Biology subjects

ten Dijke, P.

Publications and source records attributed to ten Dijke, P..

16 recordsLinked to original sources

bioRxiv2026

Label-free Isolation of Heterogeneous Breast Cancer Cell Populations via Insulator-Based Dielectrophoresis

Circulating tumor cells (CTCs) are valuable biomarkers for cancer diagnosis and monitoring, yet their isolation from blood remains challenging due to their phenotypic heterogeneity and rarity. Label-free microfluidic technologies offer a promising alternative to affinity-based approaches by exploiting intrinsic biophysical differences between cell types. Here, we developed a microfluidic platform for label-free cell separation based on insulator-based dielectrophoresis (iDEP). The microfluidic device employs an array of triangular insulating structures that generate strong electric field gradients in response to an externally applied alternating current (AC) electric field, enabling selective isolation of breast cancer cells from blood cells based on their dielectric properties. Hydrodynamic focusing is used to confine the sample stream and precisely control cell trajectories within the separation region. Numerical simulations were performed to optimize the electric field distribution and fluid flow characteristics within the device. Experimental validation using breast cancer cell lines (mesenchymal-like MDA-MB-231 cells and epithelial-like MCF-7 cells) spiked into peripheral blood mononuclear cells (PBMCs) demonstrates selective dielectrophoretic deflection of cancer cells while PBMCs largely follow the central streamline. The platform achieves recovery rates exceeding 98% and a separation purity above 65% within the optimized operating conditions. The proposed system provides a simple label-free approach to separate heterogeneous cell populations and represents a promising tool for microfluidic liquid biopsy enrichment applications.

cancer biology↗

bioRxiv2026

Transient suppression of the ECM1 gatekeeper is essential for HGF/c-MET-driven liver regeneration

Hepatocyte growth factor (HGF) is a multifunctional cytokine stored in the extracellular matrix as an inactive precursor and is essential for tissue repair. How HGF activity is dynamically regulated during regeneration remains unclear. Here, we identify extracellular matrix protein 1 (ECM1) as a physiological inhibitor of active HGF during liver regeneration. Following 70% partial hepatectomy, active HGF rapidly increases in parallel with a sharp decline in ECM1, and preventing this downregulation delays liver mass recovery. Mechanistically, ECM1 directly binds the active HGF -subunit through a mechanism dependent on residue R392, thereby suppressing c-MET-ERK-MYC signaling and hepatocyte proliferation. Loss of ECM1 permits activation of this pathway, whereas MYC overexpression rescues ECM1-mediated growth inhibition. In patients, proliferative hepatocytes localize to ECM1-negative regions. Supported by transcriptomic analyses and computational modeling, these findings identify ECM1 as an extracellular gatekeeper whose transient downregulation enables HGF-driven tissue repair. HighlightsO_LIECM1 is temporally downregulated during liver regeneration, while forced ECM1 expression delays liver mass recovery after 70% PHx by suppressing hepatocyte proliferation. C_LIO_LIRapid and transient ECM1 downregulation permits HGF-c-Met-ERK-MYC signaling to drive hepatocyte cell-cycle entry and expansion. C_LIO_LIECM1 directly binds the active HGF -subunit with residue R392 playing a critical role, revealing a novel extracellular mechanism for growth factor inhibition. C_LIO_LILoss of ECM1 marks proliferative hepatocytes in human liver repair, identifying ECM1 as a tunable regulator of regenerative capacity. C_LI

cell biology↗

bioRxiv2026

Pro-domain-dependent folding and co-receptor-mediated targeting to optimize an antagonistic TGF-β monomer for gene-based delivery

