Reversible Attachment Research Articles

Tissue adaptation to metabolic stress: insights from SUMOylation

Post-translational modification (PTM) plays a crucial role in adaptation of mammals to environmental changes, enabling them to survive in stressful situations. One such PTM is SUMO modification, which is evolutionarily conserved. It involves the covalent and reversible attachment of a small ubiquitin-like modifier (SUMO) to lysine (Lys) residues in the target protein. SUMOylation regulates various functions, including cell proliferation, differentiation, apoptosis, senescence, and maintenance of specific cellular activities. It achieves this by influencing protein-protein interactions, subcellular localization, protein stability, and DNA binding activity. Mounting evidence suggests that SUMOylation is implicated in the pathogenesis of metabolic disorders such as obesity, insulin resistance, and fatty liver. This review aims to provide an overview of the role of SUMOylation in regulating tissue adaptation to metabolic stress. Recent advancements in spectroscopic techniques have shed light on potential targets of SUMOylation and the underlying regulatory mechanisms have been elucidated, laying the theoretical foundation for the development of targeted SUMOylation interventions for metabolic syndrome while minimizing side effects.

Protein SUMOylation and Its Functional Role in Nuclear Receptor Control

Post-translational protein modifications (PTMs) significantly enhance the functional diversity of proteins and are therefore important for the expansion and the dynamics of the cell’s proteome. In addition to structurally simpler PTMs, substrates also undergo modification through the reversible attachment of small proteins. The best understood PTM of this nature to date is the covalent conjugation of ubiquitin and ubiquitin-like proteins (UBLs) to their substrates. The protein family of small ubiquitin-like modifier (SUMO) is one of these UBLs that has received increasing scientific attention. The pathway of SUMOylation is highly conserved in all eukaryotic cells and is crucial for their survival. It plays an essential role in many biological processes, such as the maintenance of genomic integrity, transcriptional regulation, gene expression, and the regulation of intracellular signal transduction, and thereby influences DNA damage repair, immune responses, cell cycle progression, and apoptosis. Several studies have already shown that in this context protein SUMOylation is involved in the control mechanisms of various cellular receptors. This article unites data from different studies focusing on the investigation of the strictly conserved three-step enzyme cascade of protein SUMOylation and the functional analysis of the involved proteins E1, E2, and E3 and SUMOylation target proteins. Furthermore, this review highlights the role of nuclear receptor SUMOylation and its importance for the cellular functionality and disease development arising from defects in correct protein SUMOylation.

Base-Filling in Double-Helical Nucleic Acids.

Base-filling, i. e., post-synthetic furnishing of an oligonucleotide scaffold with base moieties or their analogues, is an interesting alternative to the conventional approach of sequential coupling of building blocks (modified or otherwise). Reversible attachment of the base moieties is particularly attractive as it allows the use of dynamic combinatorial chemistry and usually leads to higher fidelity. This concept article summarizes the various backbones and coupling reactions used for base-filling over the past fifteen years, discusses the impact of base stacking and pairing on efficiency and fidelity and highlights potential and realized applications.

Disulfide Tethering to Map Small Molecule Binding Sites Transcriptome-wide.

We report the development of Tether-seq, a transcriptome-wide screen to probe RNA-small molecule interactions using disulfide tethering. This technique uses s4U metabolic labeling to provide sites for reversible and covalent attachment of small molecule disulfides to the transcriptome. By screening under reducing conditions, we identify interactions that are stabilized by binding over those driven by the reactivity of the RNA sites. When applied to cellular RNA, Tether-seq with a disulfide analogue of risdiplam, an FDA-approved drug that targets RNA to treat spinal muscular atrophy (SMA), revealed a number of potential binding sites, most prominently at a site within the cytochrome C oxidase 1 (COX1) transcript. Structure probing by SHAPE-MaP revealed a structured motif and confirmed binding to the lead molecule. This work demonstrates that these screens have the power to identify binding sites throughout the transcriptome and provide invaluable insight into the thermodynamic properties that define small molecule binding.

In vitro study to analyze reverse torque values of attachment screws with multiple loosening/tightening preload cycles.

