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| Status |
Public on Feb 04, 2021 |
| Title |
Substrate Identification Using a Chemical Genetics Approach Reveals a Role for PARP-7-Mediated MARylation in Controlling Microtubule Stability in Ovarian Cancer Cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
ADP-ribosylation (ADPRylation) is a regulatory posttranslational modification of proteins that results in the covalent attachment of ADP-ribose (ADPR) moieties on target proteins. PARP-7 (TiPARP) is a monoADPR transferase whose proteins substrates and biological activities are poorly understood. Here, we describe a chemical genetics approach that has allowed us to identify substrates of PARP-7 and explore their biological functions. We observed reduced levels of PARP7 mRNA in ovarian cancer patient samples versus normal tissue, but found nonetheless that PARP-7 is required for a number of cancer-related biological endpoints in ovarian cancer cells, including growth, migration, and invasion. Global gene expression and gene ontology analyses in ovarian cancer cells subjected to siRNA-mediated knockdown of PARP7 revealed an enrichment of genes encoding proteins with roles in cell-cell adhesion, cell cycle arrest, apoptosis, and gene regulation. To identify the ADPRylated substrates that underlie PARP-7-mediated ovarian cancer cell phenotypes, we developed an NAD+ analog-sensitive approach for PARP-7 comprising a PARP-7 NAD+ binding pocket mutant (S563G) paired with the NAD+ analog 8-Bu(3-yne)T-NAD+. When coupled with mass spectrometry, this approach allowed us to identify the PARP-7 ADP-ribosylated proteome in ovarian cancer cells, including components of the cell-cell adhesion and cytoskeleton organization machinery. Specifically, we found that PARP-7 monoADPRylates α-tubulin to promote microtubule instability, which may play a key role in the regulating ovarian cancer cell growth and motility. Collectively, our results point to an extensive PARP-7 ADP-ribosylated proteome with important roles in cancer-related cellular phenotypes.
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| Overall design |
Using RNA-seq we evaluated the expression changes of mRNAs in OVCAR4 cells after siRNA mediated PARP7 knockdown.
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| Contributor(s) |
Lavanya PP, Bryan G, Sridevi C, Tulip N, Mikayla S, Dan H, Jayanthi L, Lee KW |
| Citation(s) |
33475085 |
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| Submission date |
Jun 26, 2020 |
| Last update date |
Feb 04, 2021 |
| Contact name |
W. Lee Kraus |
| E-mail(s) |
lee.kraus@utsouthwestern.edu
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| Organization name |
UT Southwestern Medical Center
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-8511 |
| Country |
USA |
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| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA642215 |
| SRA |
SRP269034 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE153395_SiRNA1.Forward.bw |
174.5 Mb |
(ftp)(http) |
BW |
| GSE153395_SiRNA1.Reverse.bw |
167.8 Mb |
(ftp)(http) |
BW |
| GSE153395_SiRNA2.Forward.bw |
186.8 Mb |
(ftp)(http) |
BW |
| GSE153395_SiRNA2.Reverse.bw |
180.0 Mb |
(ftp)(http) |
BW |
| GSE153395_SiRNA3.Forward.bw |
169.4 Mb |
(ftp)(http) |
BW |
| GSE153395_SiRNA3.Reverse.bw |
162.9 Mb |
(ftp)(http) |
BW |
| GSE153395_SiRNANeg.Forward.bw |
169.5 Mb |
(ftp)(http) |
BW |
| GSE153395_SiRNANeg.Reverse.bw |
163.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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