Transforming growth factor-beta (TGF-{beta}), a potent promoter of extracellular matrix deposition and suppressor of infiltrating immunity, has arisen as an attractive target for improving outcomes in tissue fibrosis and cancer immune therapy. Despite the promise of TGF-{beta} inhibitors for attenuating the progression of fibrotic disorders or as adjuncts for cancer immunotherapy, current systemically administered inhibitors that target the ligand or receptors have significant on-target liabilities, including cardiotoxicity and development of pre-malignant cutaneous squamous lesions. Recently, an engineered mini monomer of TGF-{beta} (mmTGF-{beta}), which potently and specifically inhibits TGF-{beta} activity, was shown to strongly synergize with checkpoint inhibitors to suppress cancer progression in an aggressive model of melanoma when genetically delivered using an engineered form of vaccinia virus that preferentially infects cancer cells. Despite these promising results, however, a significant fraction of the mmTGF-{beta} was found to misfold, likely due to mispairing of the cysteines that comprise its cystine knot. Here, we demonstrate that inclusion of a modified form of the TGF-{beta} pro-domain that lacks its dimerization motif, the bowtie knot, dramatically improves both the folding and inhibitory activity upon secretion by mammalian cells, thus overcoming one of the major limitations of genetically delivering mmTGF-{beta}. Furthermore, we show that fusion of mmTGF-{beta} to a CD44 binding domain enhances the inhibitory potential of mmTGF-{beta} on immune cells, and on other cell types which express CD44, by more than 30-fold compared to cells negative for CD44. Together, these modifications provide a framework for further enhancing the efficacy and safety of mmTGF-{beta} for cancer immune therapy, and possibly also tissue fibrosis, when delivered genetically using vaccinia, or other related approaches.

bioengineering↗

bioRxiv2026

Decoding the Mechanism of Action of a Parasite TGFβAntagonist Inspires the Creation of Cell-type-specific TGFβ Modulators

Heligmosomoides polygyrus, a mouse parasite, modulates host immunity by secreting modular transforming growth factor-{beta} (TGF{beta}) mimics (TGMs). The agonist TGM1 interacts with TGFBR1, TGFBR2, and the co-receptor CD44 through domains D1/2, D3, and D4/5, respectively. In contrast, the antagonist TGM6, which lacks D1/2, but retains TGFBR2 binding through D3, targets different subsets of cells compared to TGM1. The TGM6 co-receptor is unknown. Using X-ray crystallography and binding studies, we show that TGM6 preferentially binds mouse TGFBR2 over human TGFBR2, and that this is essential for its antagonistic function. We identified low-density lipoprotein receptor-related protein 1 (LRP1) and betaglycan (TGFBR3) as co-receptors for TGM6. LRP1 enhances TGM6 efficacy and is vital for its specific antagonistic effects by promoting TGFBR2 degradation, while betaglycan counteracts TGM6 in a TGFBR2-dependent manner. The modular organization of TGMs enabled us to rationally design TGM1/6 chimeras or TGM-D3 fusion with an affibody that recognizes a specific cell-surface receptor, thereby altering cell-type specificity and functionality. Furthermore, we developed a TGFBR2 nanobody that, on its own, has no inhibitory effect but, when fused to a receptor antibody, antagonizes TGF{beta} signaling in a cell-selective manner. Thus, we designed programmable agents that modulate TGF{beta} signaling only in target co-receptor-expressing cells.

biochemistry↗

bioRxiv2024

Novel high throughput 3D ECM remodeling assay identifies MEK as key driver of fibrotic fibroblast activity

In fibrotic tissues, activated fibroblasts remodel the collagen-rich extracellular matrix (ECM). Intervening with this process represents a candidate therapeutic strategy to attenuate disease progression. Models that generate quantitative data on 3D fibroblast-mediated ECM remodeling with the reproducibility and throughput needed for drug testing are lacking. Here, we develop a model that fits this purpose and produces combined quantitative information on drug efficacy and cytotoxicity. We use microinjection robotics to design patterns of fibrillar collagen-embedded fibroblast clusters and apply automated microscopy and image analysis to quantify ECM remodeling between-, and cell viability within clusters of TGF{beta}-activated primary human skin or lung fibroblasts. We apply this assay to compound screening and reveal actionable targets to suppress fibrotic ECM remodeling. Strikingly, we find that after an initial phase of fibroblast activation by TGF{beta}, canonical TGF{beta} signaling is dispensable and, instead, non-canonical activation of MEK-ERK signaling drives ECM remodeling. Moreover, we reveal that higher concentrations of two TGF{beta} receptor inhibitors while blocking canonical TGF{beta} signaling, in fact stimulate this MEK-mediated profibrotic ECM remodeling activity.

