scholarly journals A new reporter cell line for studies with proteasome inhibitors in Trypanosoma brucei

2018 ◽  
Author(s):  
Danielle MN Moura ◽  
Osvaldo P de Melo Neto ◽  
Mark Carrington
Keyword(s):  
Cell Line ◽  
Trypanosoma Brucei ◽  
Fluorescent Protein ◽  
Proteasome Inhibitors ◽  
Reporter Cell ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Green Fluorescent ◽  
Inhibitor Treatment

AbstractA Trypanosoma brucei cell line is described that produces a visual readout of proteasome activity. The cell line contains an integrated transgene encoding an ubiquitin-green fluorescent protein (GFP) fusion polypeptide responsive to the addition of proteasome inhibitors. A modified version of T. brucei ubiquitin unable to be recognized by deubiquitinases (UbG76V) was fused to eGFP and constitutively expressed. The fusion protein is unstable but addition of the proteasome inhibitor lactacystin stabilizes it and leads to visually detectable GFP. This cell line can be widely used to monitor the efficiency of inhibitor treatment through detection of GFP accumulation in studies involving proteasome-mediated proteolysis, screening of proteasome inhibitors or other events related to the ubiquitin-proteasome pathway.

Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Kellie R Machlus ◽  
Prakrith Vijey ◽  
Thomas Soussou ◽  
Joseph E Italiano
Keyword(s):  
Protein Degradation ◽  
Proteasome Inhibitors ◽  
Aurora Kinase ◽  
Proteasome Activity ◽  
Major Pathway ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Proplatelet Formation

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

The Ubiquitin Proteasome Pathway from Bench to Bedside

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 220-225 ◽  
Author(s):  
Robert Z. Orlowski
Keyword(s):  
Phase I ◽  
Hematological Malignancies ◽  
Expression Profiles ◽  
Proteasome Inhibitors ◽  
Rational Approach ◽  
Significant Activity ◽  
Ubiquitin Proteasome Pathway ◽  
Gene And Protein Expression ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

Abstract The validation of the ubiquitin-proteasome pathway as a target for therapy of hematological malignancies stands out as one salient example of the ability to translate laboratory-based findings from the bench to the bedside. Preclinical studies showed that proteasome inhibitors had significant activity against models of non-Hodgkin lymphoma and multiple myeloma, and identified some of the relevant mechanisms of action. These led to phase I through III trials of the first clinically available proteasome inhibitor, bortezomib, which confirmed its activity as a single agent in these diseases. Modulation of proteasome function was then found to be a rational approach to achieve both chemosensitization in vitro and in vivo, as well as to overcome chemotherapy resistance. Based on these findings, first-generation bortezomib-based regimens incorporating traditional chemotherapeutics such as alkylating agents, anthracyclines, immunomodulatory agents, or steroids have been evaluated, and many show promise of enhanced clinical anti-tumor efficacy. Further studies of the pro-and anti-apoptotic actions of proteasome inhibitors, and of their effects on gene and protein expression profiles, suggest that novel agents, such as those targeting the heat shock protein pathways, are exciting candidates for incorporation into these combinations. Phase I trials to test these concepts are just beginning, but have already shown some encouraging results. Finally, novel proteasome inhibitors are being developed with unique properties that may also have therapeutic applications. Taken together, these studies demonstrate the power of rational drug design and development to provide novel, effective therapies for patients with hematological malignancies.

scholarly journals Image-Based Screening for the Identification of Novel Proteasome Inhibitors

2007 ◽  
Vol 12 (2) ◽  
pp. 203-210 ◽  
Author(s):  
Linda Rickardson ◽  
Malin Wickström ◽  
Rolf Larsson ◽  
Henrik Lövborg
Keyword(s):  
Cell Line ◽  
Fluorescent Protein ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
Cancer Drug ◽  
Screening Assay ◽  
Hek 293 ◽  
Compound Libraries ◽  
Human Embryo Kidney ◽  
Green Fluorescent

The proteasome is a new, interesting target in cancer drug therapy, and the proteasome inhibitor bortezomib has shown an effect in myeloma patients. It is of interest to efficiently discover and evaluate new proteasome inhibitors. The authors describe the development of an image-based screening assay for the identification of compounds with proteasome-inhibiting activity. The stably transfected human embryo kidney cell line HEK 293 ZsGreen Proteasome Sensor Cell Line expressing the ZsProSensor-1 fusion protein was used for screening and evaluation of proteasome inhibitors. Inhibition of the proteasome leads to accumulation of the green fluorescent protein ZsGreen, which is measured in the ArrayScan® High Content Screening system, in which cell morphology is studied simultaneously. When screening the LOPAC1280 substance library, several compounds with effect on the proteasome were found; among the hits were disulfiram and ammonium pyrrolidinedithiocarbamate (PDTC). Cytotoxic analysis of disulfiram and PDTC showed that the compounds induced cytotoxicity in the myeloma cell line RPMI 8226. The average Z' value for the assay was 0.66. The results indicate that the assay rapidly identifies new proteasome-inhibiting substances, and it will be further used as a tool for image-based screening of other chemically diverse compound libraries.

