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. 2021 Feb 27;24(3):102242. doi: 10.1016/j.isci.2021.102242

Dissociation of DNA damage sensing by endoglycosidase HPSE

Alex Agelidis 1,2, Rahul K Suryawanshi 2, Chandrashekhar D Patil 2, Anaamika Campeau 3,4, David J Gonzalez 3,4, Deepak Shukla 1,2,5,∗
PMCID: PMC7957091  PMID: 33748723

Summary

Balance between cell proliferation and elimination is critical in handling threats both exogenous and of internal dysfunction. Recent work has implicated a conserved but poorly understood endoglycosidase heparanase (HPSE) in the restriction of innate defense responses, yet biochemical mediators of these key functions remained unclear. Here, an unbiased immunopurification proteomics strategy is employed to identify and rank uncharacterized interactions between HPSE and mediators of canonical signaling pathways linking cell cycle and stress responses. We demonstrate with models of genotoxic stress including herpes simplex virus infection and chemotherapeutic treatment that HPSE dampens innate responses to double-stranded DNA breakage by interfering with signal transduction between initial sensors and downstream mediators. Given the long-standing recognition of HPSE in driving late-stage inflammatory disease exemplified by tissue destruction and cancer metastasis, modulation of this protein with control over the DNA damage response imparts a unique strategy in the development of unconventional multivalent therapy.

Subject areas: Immunology, Molecular Physiology, Proteomics

Graphical abstract

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Highlights

  • •

    HPSE binds key proteins at interface of DNA damage signaling and IFN responses

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    Nuclear translocation of DNA damage transducer ATM is enhanced in absence of HPSE

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    Cells lacking HPSE display enhanced sensitivity to DNA damage-induced death

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    HPSE interfaces with regulators of DNA damage response to influence cell fate

Immunology; Molecular Physiology; Proteomics

Introduction

Although discovered decades ago as a key extracellular component serving as an attachment point for numerous growth factors, cellular signals, and microbes, the glycosaminoglycan heparan sulfate (HS) continues to be assigned new functions in the maintenance of cellular homeostasis and regulation of disease (Aquino et al., 2010; Xu and Esko, 2014; Zhang et al., 2014). Despite its prevalence across cell types and the existence of many situational structural modifications, our understanding of this sugar molecule and its regulatory enzymes is far from complete. One such enzyme, heparanase (HPSE), is known to be the only mammalian protein capable of splitting chains of HS into smaller subunits (Rabelink et al., 2017). Through its action on cell surface and intracellular HS, HPSE is now known to contribute to disease processes including inflammation, destruction of tissue architecture, and metastasis (Hong et al., 2012; Kundu et al., 2016; Vlodavsky et al., 2018, 2020). More recent work has demonstrated an important contribution of HPSE to microbial pathogenesis, particularly through the promotion of viral egress and the cultivation of an inflammatory milieu conducive to viral spread. Initial studies in this realm were performed in herpes simplex virus 1 (HSV-1), and multiple investigations proved similar actions of this protein across viral families including dengue virus, hepatitis C virus, adenovirus, and human papillomavirus (Agelidis and Shukla, 2020; Agelidis et al., 2017; Guo et al., 2017; Hadigal et al., 2015; Puerta-Guardo et al., 2016; Thakkar et al., 2017). As a prototypic DNA virus that uses HS for initial cellular attachment, HSV-1 has been used as a model cellular perturbation to study the various roles of HPSE in driving pathogenesis. HSV-1 most commonly causes vesicular mucoepithelial eruptions of the oral or genital areas; however, these can progress to more serious sequelae in some individuals, such as encephalitis, vertical transmission to neonates, or ocular keratitis. Our poor grasp of why certain individuals progress to these more serious consequences, along with the fact that all clinical trials of vaccines directed against viral components have failed, points to the importance of a more comprehensive understanding of host factors in driving infection (Belshe et al., 2012; Stanberry et al., 2002). In studying this widespread pathogen that has infected a majority of the global population, investigators now understand that the virus' ability to evade host defense responses has played a large part in its genetic success (Chan and Gack, 2016; Orzalli and Knipe, 2014). Evasion of the DNA damage response is one such mechanism. HSV-1 and other viruses have established through their natural drive to propagate over ages of evolution (Christensen and Paludan, 2017). Likewise, dampening of natural antiviral immune responses including production of type I interferons allows viral replication to proceed undetected. Interestingly, a number of recent publications have described robust associations between these two pathways: cellular sensing of DNA damage can cause robust induction of type I interferon; however, the molecular mechanisms that connect these systems remain to be characterized (Dunphy et al., 2018; Hartlova et al., 2015; Yu et al., 2015). Here we demonstrate that HPSE serves as an important intermediary between DNA damage and interferon production, an intersection with extensive implications in the development of human disease.

