<i>In vivo</i> imaging of proteasome inhibition using a proteasome‐sensitive fluorescent reporter

Momose, Isao; Tatsuda, Daisuke; Ohba, Seigo; Masuda, Tohru; Ikeda, Daishiro; Nomoto, Akio

doi:10.1111/j.1349-7006.2012.02352.x

Cited by 15 publications

(14 citation statements)

References 26 publications

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“…b). To examine proteasome inhibition in living cells, we used stably transfected HEK293 cells (HEK293PS) that are continuously expressing the ZsProSensor‐1 protein, a proteasome‐sensitive fluorescent reporter . The ZsProSensor‐1 protein is a fusion of the green fluorescent protein ZsGreen and mouse ornithine decarboxylase, which can be degraded by the proteasome without being ubiquitinated .…”

Section: Resultsmentioning

confidence: 99%

“…To examine proteasome inhibition in living cells, we used stably transfected HEK293 cells (HEK293PS) that are continuously expressing the ZsProSensor-1 protein, a proteasome-sensitive fluorescent reporter. (28) The ZsProSensor-1 protein is a fusion of the green fluorescent protein ZsGreen and mouse ornithine decarboxylase, which can be degraded by the proteasome without being ubiquitinated. (29) In these cells, the fluorescent protein was undetectable by fluorescence microscopy because of rapid degradation by the proteasome under steady-state conditions, but detectable in the presence of proteasome inhibitors.…”

Section: Resultsmentioning

confidence: 99%

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Antitumor effects of tyropeptin‐boronic acid derivatives: New proteasome inhibitors

et al. 2014

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The proteasome degrades numerous regulatory proteins that are critical for tumor growth. Thus, proteasome inhibitors are promising antitumor agents. New proteasome inhibitors, such as tyropeptins and tyropeptin-boronic acid derivatives, have a potent inhibitory activity. Here we report the antitumor effects of two new tyropeptin-boronic acid derivatives, AS-06 and AS-29. AS-06 and AS-29 significantly suppress the degradation of the proteasome-sensitive fluorescent proteins in HEK293PS cells, and induce the accumulation of ubiquitinated proteins in human multiple myeloma cells. We show that these derivatives also suppress the degradation of the NF-κB inhibitor IκB-α and the nuclear translocation of NF-κB p65 in multiple myeloma cells, resulting in the inhibition of NF-κB activation. Furthermore, we demonstrate that AS-06 and AS-29 induce apoptosis through the caspase-8 and caspase-9 cascades. In a xenograft mouse model, i.v. administration of tyropeptin-boronic acid derivatives inhibits proteasome in tumors and clearly suppresses tumor growth in mice bearing human multiple myeloma. Our results indicate that tyropeptin-boronic acid derivatives could be lead therapeutic agents against human multiple myeloma.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Antitumor effects of tyropeptin‐boronic acid derivatives: New proteasome inhibitors

et al. 2014

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“…Therefore, we developed a system for the in vivo fluorescence imaging of proteasome activity in the tumors of living mice using a proteasome-sensitive fluorescent reporter, ZsProSensor-1. 80 This reporter is a fusion protein containing a fluorescent protein, ZsGreen, and mouse ornithine decarboxylase that is degraded by the proteasome without ubiquitin conjugation. 81 In stably transfected cells expressing ZsProSensor-1, the fluorescent reporter is rapidly degraded under steady-state conditions, whereas it is stabilized in the presence of proteasome inhibitors such as AS-29 and accumulates in the cells (Figure 6b).…”

Section: Prospectmentioning

confidence: 99%

“…Intravenous administration of bortezomib significantly suppressed tumor growth and increased fluorescent protein in the tumors, while oral administration did not exert antitumor effects and did not induce the accumulation of fluorescent protein in the tumors. 80 Moreover, oral administration of delanzomib, an orally active proteasome inhibitor, 82,83 markedly reduced tumor growth and emitted a fluorescent signal in the tumors. Therefore, the use of this in vivo imaging system can easily explore new orally active proteasome inhibitors.…”

Section: Prospectmentioning

confidence: 99%

Tyropeptins, proteasome inhibitors produced by Kitasatospora sp. MK993-dF2

Momose

Watanabe

2017

J Antibiot

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Tyropeptins are new proteasome inhibitors isolated from the culture broth of Kitasatospora sp. MK993-dF2. Tyropeptins permeate cell membranes, inhibit intracellular proteasomes and reduce the degradation of ubiquitinated proteins in mammalian cells. We performed structure-based drug design and structure-activity relationship studies on tyropeptin derivatives to obtain valuable information of derivatives. Among the synthesized tyropeptin derivatives, some boronic acid derivatives exhibited potent antitumor effects against human multiple myeloma. In this review, we summarize the discovery of tyropeptins and the development of tyropeptin derivatives.

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“…Stable expression of the ODC-GFP reporter in mouse models has been used to evaluate proteasome activity. This system has been shown capable of detecting Bortezomib inhibition of proteasome activity in tumor cells, indicating that it could be used in the future for library-based screens for proteasome inhibitors (26). Affinity tags combined with the ODC degron produced a reporter used in Saccharomyces cerevisiae .…”

Section: Measuring Proteasome Activitymentioning

confidence: 99%

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

Melvin

Woss

Park

et al. 2013

Cell Biochem Biophys

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The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS have provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequences fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes (DUBs). This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, recent work is presented highlighting the development of novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.

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In vivo imaging of proteasome inhibition using a proteasome‐sensitive fluorescent reporter

Cited by 15 publications

References 26 publications

Antitumor effects of tyropeptin‐boronic acid derivatives: New proteasome inhibitors

Antitumor effects of tyropeptin‐boronic acid derivatives: New proteasome inhibitors

Tyropeptins, proteasome inhibitors produced by Kitasatospora sp. MK993-dF2

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

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