Substrate Identification Using a Chemical Genetics Approach Reveals a Role for PARP-7-Mediated MARylation in Controlling Microtubule Stability in Ovarian Cancer Cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA642215 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12100576.fastq.gz | Fastqsanger.gz | |||
| SRR12100577.fastq.gz | Fastqsanger.gz | |||
| SRR12100578.fastq.gz | Fastqsanger.gz | |||
| SRR12100579.fastq.gz | Fastqsanger.gz | |||
| SRR12100580.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 8 |
Similar Datasets
Project description: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.
2021-02-04 | GSE153395 | GEO
Project description: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 alpha-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.
2020-12-17 | MSV000086611 | MassIVE
Project description:Poly (ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyze ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP- ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP13 (a critical regulator of the antiviral innate immune response) is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.
2021-07-21 | PXD020323 | Pride
Project description:PARP inhibition and the effect on Arabidopsis thaliana high light tolerance
2012-05-01 | E-MTAB-896 | biostudies-arrayexpress
Project description:Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme; however, recently metabolic properties had been attributed to it. Hereby, we examined the metabolic consequences of PARP-2 ablation in liver. Microarray analysis of PARP-2 knockdown HepG2 cells revealed the dysregulation of lipid and cholesterol metabolism genes. Induction of cholesterol biosynthesis genes stemmed from the enhanced expression of sterol-regulatory element binding protein (SREBP)-1. We revealed that PARP-2 is a suppressor of the SREBP-1 promoter, therefore ablation of PARP-2 induces SREBP-1 expression and consequently cholesterol synthesis. PARP-2-/- mice had higher SREBP-1 expression that was translated into enhanced hepatic and serum cholesterol levels. PARP-2 silencing was performed employing shPARP-2 (small hairpin) and scPARP-2 (scrambled) shRNA by lentiviral delivery (Sigma) using 40 MOI lentiviruses coding shRNA sequence against PARP-2.
2013-12-31 | E-GEOD-43981 | biostudies-arrayexpress
Project description:The PARP family of proteins comprises 17 members, about two thirds of which are active mono- or poly(ADP-ribosyl)transferase enzymes that transfer the ADP-ribose moiety of NAD+ onto target proteins. In many cases, ADP-ribosylation, which plays critical roles in human diseases (e.g., cancer, heart disease, and neuropathies) is associated with abrogation of the molecular functions of the target. Discerning ADP-ribosylation events mediated by a specific PARP is challenging, since all PARPs use the same substrate (i.e., NAD+) and the available inhibitors lack the specificity needed to make such conclusions. In order to identify the direct and specific targets of individual PARP family members, we have developed a chemical and genetic approach known as analog sensitivity, in which alteration of a single conserved amino acid in the active site of the PARP protein creates a pocket that allows use of an unnatural NAD+ analog containing a steric moiety. We have functionalized the steric moiety with alkyne for use in click chemistry reactions. This approach, which is transferable to other PARP family members, creates substrate specificity where none previously existed, allowing PARP-specific post-translational modification followed by target visualization, or isolation of ADP-ribosylated targets using click chemistry techniques. We have used this technology in conjunction with mass spectrometry to identify hundreds of targets both unique to, as well as shared among, the nuclear PARP proteins, PARP-1, PARP-2, and PARP-3. We have also determined the genome-wide distribution of PARP-1-specific ADP-ribosylation by coupling this analog-sensitive PARP approach with chromatin cross-linking in method that we call “Click-ChIP-seq”. We observed that PARP-1-specific ADP-ribosylation is enriched at transcriptionally active promoters in proximity to sites of PARP-1 enrichment. In addition, we observed that NELF, an important regulator of RNA Polymerase II (Pol II) pausing, is not only a target of ADP-ribosylation by PARP-1 but also spatially correlated with chromatin-associated ADP-ribose and PARP-1 in Click-ChIP-seq and ChIP-seq assays, respectively. Given these observations, we hypothesized that ADP-ribosylation might modulate NELF function and result altered Pol II pausing. We have explored this possibility using global run-on coupled with deep sequencing (GRO-seq) in MCF-7 cells in which PARP-1 was depleted by RNAi-mediated knockdown. PARP-1 depletion caused an increase in Pol II pausing genome-wide. Taken together, these results suggest the intriguing possibility that ADP-ribosylation of NELF by PARP-1 may be an important and heretofore unknown step in the release of Pol II into productive elongation.