cell biology↗

bioRxiv2024

Automated microinjection for zebrafish xenograft models

Zebrafish xenograft models have been increasingly recognized for their ability to predict patient responses to cancer therapeutics, suggesting their potential as diagnostic tools in clinical settings. However, these models require the precise microinjection of cancer cell suspensions in many small and fragile zebrafish larvae. Manual injections are so challenging that, even after months of training, variability in experimental results persists among researchers. This limits the uptake and deployment of zebrafish xenograft models for clinical use and drug discovery. To address this challenge, we have designed, built, and validated an automated microinjection robot. Combined results of injections into the vasculature, perivitelline space, and hindbrain ventricle demonstrated an average injection success rate of approximately 60%, with a larvae survival rate exceeding 70%, comparable to manual injections using a traditional micromanipulator. Notably, the full automated mode was twice as fast as manual injections. This automation of the microinjection process significantly reduces the need for extensive personnel training while it enhances reproducibility, efficiency, and accuracy, paving the way for more extensive use of zebrafish xenograft models in drug discovery and patient diagnostics.

cancer biology↗

bioRxiv2024

EGF/STAT1 signals to maintain ECM1 expression in hepatic homeostasis are disrupted by IFNγ/NRF2 in chronic liver disease

In healthy livers, extracellular matrix protein 1 (ECM1) is essential for liver homeostasis by keeping latent transforming growth factor-{beta} (LTGF-{beta}) quiescent. Upon hepatocyte damage, ECM1 is downregulated, facilitating LTGF-{beta} activation and fibrogenesis. However, little is known about how hepatic ECM1 is regulated. Here we found in healthy hepatocytes, EGF/EGFR signaling sustains ECM1 expression through phosphorylating STAT1 at S727, enhancing its binding to the ECM1 promoter and boosting gene transcription. During liver inflammation, accumulating IFN{gamma} disrupts this process by downregulating EGFR and inhibiting EGF/EGFR/STAT1-mediated ECM1 promoter binding. Mechanistically, IFN{gamma}-induced STAT1 phosphorylation at Y701 impairs the binding of p-STAT1 S727 to the ECM1 promoter. Additionally, IFN{gamma} induces NRF2 nuclear translocation, which repressively binds to the ECM1 promoter, further reducing its expression. These findings were confirmed in several chronic liver disease (CLD) mouse models. Moreover, AAV8-ECM1 significantly attenuates liver fibrosis and injuries in Western diet (WD)-fed mice. Notably, in patients with CLD, ECM1 levels align with EGFR expression, while NRF2 and LTGF-{beta} activation show a negative correlation with both.

cell biology↗

bioRxiv2023

TGM6, a helminth secretory product, mimics TGF-β binding to TβRII to antagonize TGF-β signaling in fibroblasts