To determine the number of torque/reverse torque cycles attachment screws can withstand before fracture, and to record the magnitude of reverse torque values for each of the 10 cycles of screw tightening and loosening. The testing assemblies consisted of 30 temporary cylinder/multiunit abutment/internal connection implants (10 each from Dentsply Sirona Astra, Biohorizons, and Zimmer Biomet Dental). The assemblies were inserted into resin blocks, and temporary cylinders were connected to the multiunit abutments with new attachment screws and torqued to the recommended preload (15 Ncm Dentsply Sirona Astra; 15 Ncm Biohorizons; 10 Ncm Zimmer Biomet Dental) using a digitaltorquegaugeinstrument (Model 3, MARK-10 Corporation, New York, USA) at room temperature without lubrication. After 24 h, the attachment screws were reverse torqued with the same instrument and the values were recorded. The torque/reverse torque cycles were repeated every 24 h for 10 days. Data were analyzed using mixed model analysis. A second test included torquing the used screws (30) until fracture or stripping occurred. The reverse torque values were recorded and compared with new screws that were torqued until fracture or stripping. A sample of used and new attachment screws from each assembly were viewed under both laser microscope (LEXT OLS4000, 3D Measuring Laser Microscope, Olympus) and scanning electron microscope (JEOL-JSM6510, Tokyo, Japan). Data were analyzed using t-test. Following 10 cycles of torquing and reverse torquing, the reverse torque values measured on day 10 were lesser compared with the reverse torque value measured on day 1 for 29/30 attachment screws (96.6%). According to the t-test used for the second test, the p-values among all three groups were ≥0.9 which were not statistically significant. The results of this study indicated that after 10 cycles of torquing and reverse torquing attachment screws, the reverse torque values decreased. There were no statistical differences between the magnitude of torque necessary to fracture new and used attachment screws.

Fabrication and Characterization of Collagen–Magnetic Particle Composite Microbeads for Targeted Cell Adhesion and Proliferation

The integration of the biocompatibility of collagen and the remote-control ability of magnetic elements serves as both a cell scaffold and an actuator. We studied the preparation, characterization, and potential applications of collagen–magnetic particle composite microbeads (CMPMBs). The interplay among collagen concentration, particle size, and surface roughness was found to influence cell adhesion and proliferation. Adsorption and desorption tests showed the reversible attachment of the particles to magnetic sheets, enabling precise spatial control and targeted cell delivery. The particles demonstrated their utility as cell carriers, supporting cell migration and proliferation. These findings showcase the potential of CMPMBs as a promising platform for advanced cell delivery and tissue regeneration applications. The ability to fine-tune particle properties and manipulate them using magnetic fields offers new possibilities for creating complex tissue constructs and controlling cellular behavior, which could contribute to the development of more effective regenerative therapies and tissue engineering approaches.

Nanobubble transport in porous media: Towards agro- and environmental applications

Nanobubbles have been increasingly used in various applications involving porous media, such as groundwater remediation and irrigation. However, the fundamental scientific knowledge regarding the interactions between nanobubbles and the media is still limited. The interactions can be repulsive, attractive, or inert, and can involve reversible or irreversible attachment as well as destructive mechanisms. Specifically, the stability and mobility of nanobubbles in porous media is expected to be dependent on the dynamic conditions and the physicochemical properties of the porous media, solutions, and nanobubbles themselves. In this study, we investigated how changes in solution chemistry (pH, ionic strength, and valence) and media characteristics (size and wettability) affect the size and concentration of nanobubbles under dynamic conditions using column experiments. Quartz crystal microbalance with dissipation monitoring provided a deeper understanding of irreversible and elastic nanobubbles’ interactions with silica-coated surfaces. Our findings suggest that nanobubbles are less mobile in solutions of higher ionic strength and valence, acidic pH and smaller porous media sizes, while the wettability of porous media has a negligible influence on the retention of nanobubbles. Overall, our findings provide insights into the underlying mechanisms of nanobubble interactions and suggest potential strategies to optimize their delivery in various applications.

Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Modular and automated synthesis of oligonucleotide-small molecule conjugates for cathepsin B mediated traceless release of payloads.

The reversible attachment of small molecules to oligonucleotides provides versatile tools for the development of improved oligonucleotide therapeutics. However, cleavable linkers in the oligonucleotide field are scarce, particularly with respect to the requirement for traceless release of the payload in vivo. Herein, we describe a cathepsin B-cleavable dipeptide phosphoramidite, Val-Ala(NB) for the automated synthesis of oligonucleotide-small molecule conjugates. Val-Ala(NB) was protected by a photolabile 2-nitrobenzyl group to improve the stability of the peptide linker during DNA synthesis. Intracellular cathepsin B digests the dipeptide efficiently, releasing the payload-phosphate which is converted to the free payload by endogenous phosphatase enzymes. With the advantages of modular synthesis and stimuli-responsive drug release, we believe Val-Ala(NB) will be a potentially valuable cleavable linker for use in oligonucleotide-drug conjugates.

Nanofiber embedded bioinspired strong wet friction surface.