2016-06-09 | GSE74142 | GEO
Project description:The PARP family of proteins comprises 17 members, about two thirds of which are active mono- or poly(ADP-ribosyl)transferase enzymes that transfer the ADP-ribose moiety of NAD+ onto target proteins. In many cases, ADP-ribosylation, which plays critical roles in human diseases (e.g., cancer, heart disease, and neuropathies) is associated with abrogation of the molecular functions of the target. Discerning ADP-ribosylation events mediated by a specific PARP is challenging, since all PARPs use the same substrate (i.e., NAD+) and the available inhibitors lack the specificity needed to make such conclusions. In order to identify the direct and specific targets of individual PARP family members, we have developed a chemical and genetic approach known as analog sensitivity, in which alteration of a single conserved amino acid in the active site of the PARP protein creates a pocket that allows use of an unnatural NAD+ analog containing a steric moiety. We have functionalized the steric moiety with alkyne for use in click chemistry reactions. This approach, which is transferable to other PARP family members, creates substrate specificity where none previously existed, allowing PARP-specific post-translational modification followed by target visualization, or isolation of ADP-ribosylated targets using click chemistry techniques. We have used this technology in conjunction with mass spectrometry to identify hundreds of targets both unique to, as well as shared among, the nuclear PARP proteins, PARP-1, PARP-2, and PARP-3. We have also determined the genome-wide distribution of PARP-1-specific ADP-ribosylation by coupling this analog-sensitive PARP approach with chromatin cross-linking in method that we call “Click-ChIP-seq”. We observed that PARP-1-specific ADP-ribosylation is enriched at transcriptionally active promoters in proximity to sites of PARP-1 enrichment. In addition, we observed that NELF, an important regulator of RNA Polymerase II (Pol II) pausing, is not only a target of ADP-ribosylation by PARP-1 but also spatially correlated with chromatin-associated ADP-ribose and PARP-1 in Click-ChIP-seq and ChIP-seq assays, respectively. Given these observations, we hypothesized that ADP-ribosylation might modulate NELF function and result altered Pol II pausing. We have explored this possibility using global run-on coupled with deep sequencing (GRO-seq) in MCF-7 cells in which PARP-1 was depleted by RNAi-mediated knockdown. PARP-1 depletion caused an increase in Pol II pausing genome-wide. Taken together, these results suggest the intriguing possibility that ADP-ribosylation of NELF by PARP-1 may be an important and heretofore unknown step in the release of Pol II into productive elongation.
2016-06-09 | GSE74141 | GEO
Project description:Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-aminoquniolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for development of cancer-specific therapeutics.
2020-08-15 | GSE148458 | GEO
Project description:Poly(ADP-ribose) polymerase 7 (PARP7) has emerged as a critically important member of a large enzyme family that catalyze mono-ADP-ribosylation (MARylation) in mammalian cells. Unlike other PARPs, PARP7 is expressed in many different cell types and is upregulated by diverse stimuli, such as toxins, growth factors, steroid hormones, and immunomodulatory ligands. Recent evidence by several independent studies show that PARP7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP7 regulates this process as well as other cellular processes. This is because the protein targets, and the specific amino acid sites, of PARP7 MARylation are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation (ADPr) site profiling for identifying the direct targets and sites of PARP7-mediated MARylation. We found that the inactive PARP family member, PARP13—a critical regulator of the innate antiviral immune response—is a major target of PARP7, and is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide MARylation analysis reveals cysteine as a major ADPr acceptor of PARP7. These studies not only provide insight into PARP7 targeting and MARylation site preference, but also serve as an important resource for further understanding PARP7 function in the innate immune response and other pathways.
2021-11-04 | PXD020340 | Pride
Project description:To address the global impact of PARP-1 on alternative splicing, we isolated total RNA in two biological replicates from control (non-treated), PARP-1 siRNA- and PJ-34-treated cells. Sequencing of these RNAs on an Illumina HiSeq 2500, yielded >56 million 100-bp paired-end RNA-seq reads. First we tested if PARP-1 KD was effective at the RNA-seq level. Read aligning to the entire gene body of PARP-1 shown a reduction in PARP-1 expression of about 1.5-fold (P-value < 0.0003), confirming that indeed PARP-1 was depleted after PARP-1 siRNA treatment. We next used these RNA-seq data sets (control, PARP-1 KD and PARylation inhibited) to assess whether these treatments resulted in changes in i) gene expression and ii) alternative splicing.
2015-12-01 | GSE56073 | GEO