The murine helminth parasite Heligmosomoides polygyrus expresses a family of proteins structurally related to TGF-{beta} Mimic 1 (TGM1), a secreted five domain protein that activates the TGF-{beta} pathway and converts naive T lymphocytes to immunosuppressive Tregs. TGM1 signals through the TGF-{beta} type I and type II receptors, T{beta}RI and T{beta}RII, with domains 1-2 and 3 binding T{beta}RI and T{beta}RII, respectively, and domains 4-5 binding CD44, a co-receptor abundant on T cells. TGM6 is a homologue of TGM1 that is co-expressed with TGM1, but lacks domains 1 and 2. Herein, we show that TGM6 binds T{beta}RII through domain 3, but does not bind T{beta}RI, or other type I or type II receptors of the TGF-{beta} family. In TGF-{beta} reporter assays in fibroblasts, TGM6, but not truncated TGM6 lacking domains 4 and 5, potently inhibits TGF-{beta}- and TGM1-induced signaling, consistent with its ability to bind T{beta}RII but not T{beta}RI or other receptors of the TGF-{beta} family. However, TGM6 does not bind CD44 and is unable to inhibit TGF-{beta} and TGM1 signaling in T cells. To understand how TGM6 binds T{beta}RII, the X-ray crystal structure of the TGM6 domain 3 bound to T{beta}RII was determined at 1.4 [A]. This showed that TGM6 domain 3 binds T{beta}RII through an interface remarkably similar to the TGF-{beta}:T{beta}RII interface. These results suggest that TGM6 has adapted its domain structure and sequence to mimic TGF-{beta} binding to T{beta}RII and function as a potent TGF-{beta} and TGM1 antagonist in fibroblasts. The coexpression of TGM6, along with the immunosuppressive TGMs that activate the TGF-{beta} pathway, may prevent tissue damage caused by the parasite as it progresses through its life cycle from the intestinal lumen to submucosal tissues and back again.

biochemistry↗

bioRxiv2023

The helminth TGF-beta mimic TGM4 is a modular ligand that binds CD44, CD49d and TGF-beta receptors to preferentially target myeloid cells

The murine helminth parasite Heligmosomoides polygyrus expresses a family of modular proteins which, replicating the functional activity of the immunomodulatory cytokine TGF-{beta}, have been named TGM (TGF-{beta} Mimic). Multiple domains bind to different receptors, including TGF-{beta} receptors T{beta}RI (ALK5) and T{beta}RII through domains 1-3, and prototypic family member TGM1 binds the cell surface co-receptor CD44 through domains 4-5. This allows TGM1 to induce T lymphocyte Foxp3 expression, characteristic of regulatory (Treg) cells, and to activate a range of TGF-{beta}-responsive cell types. In contrast, a related protein, TGM4, targets a much more restricted cell repertoire, primarily acting on myeloid cells, with less potent effects on T cells and lacking activity on other TGF-{beta}-responsive cell types. TGM4 binds avidly to myeloid cells by flow cytometry, and can outcompete TGM1 for cell binding. Analysis of receptor binding in comparison to TGM1 reveals a 10-fold higher affinity than TGM1 for TGF{beta}R-I (T{beta}RI), but a 100-fold lower affinity for T{beta}RII through Domain 3. Consequently, TGM4 is more dependent on co-receptor binding; in addition to CD44, TGM4 also engages CD49d (Itga4) through Domains 1-3, as well as CD206 and Neuropilin-1 through Domains 4 and 5. TGM4 was found to effectively modulate macrophage populations, inhibiting lipopolysaccharide-driven inflammatory cytokine production and boosting interleukin (IL)-4-stimulated responses such as Arginase-1 in vitro and in vivo. These results reveal that the modular nature of TGMs has allowed the fine tuning of the binding affinities of the T{beta}R- and co-receptor binding domains to establish cell specificity for TGF-{beta} signalling in a manner that cannot be attained by the mammalian cytokine.

immunology↗

bioRxiv2021

Spatial proteogenomics reveals distinct and evolutionarily-conserved hepatic macrophage niches

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here, we present a spatial proteogenomic atlas of the healthy human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and bile-duct macrophages. We also uncover the respective spatially-resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that bile-duct macrophages are induced by local lipid exposure, while Kupffer cells crucially depend on their crosstalk with hepatic stellate cells via the evolutionarily-conserved ALK1-BMP9/10 axis.

immunology↗

bioRxiv2021

Dissection of N-, O- and glycosphingolipid glycosylation changes in PaTu-S pancreatic adenocarcinoma cells upon TGF-β challenge