Robust and reversible wet attachments are important for medical engineering and wearable electronics. Although ultrastrong capillarity from interfacial nano-thick liquid bridges creates tree frog's strong wet friction, its unstable nano-liquid characteristic challenges further wet friction enhancement. Here, unique hierarchical micro-nano fibrous pillars have been discovered on Chinese bush crickets exhibiting a robust wet friction ~3.8 times higher than tree frog's bulk pillar. By introducing a nano-fibrous pillar array covered with thin films (NFPF), the pillar's separation position switches from the rear to front side compared with bulk pillars, indicating the interfacial contact stress shifting from compressing to stretching. This largely decreases the interfacial separation stress to form more stable and larger nano-liquid bridges. The NFPF array with self-splitting of interfacial liquid and contact stress further guards such interfacial stress shifting to ensure a ~1.9 times friction enhancement. Last, the theories are established, and the applications on wearable electronics are validated.

Unsaturated Transport and Targeted Binding of Pluronic-Coated Nanoparticles: Lysimeter Experiments

Efficient delivery of engineered nanoparticles (NPs) to a non-aqueous-phase liquid (NAPL) target zone located above the water table requires an understanding of their transport and binding characteristics. In this investigation, NPs coated with a tunable amphiphilic copolymer were employed in a series of experiments using a 1.4-m-long lysimeter. A crude oil zone was emplaced in the lysimeter to evaluate NP binding capabilities to a representative NAPL. Experimental observations were supported by a reactive transport model. NPs coated with a polymer concentration that promotes enhanced binding to crude oil were successfully delivered and retained in the NAPL zone at concentrations about three times higher than elsewhere in the lysimeter and consistent with the distribution of total petroleum hydrocarbons. Model simulations were able to reproduce the observed asymmetrical NP breakthrough curves and retention profile. The estimated attachment rate coefficient was two orders of magnitude higher for the NAPL zone than elsewhere, supporting the observed preferential binding to the crude oil. Depth-dependent straining was used in the model to capture the NP retention observed near the top of the lysimeter, presumably due to film straining caused by the increased capillary pressure. In addition to the reversible attachment and straining mechanisms, model simulations also indicated that loss of aqueous NP mass was required deeper in the lysimeter to provide a reasonable fit to the observed NP data. Due to increased contact with sediments, the polymer structure that coats the NPs may be slowly removed, leading to aggregation and reduced mobility because of physical trapping. Findings of this study revealed that despite the demonstrated targeted binding capability of these NPs in unsaturated systems, their delivery to a target NAPL zone distant from an injection location may be a challenge due to possible aggregation over longer travel distances and thus an important design consideration.Practical ApplicationsSoil and groundwater contamination by petroleum hydrocarbons is a global issue due to the harm it causes to the environment and human health. Hence, developing novel in situ technologies is necessary due to limitations associated with ex situ remediation. One emerging technology called nanoremediation involves injecting nanosize particles into the impacted area to interact with contaminants effectively due to their small size and large surface area. By modifying their surface, particles can transport through soil and stick to specific contaminants, which is called targeted delivery. To better understand this technology, it should be studied under conditions similar to those in the field. Hence, we investigated targeted delivery of engineered nanoparticles in a large 1.4-m-long column packed with natural soil, representative of the unsaturated zone above a shallow aquifer. Our findings demonstrated preferential nanoparticle attachment to the crude oil–impacted sediment which provides the foundation to advance the use of these nanoparticles as a treatment agent and increases treatment efficiency. However, the noted increase in nanoparticle retention over longer travel distances is suggestive of their stability issues in unsaturated porous media, which may pose a limitation for the delivery of these NPs to a target zone distal from the injection location.

Parametrization and thermodynamic scaling of pair correlation functions for the fractional quantum Hall effect

The calculation of pair correlations and density profiles of quasiholes are routine steps in the study of proposed fractional quantum Hall states. Nevertheless, the field has not adopted a standard way to present the results of such calculations in an easily reproducible form. We develop a polynomial expansion that allows for easy quantitative comparison between different candidate wavefunctions, as well as reliable scaling of correlation and quasihole profiles to the thermodynamic limit. We start from the well-known expansion introduced by Girvin [PRB, 30 (1984)] (see also [Girvin, MacDonald and Platzman, PRB, 33 (1986)]), which is physically appealing but, as we demonstrate, numerically unstable. We orthogonalize their basis set to obtain a new basis of modified Jacobi polynomials, whose coefficients can be stably calculated. We then apply our expansion to extract pair correlation expansion coefficients and quasihole profiles in the thermodynamic limit for a wide range of fractional quantum Hall wavefunctions. These include the Laughlin series, composite fermion states with both reverse and direct flux attachment, the Moore-Read Pfaffian state, and BS hierarchy states. The expansion procedure works for both abelian and non-abelian quasiholes, even when the density at the core is not zero. We find that the expansion coefficients for all quantum Hall states considered can be fit remarkably well using a cosine oscillation with exponentially decaying amplitude. The frequency and the decay length are related in an intuitive, but not elementary way to the filling fraction. Different states at the same filling fraction can have distinct values for these parameters. Finally, we also use our scaled correlation functions to calculate estimates for the magneto-roton gaps of the various states.

