC-PEG2-TAB2-ZnF4 (12), was prepared on pre-swollen rink amide resin (0.1 mmol) as described in the general procedure to give FITC-PEG2-TAB2-ZnF4 (12). The crude peptide 12 was purified by preparative HPLC using C18 column with a gradient of 0-60% buffer B over 30 min to afford the corresponding peptide FITC-PEG2-TAB2-ZnF4 (12) in ~26 % isolated yield.

MBL:
Ub analogues labelled with Cy5 (5), TAMRA (10) and Fluorescein (11) were delivered along with mix (5 + 10 + 11) into the U2OS cells and treated with Hoechst before imaging as described in the general procedure (Figure 6 a). We compared BL and MBL using EGFP, 5 and 7 in same cells with Hoechst staining (Figure 6b).

Live cell MBL of three different proteins:
Three different proteins labelled with fluorophores of different emission wavelength such as Ub labelled with Cy5, SUMO-2 labelled with TAMRA and TAB2-ZnF4 labelled with FITC were 21 delivered separately as well as mix (5 + 7 + 12) into the U2OS wild type cells and treated with Hoechst before imaging as described in the general procedure.

Fluorescent gel analysis:
For SDS-PAGE analysis of three-protein delivery, the proteins 5, 7 and 10 were mixed and loaded into U2OS cell cultured on an 8-well plate (according to the general procedure).
Following the loading procedure, cells were incubated for 60 min at 37 °C under 5% CO2 and then treated with and without 10 µm of MG-132 in full-DMEM for overnight. Then the medium was aspirated and washed with (PBS X 2) and lysed with Fluorescent reducing buffer and heated for 5 min at 95 °C and ran the gel using 4-12% MOPS gel. The gel was imaged using Fusion FX6 ECL and fluorescent detection system.
22 Figure S8. Fluorescent gel analysis using 4-12% MOPS gel with lysates from U2OS cells 5, 7, 12 proteins treated with and without MG-132, In each fluorophore line 1 represents the mixed lysate and line 2 represents the mixed lysate treated with 10 µm of MG-132 for overnight.
Cells were washed with PBS X 2 and proceeded for fixation procedure (as described in the general procedure). Fixed cells were stained with Anti-NBR1 (1:150) followed by labelling with secondary antibody, AF488 conjugate (1:1000). Cells were imaged in both laser scanning confocal microscopy (LSCM) and super resolution microscopy via structure illuminated matrix (SIM 2 ).

Live cell imaging of Cy5-Ub (5) and TAMRA-SUMO-2 (7) in U2OS ±parkin cells
To measure the involvement of Ub (5) and sumo-2 (7) analogues in mitophagy, we loaded 5 + 7 into live cells, with and without parkin expression, via MBL and induced mitophagy using 20 µM CCCP for 4 h. Following CCCP treatment, the cells were treated with lysotracker-blue and mitotracker green as described in the general procedure.
We loaded them into the U2OS + parkin cells and treated with and without CCCP for 4 h and stained with mitotracker green and imaged at various time points.
27 Figure S12. Representative zoomed SIM 2 images of U2OS +parkin cells loaded with 5 and 7 without CCCP treatment at different time points. Scale bars are 10µm.