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-{beta} (TGF-{beta}) plays a key role in tumor progression, which is often associated with aberrant glycosylation. How PDAC cells respond to TGF-{beta} and the role of glycosylation therein is, however, not well known. Here, we investigated the TGF-{beta}-mediated response and glycosylation changes in SMAD4-deficient PaTu-8955S (PaTu-S) cell line. PaTu-S cells responded to TGF-{beta} by upregulating SMAD2 phosphorylation and target gene expression. TGF-{beta} induced expression of the mesenchymal marker N-cadherin, but did not significantly affect epithelial marker E-cadherin expression. The differences of N-glycans, O-glycans and glycosphingolipid (GSL) glycans in PaTu-S cells with TGF-{beta} stimulation were examined. TGF-{beta} treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in line with TGF-{beta}-induced changes in the expression of glycosylation-related genes. In addition, we observed differences in O- and GSL-glycosylation profiles after TGF-{beta} treatment, including lower levels of sialylated Tn antigen, and neoexpression of globosides. Furthermore, SOX4 expression was upregulated upon TGF-{beta} stimulation, and its depletion blocked the TGF-{beta}-induced N-glycomic changes. Thus, our study provides a mechanism by which TGF-{beta}-induced N-glycosylation changes in SOX4 dependent and SMAD4 independent manner in pancreatic cancer cells. Our results open up avenues to study the relevance of glycosylation in TGF-{beta} signaling in SMAD4 inactivated PDAC.

cancer biology↗

bioRxiv2020

Breast cancer dormancy is associated with a 4NG1 state and not senescence

Reactivation of dormant cancer cells can lead to cancer relapse, metastasis and patient death. Dormancy is a non-proliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and proliferative D2A1 breast cancer cell line models in vivo and in vitro, and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro, we show that D2.OR cells are polyploid ER+/Her2+ cells, and in response to a 3D environment are growth arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible, and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy.

cell biology↗

bioRxiv2020

TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

Bladder cancer is one of the most prevalent cancer types in the world, frequently exhibiting invasion and metastasis and therefore associated with poor prognosis. It is a progressive disease with high recurrence rates even after surgery, which calls for the urgent need for early intervention and diagnosis. The E3 ubiquitin ligase TNF Receptor Associated Factor 4 (TRAF4) has been largely implicated as a tumour-promoting element in a wide range of cancers. Over-expression and amplification of this gene product has been a common observation in breast and other metastatic tumours. In contrast, we observed that expression of TRAF4 negatively correlated with overall patient survival. Moreover, its expression was repressed at epigenetic levels in aggressive bladder cancer cells. We also describe an ERK kinase phosphorylation site on TRAF4 that inhibits its stability and localization to plasma membrane in such cells. Furthermore, knockdown of TRAF4 in epithelial bladder cancer cell lines leads to gain of mesenchymal genes and a loss of epithelial integrity. Reciprocally, stable over-expression of TRAF4 in mesenchymal cells leads to decreased migratory and invasive properties. Transcriptomic analysis upon TRAF4 mis-expression in bladder cancer cell lines revealed that higher TRAF4 expression enhanced BMP/SMAD and dampened NF-{kappa}B signalling pathways. Importantly, this notion was confirmed in bladder cancer patient material. Mechanistically, we showed that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signalling, for proteasomal degradation in bladder cancer cells. We show that genetic and pharmacological inhibition of SMURF1 or its function inhibited migration of aggressive (mesenchymal) bladder cancer cells.

cancer biology↗

bioRxiv2020

Cercosporamide inhibits bone morphogenetic protein receptor type I kinase activity in zebrafish

