The CRAPome: a contaminant repository for affinity purification–mass spectrometry data

Mellacheruvu, Dattatreya; Wright, Zachary C.; Couzens, Amber L.; Lambert, Jean-Philippe; St‐Denis, Nicole; Li, Tuo; Miteva, Yana; Hauri, Simon; Sardiu, Mihaela E.; Low, Teck Yew; Halim, Vincentius A.; Bagshaw, Richard D.; Hubner, Nina C.; Al-Hakim, Abdallah; Bouchard, André; Faubert, Denis; Fermin, Damian; Dunham, Wade H.; Goudreault, Marilyn; Lin, Zhen Yuan; Badillo, Beatriz Gonzalez; Pawson, Tony; Durocher, Daniel; Coulombe, Benoit; Aebersold, Ruedi; Superti‐Furga, Giulio; Colinge, Jacques; Heck, Albert J. R.; Choi, Hyungwon; Gstaiger, Matthias; Mohammed, Shabaz; Cristea, Ileana M.; Bennett, Keiryn L.; Washburn, Michael P.; Raught, Brian; Ewing, Rob M.; Gingras, Anne‐Claude; Nesvizhskii, Alexey I.

doi:10.1038/nmeth.2557

Cited by 1,503 publications

(1,491 citation statements)

References 51 publications

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“…PICh nominated ∼80 proteins that were enriched more than twofold at deprotected mouse telomeres (Supplemental Table S1). Comparison with the CRAPome (Mellacheruvu et al 2013) showed that a large fraction of the candidates is likely contaminants (Supplemental Table S1), as had been noted previously (Bartocci et al 2014). One protein, PHF11, was not an obvious contaminant.…”

Section: Identification Of Phf11 As a Ddr Factorsupporting

confidence: 59%

PHF11 promotes DSB resection, ATR signaling, and HR

et al. 2017

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Resection of double-strand breaks (DSBs) plays a critical role in their detection and appropriate repair. The 3 ′ ssDNA protrusion formed through resection activates the ATR-dependent DNA damage response (DDR) and is required for DSB repair by homologous recombination (HR). Here we report that PHF11 (plant homeodomain finger 11) encodes a previously unknown DDR factor involved in 5 ′ end resection, ATR signaling, and HR. PHF11 was identified based on its association with deprotected telomeres and localized to sites of DNA damage in S phase. Depletion of PHF11 diminished the ATR signaling response to telomere dysfunction and genome-wide DNA damage, reduced end resection at sites of DNA damage, resulted in compromised HR and misrejoining of S-phase DSBs, and increased the sensitivity to DNA-damaging agents. PHF11 interacted with the ssDNA-binding protein RPA and was found in a complex with several nucleases, including the 5 ′ dsDNA exonuclease EXO1. Biochemical experiments demonstrated that PHF11 stimulates EXO1 by overcoming its inhibition by RPA, suggesting that PHF11 acts (in part) by promoting 5 ′ end resection at RPA-bound sites of DNA damage. These findings reveal a role for PHF11 in DSB resection, DNA damage signaling, and DSB repair.[Keywords: PHF11; EXO1; RPA; ATR; DSB; resection; homologous recombination] Supplemental material is available for this article.Received October 9, 2016; revised version accepted December 22, 2016.Nuclear double-strand breaks (DSBs) activate the ATM and ATR kinase-dependent DNA damage response (DDR) pathways (for review, see Ciccia and Elledge 2010). Whereas the ATM kinase responds to the presence of dsDNA ends, activation of the ATR kinase requires the presence of ssDNA that is bound by RPA. In addition, activation of the ATR kinase requires the loading of the 9-1-1 complex on the double-stranded-single-stranded junction formed after 5 ′ end resection. Resection of the 5 ′ end of DSBs is therefore a critical step in the activation of ATR signaling. DSB resection is also required for the initiation of several DNA repair pathways, including homologous recombination (HR) and single-strand annealing (SSA) (for review, see Symington and Gautier 2011). HR can restore the original DNA sequence using the sister chromatid as a template for repair. After 5 ′ end resection, HR is initiated by the BRCA2-mediated loading of the Rad51 recombinase onto the ssDNA. In the absence of HR, S-phase DSBs can become a substrate for more error-prone DSB repair, including SSA and classical or alternative nonhomologous end-joining (NHEJ). DSB resection is initiated through the agency of BRCA1, the MRE11/RAD50/NBS1 (MRN) complex, and CtIP (for review, see Cejka 2015). The outcome is a ssDNA end with a short (∼20-nucleotide [nt]) 3 ′ overhang that is a substrate for further nucleolytic attack by EXO1, a dsDNA exonuclease that degrades only the 5 ′ strand to leave a 3 ′ overhang that can be thousands of nucleotides in length. In addition, an overlapping pathway involving the DNA2 nuclease acting in conjun...

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Section: Identification Of Phf11 As a Ddr Factorsupporting

confidence: 59%

PHF11 promotes DSB resection, ATR signaling, and HR

et al. 2017

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“…Following concentration, the complexes were subjected to SDS–PAGE, excised and further processed by trypsin digestion for LC‐MS/MS analysis (Fig EV1B). Amongst the strongest LGP2 interactors in both the type I IFN and poly(I:C)‐treated samples, we identified the endoribonuclease Dicer, as well as several known co‐factors of Dicer, including TRBP (HIV TAR RNA‐binding protein), PACT (protein activator of PKR) and PKR (protein kinase R; Fig 1B), all of which appear at negligible frequency in the “Contaminant Repository for Affinity Purification” databases for FLAG IPs (Mellacheruvu et al , 2013). …”

Section: Resultsmentioning

confidence: 99%

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

et al. 2018

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In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.

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“…To eliminate background contaminants the CRAPome database was used (30). CRAPome is a repository of negative controls generated by previous proteomics experiments and calculates a score for the interaction data as 1-number of experiments the protein is found in the database/total number of experiments in the database.…”

Section: Bioinformatic Analysis Of Drug Pulldown Datamentioning

confidence: 99%

“…We computed the probability of true interaction using the SAINT algorithm (29) and opposed it to the magnitude of spectral count reduction upon competition with the free compound and we also considered the frequency of appearance in negative control experiments found in the CRAPome database (30). Altogether, this allowed us to capture the core target spectrum of PKC412 in Ewing sarcoma cell line (Fig.…”

Section: Identification Of a Ewing Sarcoma Specific Synergy Between Pmentioning

confidence: 99%

Combinatorial Drug Screening Identifies Ewing Sarcoma–specific Sensitivities

Radic-Sarikas

Tsafou

Emdal

et al. 2017

Molecular Cancer Therapeutics

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Abbreviations: EWS, Ewing sarcoma breakpoint region 1, at chromosome 22; ETS, E-twenty-six family; FLI1, Friend leukemia integration 1 transcription factor; INSR, insulin receptor; IGF1, Insulin-like growth factor 1; IGF1R, insulin-like growth factor 1 receptor; CI, combination index; MS, mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; kd, knockdown. AbstractImprovements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma -specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1. We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1 dependent manner.3

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The CRAPome: a contaminant repository for affinity purification–mass spectrometry data

Cited by 1,503 publications

References 51 publications

PHF11 promotes DSB resection, ATR signaling, and HR

PHF11 promotes DSB resection, ATR signaling, and HR

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

Combinatorial Drug Screening Identifies Ewing Sarcoma–specific Sensitivities

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