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Binder design The β-cat_SnP_7 peptide 10 , β-cat_SnP_8 peptide 10 , P60D2A peptide 27 , and YFP nanobodies 13 were described in previous works and obtained from respective manuscript metadata. Binding peptides designed in this study were either generated by the previously-described SaLT&PepPr algorithm 10 ( https://huggingface.co/ubiquitx/saltnpeppr ) via input of an interacting partner sequence, by the de novo PepPrCLIP algorithm 12 ( https://huggingface.
co/ubiquitx/pepprclip ) via input of the target protein sequence, or by the target sequence-conditioned PepMLM algorithm 11 ( https://huggingface.co/ChatterjeeLab/PepMLM-650M ) 11 . All binder sequences can be found in Supplementary Table 2 .
Plasmid generation All duAb plasmids were generated from the standard pcDNA3 vector, harboring a cytomegalovirus (CMV) promoter. An Esp3I restriction site was introduced immediately upstream of the OTUB1 catalytic domain and flexible GSGSG linker via the KLD Enzyme Mix (NEB, Cat # M0554S) following PCR amplification with mutagenic primers (Azenta). For duAb assembly, oligos for candidate peptides were annealed and ligated via T4 DNA Ligase (NEB, Cat # M0202S) into the Esp3I-digested duAb backbone.
Assembled constructs were transformed into 50 μL NEB Turbo Competent Escherichia coli cells (NEB, Cat # C2984H), and plated onto LB agar supplemented with the appropriate antibiotic for subsequent sequence verification of colonies and plasmid purification (Azenta). Cell culture The HEK293T and HeLa cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco Cat # 11995073) supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin (Gibco, Cat # 15140122), and 10% fetal bovine serum (FBS, Gibco, Cat # A5670402). The hepatocellular carcinoma cell line, HepG2, was maintained in Eagle’s Minimum Essential Medium (EMEM, Sigma Aldrich Cat # M2279-500ML) supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS).
The alveolar rhabdomyosarcoma cell line, RH4, was maintained in RPMI 1640 supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS). The RH4 cell line was a generous gift from Dr. Corinne Linardic.
For duAb screening in reporter cell lines, pcDNA-duAb (500 ng) plasmids were transfected into cells as triplicates (2 × 10 5 /well in a 24-well plate) with Lipofectamine 2000 (Invitrogen, Cat # 11668027) in Opti-MEM (Gibco, Cat # 31985062). After 2 days post transfection, 4 μM PR-619 (DUB inhibitor, MedChemExpress, Cat # HY-13814) was added to applicable cells (with equivalent volume of media added to non-treated cells), and subsequently cells were harvested within 24 hours post-treatment and analyzed on a Attune NxT Flow Cytometer (ThermoFisher) for GFP fluorescence (488-nm laser excitation, 530/30 filter for detection) and mCherry fluorescence (561-nm laser excitation, 620/15 filter for detection). 10,000 events were gated for data analysis based on default FSC/SSC parameters for the analyzed cells.
Cells expressing eGFP and mCherry were gated, and these were normalized to a sample transfected with a non-targeting duAb using the FlowJo software ( https://flowjo.com/ ). Representative flow cytometry gating strategies are indicated in Supplementary Fig.
1 . For endogenous target screening in cell lines, pcDNA-duAb (800 ng) plasmids were transfected into cells as duplicates (3 × 10 5 /well in a 12-well plate) with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco). Cells were harvested after 72 h for subsequent cell fractionation and immunoblotting.
Lentiviral production For target-reporter packaging, HEK293T cells were seeded in a 6-well plate and transfected at ~50% confluency. For each well, 0.5 μg pMD2.
G (Addgene #12259), 1.5 μg psPAX2 (Addgene #12260) and 0.5 μg of the target-mCherry reporter transfer vector were transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.
The medium was exchanged 8 hours post transfection, and the viral supernatant was harvested at 48 and 72 hours post transfection. The viral supernatant was concentrated to 100x in 1× DPBS using Lenti-X Concentrator (Clontech, Cat # 631232) according to the manufacturer’s instructions, and stored at −80 °C for further use. Target-mCherry reporter monoclonal cell line generation For target-reporter cell line generation, 1 × 10 5 HEK293T cells were mixed with 20 μL of the concentrated virus in a 6-well plate.