Zebrafish models are well established tools for investigating underlying mechanisms of diseases. Here, we identified cercosporamide, a metabolite from the fungus Ascochyta aquiliqiae, as a potent bone morphogenetic protein (BMP) type I receptor kinase inhibitor through a zebrafish embryo phenotypic screen. The developmental defects in zebrafish, including lack of the ventral fin induced by cercosporamide was strikingly similar as the phenotypes caused by renowned small molecule BMP type I receptor kinase inhibitors and inactivating mutations in zebrafish BMP receptors. In mammalian cell-based assays, cercosporamide blocked BMP/SMAD-dependent transcriptional reporter activity and BMP-induced SMAD1/5-phosphorylation. Biochemical assays with a panel of purified recombinant kinases demonstrated that cercosporamide directly inhibited kinase activity of BMP type I receptors (also called activin receptor-like kinases (ALKs)). In mammalian cells, cercosporamide selectively inhibited constitutively active BMP type I receptor-induced SMAD1/5 phosphorylation. Importantly, cercosporamide rescued the developmental defects caused by constitutively active Alk2 in zebrafish embryos. Taken together, we believe cercosporamide may be the first of a new class of molecules with potential to be developed further for clinical use against diseases that are causally linked to overactivation of BMP receptor signaling, including Fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma.View Full Text

pharmacology and toxicology↗

bioRxiv2019

Transforming growth factor-beta signaling via ALK1 and ALK5 regulates distinct functional pathways in vein graft intimal hyperplasia

RationaleTransforming growth factor-beta (TGF{beta}) is tightly regulated at multiple levels, with regulation at the receptor level now recognized as a key determinant of the cellular response to this pleiotropic cytokine. TGF{beta} promotes saphenous vein graft neointima formation after coronary artery bypass graft (CABG) surgery, inducing smooth muscle cell (SMC) hyperplasia and fibrosis by signaling via activin receptor-like kinase 5(ALK5). However, the role of the alternate TGF{beta} receptor ALK1 remains completely unknown. ObjectiveTo define the receptor pathways activated by TGF{beta} in SMCs and their mechanistic importance during CABG neointima formation. Methods and resultsRadioligand co-IP assays revealed direct interactions between TGF{beta}, ALK5 and ALK1 in primary saphenous vein graft SMC (HSVSMC) from patients undergoing CABG. Knockdown and pharmacological inhibition of ALK5 or ALK1 in HSVSMC significantly attenuated TGF{beta}-induced phosphorylation of receptor-regulated (R)-Smads 2/3 and 1/5, respectively. Microarray profiling followed by qRT-PCR validation showed that TGF{beta} induced distinct transcriptional networks downstream of ALK5 or ALK1, associated with HSVSMC contractility and migration, respectively and confirmed using migration assays as well as qRT-PCR and western blot assays of contractile SMC markers. scRNAseq analysis of TGF{beta}-treated HSVSMC identified distinct subgroups of cells showing ALK5 or ALK1 transcriptional responses, while RNA velocity analyses indicated divergence in differentiation towards ALK5 or ALK1-dominant lineages. ALK1, ALK5 and their downstream effectors pSmad1/5 and pSmad2/3 were localized to SMA+ neointimal SMCs in remodelled mouse vein grafts. Pharmacological inhibition or genetic ablation of Smad1/5 substantially reducing neointima formation following acute vascular injury. Notably, expression and activation of ALK1, ALK5 and their respective downstream R-Smads was already evident in hyperplastic saphenous veins prior to grafting. ConclusionsWhilst canonical TGF{beta} signaling via ALK5 promotes a contractile HSVSMC phenotype, transactivation of ALK1 by TGF{beta} induces neointima formation by driving cell migration. Restoring the balance between ALK1 and ALK5 in HSVSMC may represent a novel therapeutic strategy for vein graft failure.

cell biology↗

bioRxiv2019

A Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos

Many reagents have been emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement allowing investigations of the function of individual enzymes in a cellular context. We here report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and whole animals. The probe labels active Ubiquitin Carboxy-terminal Hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1-2% of total brain protein. UCHL1 variants have been linked with the neurodegenerative disorders Parkinsons and Alzheimers disease. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1 specific research tools exist. We show that the reagents reported here are specific for UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 irreversibly in an activity-dependent manner. Its use is demonstrated by labelling of UCHL1 both in vitro and in cells. We furthermore show that this probe can report UCHL1 activity during the development of zebrafish embryos.

biochemistry↗