Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals

Zhang, Qiang; Schepis, Antonino; Huang, Hai; Yang, Junjiao; Wen, Ming‐Shien; Torra, Joaquim; Zhang, Shaoqing; Yang, Lina; Wu, Haifan; Nonell, Santi; Dong, Zhiqiang; Kornberg, Thomas B.; Coughlin, Shaun R.; Shu, Xiaokun

doi:10.1021/jacs.8b13042

Cited by 74 publications

(99 citation statements)

References 45 publications

(64 reference statements)

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“…Given the relatively large size of our heart organoids (up to 1 mm), we decided to verify whether the formation of these chambers could be associated to internal cell death. To do this, we created a transgenic hiPSC line expressing FlipGFP, a non-uorescent engineered GFP variant which turns uorescent upon effector caspase activation and is thus a reporter for apoptosis 19 . FlipGFP organoids in control conditions exhibited no uorescence indicating that there is no signi cant programmed cell death (Suppl.…”

Section: Figure 1amentioning

confidence: 99%

Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Lewis-Israeli

Wasserman

Ball

et al. 2020

Preprint

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Congenital heart defects (CHD) constitute the most common birth defect in humans, affecting approximately 1% of all live births. Our ability to understand how these disorders originate is hindered by our limited ability to model the complexity of the human heart in vitro. There is a pressing need to develop more faithful organ-like platforms recapitulating complex in vivo phenotypes to study human development and disease in vitro. Here we report a novel method to generate human heart organoids by self-assembly using pluripotent stem cells. Our method is fully defined, highly efficient, scalable, shows high reproducibility and is compatible with screening and high-throughput approaches. Human heart organoids (hHOs) are generated using a two-step canonical Wnt signaling modulation strategy using a combination of chemical inhibitors and growth factors in completely defined culture conditions. hHOs faithfully recapitulate human cardiac development and are similar to age-matched fetal cardiac tissues at the transcriptomic, structural and cellular level. hHOs develop sophisticated internal chambers with well-organized multi-lineage cell-type regional identities reminiscent of the heart fields and the atrial and ventricular chambers, as well as the epicardium, endocardium, and coronary vasculature, and exhibit functional activity. We also show that hHOs can recreate complex metabolic disorders associated with CHD by establishing the first in vitro human model of diabetes during pregnancy (DDP) to study embryonic CHD. morphological and metabolically effects of increased glucose and insulin, showing the capability of modeling the effects of diabetes during pregnancy (DDP). Our heart organoid model constitutes a powerful novel tool for translational studies in human cardiac development and disease.

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Section: Figure 1amentioning

confidence: 99%

Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Lewis-Israeli

Wasserman

Ball

et al. 2020

Preprint

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“…In order to develop a fluorescent reporter responsive to the SARS-CoV-2 main protease, we started with the FlipGFP protein (16). FlipGFP is used to detect protease activity by expressing the GFP beta-strands b10-11 separately from, and in a conformation incompatible with, the rest of the GFP beta-barrel, b1-9 ( Fig.…”

Section: Generation Of a Fluorescent Sars-cov-2 3cl Pro Activity Repomentioning

confidence: 99%

“…When the appropriate protease is present the linker containing the cleavage site is cut. This cleavage allows GFP b11 to reorient such that GFP b10 and 11 are antiparallel and able to fit into GFP b1-9, inducing fluorescence ~100-fold over background (16). SARS-CoV-2 generates two proteases that cleave the viral polyprotein, a papain-like protease (PL pro ) and a chymotrypsin-like protease (3CL pro ).…”

Section: Generation Of a Fluorescent Sars-cov-2 3cl Pro Activity Repomentioning

confidence: 99%

Development of a fluorescence based, high-throughput SARS-CoV-2 3CL^pro reporter assay

Froggatt

Heaton

2020

Preprint

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word count: 242 22 Text word count: 3,127 23 SARS-CoV-2 3CL pro reporter assay 2 ABSTRACT 24In late 2019 a human coronavirus, now known as SARS-CoV-2, emerged, likely from a zoonotic 25 reservoir. This virus causes COVID-19 disease, has infected millions of people, and has led to 26 hundreds of thousands of deaths across the globe. While the best interventions to control and 27 ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit 28 morbidity and mortality in those already infected. At this time, only one FDA approved anti-29 SARS-CoV-2 antiviral drug, remdesivir, is available and unfortunately, its efficacy appears to be 30 limited. Thus, the identification of new and efficacious antivirals is of highest importance. In order 31 to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are 32 required. With respect to drug targets, most attention is focused on either the viral RNA-dependent 33 RNA polymerase or the main viral protease, 3CL pro . 3CL pro is an attractive target for antiviral 34 therapeutics as it is essential for processing newly translated viral proteins, and the viral lifecycle 35 cannot be completed without protease activity. In this work, we present a new assay to identify 36 inhibitors of the SARS-CoV-2 main protease, 3CL pro . Our reporter is based on a GFP-derived 37 protein that only fluoresces after cleavage by 3CL pro . This experimentally optimized reporter assay 38 allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-39 throughput compatible protocols. Using this screening approach in combination with existing drug 40 libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 41 replication and spread. 42 43 44 SARS-CoV-2 3CL pro reporter assay

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“…A protease sensor named FlipGFP was recently designed by inverting the orientation of strand β11 with respect to strand β10 so that complementation with GFP1–9 can no longer occur [104]. Insertion of a protease cleavage site reverts the orientation of β11 after protease digestion to form an antiparallel structure that binds GFP1–9 and restore the fluorescence.…”

Section: Split-fluorescent Proteinsmentioning

confidence: 99%

“…The caspase activity sensor was applied to live cell animals, zebrafish, and Drosophila. A less sensitive red version of the protease reporter was engineered from sfCherry by the same authors [104].…”

Section: Split-fluorescent Proteinsmentioning

confidence: 99%

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

Pédelacq

Cabantous

2019

IJMS

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Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

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Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals

Cited by 74 publications

References 45 publications

Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Development of a fluorescence based, high-throughput SARS-CoV-2 3CL^pro reporter assay

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

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Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals

Cited by 74 publications

References 45 publications

Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Development of a fluorescence based, high-throughput SARS-CoV-2 3CLpro reporter assay

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

Contact Info

Product

Resources

About

Development of a fluorescence based, high-throughput SARS-CoV-2 3CL^pro reporter assay