Anti-Bacterial Adhesion on Abiotic and Biotic Surfaces of the Exopolysaccharide from the Marine Bacillus licheniformis B3-15

The eradication of bacterial biofilm represents a crucial strategy to prevent a clinical problem associated with microbial persistent infection. In this study we evaluated the ability of the exopolysaccharide (EPS) B3-15, produced by the marine Bacillus licheniformis B3-15, to prevent the adhesion and biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 on polystyrene and polyvinyl chloride surfaces. The EPS was added at different times (0, 2, 4 and 8 h), corresponding to the initial, reversible and irreversible attachment, and after the biofilm development (24 or 48 h). The EPS (300 µg/mL) impaired the initial phase, preventing bacterial adhesion even when added after 2 h of incubation, but had no effects on mature biofilms. Without exerting any antibiotic activity, the antibiofilm mechanisms of the EPS were related to the modification of the (i) abiotic surface properties, (ii) cell-surface charges and hydrophobicity, and iii) cell-to-cell aggregation. The addition of EPS downregulated the expression of genes (lecA and pslA of P. aeruginosa and clfA of S. aureus) involved in the bacterial adhesion. Moreover, the EPS reduced the adhesion of P. aeruginosa (five logs-scale) and S. aureus (one log) on human nasal epithelial cells. The EPS could represent a promising tool for the prevention of biofilm-related infections.

A Recombinant Chimeric Cedar Virus-Based Surrogate Neutralization Assay Platform for Pathogenic Henipaviruses.

The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment.

Novel findings deduced from the microscopic kinetics model contest the classical nucleation theory

In this study, we revisit the microscopic kinetics model considering crystal nucleation as reversible attachment and detachment of units from growing clusters. Based on the variation of the rate constants of attaching and detaching with cluster size, we derive some findings that contest the classical nucleation theory. First, the equivalent thermodynamic parameters are deduced from the rate constants, which reveal that the interfacial free energy per area varies with cluster size and finally levels off. Second, if the crystal is not perfect, at the melting point, the nucleation barrier will be definite rather than infinitely large. Third, it is predicted that the critical nuclei size does not vary with supersaturation. Fourth, when the neighboring units from the same polymer chain are used for crystal nucleation, the attaching rate constant should decrease with cluster size, which is distinctly different from the same attaching rate constant in the nucleation of small molecular crystals. These interesting findings show that teaching the old dog (the microscopic kinetics model) new tricks could lead to new findings and deepen our understanding of crystal nucleation.

Investigation of Three-Dimensional Bacterial Adhesion Manner on Model Organic Surfaces Using Quartz Crystal Microbalance with Energy Dissipation Monitoring.

Bacterial biofilms reduce the performance and efficiency of biomedical and industrial devices. The initial step in forming bacterial biofilms is the weak and reversible attachment of the bacterial cells onto the surface. This is followed by bond maturation and secretion of polymeric substances, which initiate irreversible biofilm formation, resulting in stable biofilms. This implies that understanding the initial reversible stage of the adhesion process is crucial to prevent bacterial biofilm formation. In this study, we analyzed the adhesion processes of E. coli on self-assembled monolayers (SAMs) with different terminal groups using optical microscopy and quartz crystal microbalance with energy dissipation (QCM-D) monitoring. We found that a considerable number of bacterial cells adhere to hydrophobic (methyl-terminated) and hydrophilic protein-adsorbing (amine- and carboxy-terminated) SAMs forming dense bacterial adlayers while attaching weakly to hydrophilic protein-resisting SAMs [oligo(ethylene glycol) (OEG) and sulfobetaine (SB)], forming sparse but dissipative bacterial adlayers. Moreover, we observed positive shifts in the resonant frequency for the hydrophilic protein-resisting SAMs at high overtone numbers, suggesting how bacterial cells cling to the surface using their appendages as explained by the coupled-resonator model. By exploiting the differences in the acoustic wave penetration depths at each overtone, we estimated the distance of the bacterial cell body from different surfaces. The estimated distances provide a possible explanation for why bacterial cells tend to attach firmly to some surfaces and weakly to others. This result is correlated to the strength of the bacterium-substratum bonds at the interface. Elucidating how the bacterial cells adhere to different surface chemistries can be a suitable guide in identifying surfaces with a more significant probability of contamination by bacterial biofilms and designing bacteria-resistant surfaces and coatings with excellent bacterial antifouling characteristics.

