scholarly journals Early-onset torsion dystonia: a novel high-throughput yeast genetic screen for factors modifying protein levels of torsinAΔE

2017 ◽  
Vol 10 (9) ◽  
pp. 1129-1140 ◽  
Author(s):  
Lucía F. Zacchi ◽  
John C. Dittmar ◽  
Michael J. Mihalevic ◽  
Annette M. Shewan ◽  
Benjamin L. Schulz ◽  
...  
Keyword(s):  
High Throughput ◽  
Early Onset ◽  
Genetic Screen ◽  
Torsion Dystonia ◽  
Protein Levels ◽  
Yeast Genetic

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

2021 ◽  
pp. 247255522110262
Author(s):  
Jonathan Choy ◽  
Yanqing Kan ◽  
Steve Cifelli ◽  
Josephine Johnson ◽  
Michelle Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Time Resolved ◽  
Protein Levels ◽  
Increased Risk ◽  
Compound Screening ◽  
High Throughput Screens ◽  
Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

Identification of microRNAs that stabilize p53 in HPV-positive cancer cells

2021 ◽  
Author(s):  
Gustavo Martínez-Noël ◽  
Patricia Szajner ◽  
Rebecca E. Kramer ◽  
Kathleen A. Boyland ◽  
Asma Sheikh ◽  
...  
Keyword(s):  
Cancer Cells ◽  
High Throughput ◽  
Ubiquitin Ligase ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Human Papillomaviruses ◽  
Live Cell ◽  
Viral Oncoprotein ◽  
Protein Levels ◽  
P53 Stability

Etiologically, 5% of all cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among their cellular targets are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of the hrHPV E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high throughput screen to determine whether exogenous microRNA (miRNA) transfection had the ability to stabilize p53 in hrHPV-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. Among the miRNAs whose transfection resulted in the greatest p53 stabilization was 375-3p that has previously been reported to stabilize p53 in HeLa cells, providing validation of the screen. The top 32 miRNAs in addition to 375-3p were further assessed using a second cell-based p53 stability reporter system as well as in non-reporter HeLa cells to examine their effects on endogenous p53 protein levels, resulting in the identification of 23 miRNAs whose transfection increased p53 levels in HeLa cells. While a few miRNAs that stabilized p53 led to decreases in E6AP protein levels, all targeted HPV oncoprotein expression. We further examined subsets of these miRNAs for their abilities to induce apoptosis and determined whether it was p53-mediated. The introduction of specific miRNAs revealed surprisingly heterogeneous responses in different cell lines. Nonetheless, some of the miRNAs described here have potential as therapeutics for treating HPV-positive cancers. Importance Human papillomaviruses cause approximately 5% of all cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral oncoprotein E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of p53 through the engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 leads to apoptosis in HPV-positive cancer cells. Using a high throughput live cell assay we identified several miRNAs whose transfection stabilize p53 in HPV-positive cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

Behavioural and pharmacological examinations in a transgenic mouse model of early-onset torsion dystonia

2011 ◽  
Vol 97 (4) ◽  
pp. 647-655 ◽  
Author(s):  
Nikola Lange ◽  
Melanie Hamann ◽  
Pullani Shashidharan ◽  
Angelika Richter
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Early Onset ◽  
Transgenic Mouse Model ◽  
Torsion Dystonia

scholarly journals Mutagenomics: A Rapid, High-Throughput Method to Identify Causative Mutations from a Genetic Screen

2020 ◽  
Vol 184 (4) ◽  
pp. 1658-1673
Author(s):  
Charles Hodgens ◽  
Nicole Chang ◽  
G. Eric Schaller ◽  
Joseph J. Kieber
Keyword(s):  
High Throughput ◽  
Genetic Screen ◽  
High Throughput Method

Targeted high-throughput sequencing identifies a TARDBP mutation as a cause of early-onset FTD without motor neuron disease

2014 ◽  
Vol 35 (5) ◽  
pp. 1212.e1-1212.e5 ◽  
Author(s):  
Matthis Synofzik ◽  
Christoph Born ◽  
Axel Rominger ◽  
Nina Lummel ◽  
Ludger Schöls ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neuron Disease ◽  
High Throughput ◽  
Early Onset ◽  
High Throughput Sequencing

Expression of the early-onset torsion dystonia gene (DYT1) in human brain

1998 ◽  
Vol 43 (5) ◽  
pp. 669-673 ◽  
Author(s):  
Sarah J. Augood ◽  
John B. Penney ◽  
Ingrid K. Friberg ◽  
Xandra O. Breakefield ◽  
Anne B. Young ◽  
...  
Keyword(s):  
Human Brain ◽  
Early Onset ◽  
Torsion Dystonia

scholarly journals The early-onset torsion dystonia-associated protein, torsinA, displays molecular chaperone activity in vitro

2010 ◽  
Vol 15 (5) ◽  
pp. 605-617 ◽  
Author(s):  
Alexander J. Burdette ◽  
Perry F. Churchill ◽  
Guy A. Caldwell ◽  
Kim A. Caldwell
Keyword(s):  
Molecular Chaperone ◽  
Early Onset ◽  
Chaperone Activity ◽  
Torsion Dystonia
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