scholarly journals Proteasome Inhibitors Block a Late Step in Lysosomal Transport of Selected Membrane but not Soluble Proteins

2001 ◽  
Vol 12 (8) ◽  
pp. 2556-2566 ◽  
Author(s):  
Peter van Kerkhof ◽  
Cristina M. Alves dos Santos ◽  
Martin Sachse ◽  
Judith Klumperman ◽  
Guojun Bu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Proteasome Inhibitors ◽  
Endocytic Pathway ◽  
Endosomal Sorting ◽  
Ubiquitin Proteasome Pathway ◽  
Late Step ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Lysosomal Transport

The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.

Regulation of elastin gene transcription by proteasome dysfunction

2005 ◽  
Vol 289 (3) ◽  
pp. C766-C773 ◽  
Author(s):  
Ping-Ping Kuang ◽  
Ronald H. Goldstein
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Proteasome Inhibitors ◽  
Lung Fibroblasts ◽  
Extracellular Matrix Protein ◽  
Mrna Levels ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Elastin Gene

Elastin, a major extracellular matrix protein and the core component of elastic fiber, is essential to maintain lung structural integrity and normal physiological function. We previously found that the downregulation of elastin gene transcription by IL-1β is mediated via activation of NF-κB and CCAAT/enhancer binding protein (C/EBP)β, both targets of the ubiquitin-proteasome pathway. To further investigate the molecular mechanisms that underlie the control of elastin gene expression, we disrupted the ubiquitin-proteasome pathway with specific proteasome inhibitors. We found that specific proteasome inhibitors decreased the steady-state level of elastin mRNA in a dose-responsive manner. Run-on assay and promoter reporter study indicated that the proteasome inhibitor MG-132 repressed the rate of elastin transcription. MG-132 did not affect mRNA levels of NF-κB and C/EBPβ, or the nuclear presence of NF-κB, but markedly increased C/EBPβ isoforms, including liver-enriched transcriptional activating protein and liver-enriched transcriptional inhibitory protein. Addition of cycloheximide blocked these increases and the downregulation of elastin mRNA by MG-132. The MG-132-induced downregulation of elastin transcription was dependent on C/EBPβ expression as assessed with small interfering RNA. These results indicate that the ubiquitin-proteasome pathway plays an essential role in maintaining elastin gene expression in lung fibroblasts. Disruption of this pathway results in the downregulation of tropoelastin transcription via posttranscriptionally induced C/EBPβ isoforms.

scholarly journals The ubiquitin-proteasome pathway and proteasome inhibitors

2001 ◽  
Vol 21 (4) ◽  
pp. 245-273 ◽  
Author(s):  
Jayhyuk Myung ◽  
Kyung Bo Kim ◽  
Craig M. Crews
Keyword(s):  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner

2000 ◽  
Vol 113 (23) ◽  
pp. 4363-4371 ◽  
Author(s):  
J. Zhao ◽  
T. Tenev ◽  
L.M. Martins ◽  
J. Downward ◽  
N.R. Lemoine
Keyword(s):  
Cell Cycle ◽  
Proteasome Inhibitors ◽  
Dependent Manner ◽  
Ubiquitin Proteasome Pathway ◽  
Apoptosis Protein ◽  
Ubiquitin Proteasome ◽  
A Cell ◽  
Proteasome Pathway ◽  
Cycle Dependent

Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G(2)/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G(1) phase. It has been suggested that cell cycle-dependent expression of survivin is regulated at the transcriptional level. In this study we demonstrate involvement of the ubiquitin-proteasome pathway in post-translational regulation of survivin. Survivin is a short-lived protein with a half-life of about 30 minutes and proteasome inhibitors greatly stabilise survivin in vivo. Expression of the survivin gene under the control of the CMV promoter cannot block cell cycle-dependent degradation of the protein. Proteasome inhibitors can block survivin degradation during the G(1) phase and polyubiquitinated derivatives can be detected in vivo. Mutation of critical amino acid residues within the baculovirus IAP repeat (BIR) domain or truncation of the N terminus or the C terminus sensitises survivin to proteasome degradation. Together, these results indicate that the ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner and structural changes greatly destabilise the survivin protein.

scholarly journals A human VE-cadherin-tdTomato and CD43-green fluorescent protein dual reporter cell line for study endothelial to hematopoietic transition

2016 ◽  
Vol 17 (2) ◽  
pp. 401-405 ◽  
Author(s):  
Ho Sun Jung ◽  
Gene Uenishi ◽  
Akhilesh Kumar ◽  
Mi Ae Park ◽  
Matt Raymond ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Dual Reporter ◽  
Green Fluorescent
Sign in / Sign up

Export Citation Format

Share Document