Reversible adhesion by type IV pili leads to formation of permanent localized clusters.

The formation of long-lived, multicellular clusters is a fundamental step in the physiopathology of many disease-causing bacteria. Experiments on abiotic surfaces suggest that bacterial colonization, including initial cluster formation, requires (1) irreversible adhesion, (2) cell proliferation, and (3) a phenotypic transition. However, here we show that on infection of a polarized MDCK epithelium, Pseudomonas aeruginosa (PA) forms long-lived- i.e., permanent- bacterial clusters without requiring irreversible adhesion, cell proliferation, or a phenotypic transition. By combining experiments and a mathematical model, we reveal that the cluster formation process is mediated by type IV pili (T4P). Furthermore, we unveil how T4P quantitatively operate during adhesion, finding that it is a stochastic process that involves an activation time, requires the retraction of pili, and results in reversible attachment. We explain how such reversible attachment process leads to the formation of permanent bacterial clusters and quantify the cluster growth dynamics.

In situ control of root-bacteria interactions using optical trapping in transparent soil.

Bacterial attachment on root surfaces is an important step preceding the colonization or internalization and subsequent infection of plants by pathogens. Unfortunately, bacterial attachment is not well understood because the phenomenon is difficult to observe. Here we assessed whether this limitation could be overcome using optical trapping approaches. We have developed a system based on counter-propagating beams and studied its ability to guide Pectobacterium atrosepticum (Pba) cells to different root cell types within the interstices of transparent soils. Bacterial cells were successfully trapped and guided to root hair cells, epidermal cells, border cells, and tissues damaged by laser ablation. Finally, we used the system to quantify the bacterial cell detachment rate of Pba cells on root surfaces following reversible attachment. Optical trapping techniques could greatly enhance our ability to deterministically characterize mechanisms linked to attachment and formation of biofilms in the rhizosphere.

The Involvement of Cell-Type-Specific Glycans in Hydra Temporary Adhesion Revealed by a Lectin Screen

Hydra is a freshwater solitary polyp, capable of temporary adhesion to underwater surfaces. The reversible attachment is based on an adhesive material that is secreted from its basal disc cells and left behind on the substrate as a footprint. Despite Hydra constituting a standard model system in stem cell biology and tissue regeneration, few studies have addressed its bioadhesion. This project aimed to characterize the glycan composition of the Hydra adhesive, using a set of 23 commercially available lectins to label Hydra cells and footprints. The results indicated the presence of N-acetylglucosamine, N-acetylgalactosamine, fucose, and mannose in the adhesive material. The labeling revealed a meshwork-like substructure in the footprints, implying that the adhesive is mainly formed by fibers. Furthermore, lectins might serve as a marker for Hydra cells and structures, e.g., many labeled as glycan-rich nematocytes. Additionally, some unexpected patterns were uncovered, such as structures associated with radial muscle fibers and endodermal gland cells in the hypostome of developing buds.

Probing protein ubiquitination in live cells.

The reversible attachment of ubiquitin governs the interaction, activity and degradation of proteins whereby the type and target of this conjugation determine the biological response. The investigation of this complex and multi-faceted protein ubiquitination mostly relies on painstaking biochemical analyses. Here, we employ recombinant binding domains to probe the ubiquitination of proteins in living cells. We immobilize GFP-fused proteins of interest at a distinct cellular structure and detect their ubiquitination state with red fluorescent ubiquitin binders. With this ubiquitin fluorescent three-hybrid (ubiF3H) assay we identified HP1β as a novel ubiquitination target of UHRF1. The use of linkage specific ubiquitin binding domains enabled the discrimination of K48 and K63 linked protein ubiquitination. To enhance signal-to-noise ratio, we implemented fluorescence complementation (ubiF3Hc) with split YFP. Using in addition a cell cycle marker we could show that HP1β is mostly ubiquitinated by UHRF1 during S phase and deubiquitinated by the protease USP7. With this complementation assay we could also directly detect the ubiquitination of the tumor suppressor p53 and monitor its inhibition by the anti-cancer drug Nutlin-3. Altogether, we demonstrate the utility of the ubiF3H assay to probe the ubiquitination of specific proteins and to screen for ligases, proteases and small molecules controlling this posttranslational modification.