Media was changed 24 h post transduction. Antibiotic selection was started 36 h post transduction by adding 2 μg/mL puromycin (Sigma, Cat # P8833). Cells were harvested for sorting at 5 days post antibiotic selection, and a single cell of mCherry positive was plated in a 96-well plate.
Genomic PCR was performed after cell growth to validate the genotype of the monoclonal cell line. Proteomics HEK293T cells were maintained in DMEM supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% FBS. Target-sfGFP (1 μg) + pcDNA3-duAb (1 μg) plasmids were transfected into cells as triplicates (5 × 10 5 /well in a 6-well plate) with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco).
After 72 h post transfection, cells were harvested and washed four times with 500 μL 1× cold PBS. The cell pellets were resuspended in 300 μL Pierce RIPA buffer (ThermoFisher, Cat # 89900) and incubated on ice for 30 min. The homogenates were treated with 20% (w/v) SDS in triethylammonium bicarbonate buffer, pH 8.
5 (Sigma Aldrich, Cat # T7408), followed by probe sonication and heating at 80 °C for 5 min. The supernatants were collected after centrifugation and the concentrations were determined using detergent-compatible Bradford assay. From each sample, 20 μg was reduced and alkylated, and digested with trypsin using an S-trap micro device.
Peptide eluents were lyophilized, and after reconstitution, equal volumes of each sample were mixed to make an SPQC pool. Approximately 1 μg of each sample, and three replicates of the SPQC pool were analyzed by 1D-LCMS/MS. Samples were analyzed using a M-Class UPLC system (Waters) coupled to an Exploris 480 high resolution accurate tandem mass spectrometer (ThermoFisher) via a Nanospray Flex Ion source and processed using Spectronaut 16.
The p values were calculated by performing a Student’s t -test on log 2 fc values. The log 2 fc values were calculated by the difference of average abundances of the proteins in the presence and absence of targeting duAbs. Functional assays For the TOP-GFP assay, 2 × 10 5 HEK293T cells/well were seeded on a 24-well plate 20–24 h prior to transfection.
On the day of transfection, each well received the following plasmids: TOP-GFP plasmid (Addgene #35489) and a duAb plasmid. A total of 500 ng of plasmid DNA in a ratio of TOP-GFP:duAb plasmids = 1:1 was mixed with Lipofectamine 2000 reagent (Invitrogen) in serum-free Opti-MEM medium (Gibco) and added dropwise to each well after incubation at room temperature for 20 min. After 72 h of incubation, cells were harvested and analyzed similarly as mentioned for duAb screening.
Viable, single cells were gated, and normalized EGFP cell fluorescence was calculated as compared to a sample transfected with a non-targeting duAb, using the FlowJo software ( https://flowjo.com/ ). Preparation of lipid nanoparticles encapsulating p53-stabilizing duAbs mRNA with ARCA cap and poly(A) tail additions for p53_pMLM_4-OTUB1 was synthesized via in vitro transcription using the HiScribe T7 ARCA mRNA Kit (NEB, Cat # E2060S).
The mRNA was then concentrated and cleaned of impurities using the RNEasy MinElute Cleanup Kit (Qiagen, Cat # 74204). CleanCap® FLuc mRNA (NEB, Cat # L7602-100) was used as a control. Lipid nanoparticles (LNPs) were prepared by diluting DLIN-MC3-DMA (MedKoo Biosciences, Cat # 555308), DSPC (Avanti Polar Lipids, Cat # 850365P-500mg), Cholesterol (Sigma Aldrich, Cat # C3045), and DMG-PEG2000 (NOF American Corporation, Cat # GM-020) in ethanol using standard molar ratios 50:10:38.
5:1.5, respectively. The prepared mRNA for p53_pMLM_4-OTUB1 and luciferase were diluted in 10 mM citrate buffer (ThermoFisher, Cat # J61249.
AP) in a 2:1 volume ratio with the lipid mixture, and mRNA was loaded in a 1:20 mass ratio with the lipid, respectively, in a NanoAssemblrTM SparkTM nanoparticle formulation system (Cytiva, Cat # NIS0001). After LNP production, they were transfected into HeLa cells 48 h post-seeding and were extracted 72 h post transfection for immunoblot analysis. Cell fractionation and immunoblotting On the day of harvest, cells were detached by addition of 0.
