Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes

Withana, Nimali P.; Garland, Megan; Verdoes, Martijn; Ofori, Leslie O.; Segal, Ehud; Bogyo, Matthew

doi:10.1038/nprot.2016.004

Cited by 54 publications

(46 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To provide evidence for the specificity of B24P, skin biopsies were pretreated with aprotinin and AEBSF that effectively inhibit activity of serine proteases (Figure ). It should be mentioned that these controls should always run in parallel with the experiments as also suggested and performed previously, especially in this case that the B24P does not have a proteolytic enzyme‐selective “bait” and its specificity merits further characterization.…”

Section: Resultsmentioning

confidence: 99%

“…Activity‐based probes have a well‐recognized ability to visualize proteases in vivo . Here, we showed that activography provides similar spatial and semiquantitative information as the in situ zymography method but is advantageous in that activography allows the mapping of enzyme specificities thus opening possibilities for new applications in clinical diagnostic laboratories.…”

Section: Discussionmentioning

confidence: 95%

See 1 more Smart Citation

Activography reveals aberrant proteolysis in desquamating diseases of differing backgrounds

Zingkou

Pampalakis

Kiritsi

et al. 2019

Experimental Dermatology

View full text Add to dashboard Cite

The role of epidermal proteolysis in overdesquamation was revealed in Netherton syndrome, a rare ichthyosis due to genetic deficiency of the LEKTI inhibitor of serine proteases. Recently, we developed activography, a new histochemical method, to spatially localize and semiquantitatively assess proteolytic activities using activity‐based probes. Activography provides specificity and versatility compared to in situ zymography, the only available method to determine enzymatic activities in tissue biopsies. Here, activography was validated in skin biopsies obtained from an array of distinct disorders and compared with in situ zymography. Activography provides a methodological advancement due to its simplicity and specificity and can be readily adapted as a routine diagnostic assay. Interestingly, the levels of epidermal proteolysis correlated with the degree of desquamation independent of skin pathology. Thus, deregulated epidermal proteolysis likely represents a universal mechanism underlying aberrant desquamation.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 95%

Activography reveals aberrant proteolysis in desquamating diseases of differing backgrounds

Zingkou

Pampalakis

Kiritsi

et al. 2019

Experimental Dermatology

View full text Add to dashboard Cite

show abstract

“…antibody of interest as described previously (34). Briefly, the fresh carotid tissue was frozen in optimal-cutting-temperature compound before sectioning.…”

Section: Immunofluorescence Stainingmentioning

confidence: 99%

Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation

Withana

Saito

et al. 2016

J Nucl Med

Self Cite

View full text Add to dashboard Cite

Macrophages are cellular mediators of vascular inflammation and are involved in the formation of atherosclerotic plaques. These immune cells secrete proteases such as matrix metalloproteinases and cathepsins that contribute to disease formation and progression. Here, we demonstrate that activity-based probes (ABPs) targeting cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes can also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Methods: Macrophage-rich carotid lesions were induced in FVB mice fed on a high-fat diet by streptozotocin injection followed by ligation of the left common carotid artery. Mice with carotid atherosclerotic plaques were injected with the optical or dual-modality probes BMV109 and BMV101, respectively, via the tail vein and noninvasively imaged by optical and small-animal PET/CT at different time points. After noninvasive imaging, the murine carotid arteries were imaged in situ and ex vivo, followed by immunofluorescence staining to confirm target labeling. Additionally, human carotid plaques were topically labeled with the probe and analyzed by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunofluorescence staining to confirm the primary targets of the probe. Results: Quantitative analysis of the signal intensity from both optical and PET/CT imaging showed significantly higher levels of accumulation of BMV109 and BMV101 (P , 0.005 and P , 0.05, respectively) in the ligated left carotid arteries than the right carotid or healthy arteries. Immunofluorescence staining for macrophages in cross-sectional slices of the murine artery demonstrated substantial infiltration of macrophages in the neointima and adventitia of the ligated left carotid arteries compared with the right. Analysis of the human plaque tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that the primary targets of the probe were cathepsins X, B, S, and L. Immunofluorescence labeling of the human tissue with the probe demonstrated colocalization of the probe with CD68, elastin, and cathepsin S, similar to that observed in the experimental carotid inflammation murine model. Conclusion: We demonstrate that ABPs targeting the cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes could also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Therefore, ABPs targeting the cysteine cathepsins are potentially valuable new reagents for rapid and noninvasive imaging of atherosclerotic disease progression and plaque vulnerability.

show abstract

“…To date, quenched activity probe-based live cell fluorescence imaging has been described for a handful of proteases, such as cysteine cathepsins . The quenched fluorescent activity probes were applied for labeling of active proteases in fresh-frozen cancer tissues (Withana et al, 2016), for macrophage detection in atherosclerotic plaques (Abd-Elrahman et al, 2016), and as a diagnostic tool to image skin tumor margins (Liu et al, 2019). Recently, a small-molecule chemical toolbox was constructed for parallel imaging of human neutrophil serine proteases (NSPs).…”

Section: Introductionmentioning

confidence: 99%

Tissue-ABPP enables high-resolution confocal fluorescence imaging of serine hydrolase activity in cryosections – Application to glioma brain unveils activity hotspots originating from tumor-associated neutrophils

Aaltonen

Singha

Jakupović

et al. 2019

Preprint

View full text Add to dashboard Cite

Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. The ABPP approach utilizes cell/tissue proteomes and features the FP warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Here, we advance the ABPP methodology to glioma brain cryosections, enabling highresolution confocal fluorescence imaging of SH activity in different cell types of the tumor microenvironment, identified by using extensive immunohistochemistry on activity probe labeled sections. We name this technique tissue-ABPP to distinguish it from conventional gel-based ABPP. We show heightened SH activity in glioma vs. normal brain and unveil activity hotspots originating from tumor-associated neutrophils. Thorough optimization and validation is provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Tissue-ABPP enables a wide range of applications for confocal imaging of SH activity in any type of tissue or animal species.

show abstract

Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes

Cited by 54 publications

References 41 publications

Activography reveals aberrant proteolysis in desquamating diseases of differing backgrounds

Activography reveals aberrant proteolysis in desquamating diseases of differing backgrounds

Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation

Tissue-ABPP enables high-resolution confocal fluorescence imaging of serine hydrolase activity in cryosections – Application to glioma brain unveils activity hotspots originating from tumor-associated neutrophils

Contact Info

Product

Resources

About