05% trypsin-EDTA and cell pellets were washed twice with ice-cold 1× PBS. Cells were then lysed and subcellular fractions were isolated from lysates using a 1:100 dilution of protease inhibitor cocktail (Millipore Sigma, Cat # P8340) in Pierce RIPA buffer (ThermoFisher, Cat # 89900). Specifically, the protease inhibitor cocktail-RIPA buffer solution was added to the cell pellet, the mixture was placed at 4 °C for 30 min followed by centrifugation at 15,000 rpm for 10 min at 4 °C.
The supernatant was collected immediately to pre-chilled PCR tubes and quantified using the Pierce BCA Protein Assay Kit (ThermoFisher, Cat # 23227). Twenty micrograms lysed protein was mixed with 4× BoltTM LDS Sample Buffer (ThermoFisher, Cat # NP0007) with 5% β-mercaptoethanol (Millipore Sigma, Cat # M3148) in a 3:1 ratio and subsequently incubated at 95 °C for 10 min prior to immunoblotting, which was performed according to standard protocols. Briefly, samples were loaded at equal volumes into BoltTM Bis-Tris Plus Mini Protein Gels (ThermoFisher, Cat # NW04125BOX) and separated by electrophoresis.
iBlotTM 2 Transfer Stacks (Invitrogen) were used for membrane blot transfer, and following a 1 h room-temperature incubation in 5% milk-TBST, proteins were probed with rabbit anti-WEE1 antibody (Abcam, Cat # ab137377; diluted 1:1000), mouse anti-p53 antibody (Santa Cruz Biotechnology, Cat # sc-126; diluted 1:1000), rabbit anti-FOXO1 antibody (Cell Signaling Technology, Cat # 2880S; diluted 1:1000), rabbit anti-Cl-PARP-1 (Cell Signaling Technology, Cat # 5625 T, diluted 1:750), rabbit anti-Vinculin (Invitrogen, Cat # 700062; diluted 1:2000), or mouse anti-GAPDH (Santa Cruz Biotechnology, Cat # sc-47724; diluted 1:10,000) for overnight incubation at 4 °C. The blots were washed three times with 1X TBST for 5 min each and then probed with a secondary antibody, donkey anti-rabbit IgG (H + L), horseradish peroxidase (HRP) (Abcam, Cat # ab7083, diluted 1:5000) or goat anti-mouse IgG (H + L) Poly-HRP (ThermoFisher, Cat # 32230, diluted 1:5000) for 1–2 h at room temperature. Following three washes with 1× TBST for 5 min each, blots were detected by chemiluminescence using a BioRad ChemiDocTM Touch Imaging System (Biorad).
Densitometry analysis of protein bands in immunoblots was performed using ImageJ software as described here: https://imagej.nih.gov/ij/docs/examples/dot-blot/ .
Briefly, bands in each lane were grouped as a row or a horizontal “lane” and quantified using FIJI’s gel analysis function. Intensity data for the duAb bands was first normalized to band intensity of either GAPDH or Vinculin in each lane then to the average band intensity for empty duAb vector control cases across replicates. Structure prediction All structures were predicted via the AlphaFold3 server ( https://alphafoldserver.
com/ ), and the shading was done according to AlphaFold’s confidence metric, plDDT, as follows: Very low (plDDT plDDT > 50) = Yellow, Confident (70 > plDDT > 90) = Light Blue, Very high (plDDT > 90) = Light Blue. Statistical analysis and reproducibility Sample sizes were not predetermined based on statistical methods but were chosen according to the standards of the field (three independent biological replicates for each condition), which gave sufficient statistics for the effect sizes of interest. All data were reported as average values with error bars representing standard deviation (SD).
Statistical analysis was performed using the two-tailed Student’s t -test using GraphPad Prism 10 software, with calculated p values are represented as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.
001, **** p ≤ 0.0001. The p value representations above each bar in the fold stabilization and densitometry analyses are indicative of comparisons between the control and the respective sample; all other p value notations are between the specified samples.
No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.
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