Results

Interaction between HPSE and multiple proteins in cell cycle regulation and biogenesis

Given a dearth of biochemical evidence in the literature to explain our recent finding that HPSE acts as a potent regulator of cellular stress responses (Agelidis et al., 2021), we undertook a quantitative proteomics analysis of immunopurified HPSE to investigate the molecular interactions involved (Figure 1A). Isolation of myc-tagged HPSE from human corneal epithelial cells and subsequent immunopurification-mass spectrometry (IP-MS) analysis yielded 270 proteins with a ratio of >10 versus isotype antibody pull-down, indicating that HPSE has the capacity for interaction with many more cytoplasmic and nuclear proteins than previously known (Figures 1B and S1). “GS3” signifies the coding sequence for the fully proteolytically processed form of HPSE that was used in this assay. The HPSE protein sequence was also predicted by NucPred to contain two nuclear localization sequences that may be contribute to its previously documented ability to enter the nucleus under various circumstances and interact with nucleic acids (Figure S1) (Brameier et al., 2007; Rivara et al., 2016). Gene Ontology and overlap analysis of these HPSE-binding proteins shows robust enrichments of various metabolic functions required for cellular proliferation, including “translational initiation,” “ribosome biogenesis,” and “mRNA catabolic process” (Figure 1C). Individual consideration of each portion of the Venn diagram generated a notion of which processes may be active in the presence or absence of HSV-1 infection. Further promoter motif analysis using the PASTAA algorithm indicates that binding partners of HPSE are likely regulated by several major transcription factors, including CREB1 and the related ATF1, ATF2, and ELK1, known for their roles in driving cell proliferation; E2F1, a key cell cycle regulator; and STAT1, which is heavily implicated in the production and response to type I interferon (Figure S2) (Bommareddy et al., 2018; Ivashkiv and Donlin, 2014; Mayr and Montminy, 2001; Roider et al., 2009). Support for the impact of these transcription factors with respect to HPSE actions also comes from our recent work showing that HPSE restricts multiple innate stress responses (Agelidis et al., 2021). This unique analysis thus draws an initial indirect connection between HPSE and multiple important drivers of cellular proliferation and defense.

Figure 1.

Figure 1

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Immunopurification-mass spectrometry (IP-MS) analysis ties HPSE to a dense network of proteins regulating cell cycle and stress responses

(A) Schematic of IP-MS procedure for the quantification of proteins bound to HPSE in human corneal epithelial (HCE) cells in the presence and absence of HSV-1 infection.

(B) Left: numbers of peptides identified by quantitative proteomics analysis fitting specified criteria. Right: Venn diagram of proteins with immunopurification to isotype ratio >10. ∗PSMs, peptide spectrum matches.

(C) Gene ontology (biological process) analysis performed on each portion of the Venn diagram described in (B).

HPSE binds multiple key proteins at the interface of DNA damage signaling and interferon response

Considering these newly identified HPSE interactions in conjunction with similar enrichments by an alternative STRING-based method drove us to investigate the possibility that HPSE integrates signals between the pathways of type I interferon generation and response to DNA damage (Figure 2A). Interestingly, multiple recent studies have demonstrated that innate immune responses including production of type I interferons are activated upon cellular sensing of DNA damage, yet the precise biochemical mediators of these connections remain unknown (Dunphy et al., 2018; Hartlova et al., 2015; Yu et al., 2015). In our hands, repeat immunopurifications of HPSE in human cells showed robust binding of MRE11, RAD50, NBS1, XRCC5, and XRCC6, proteins heavily implicated in DNA damage sensing and repair (Figure 2B). These sensors are known to bind particularly to regions of double-stranded DNA breaks and have been recently established as important drivers of type I interferon production and signaling (Dunphy et al., 2018; Hartlova et al., 2015; Yu et al., 2015). The association of HPSE with the DNA damage response thus appears specific to the apparatus of double-stranded DNA breaks as no interactions with proteins involved in nucleotide excision, base excision, or single-stranded DNA breaks were identified by the IP-MS approach.

Figure 2.

Figure 2

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HPSE binds multiple key proteins at the interface of DNA damage signaling and interferon response

(A) Proteins and corresponding Gene Ontology terms overrepresented in HPSE IP-MS. Network analysis performed with ClueGO in Cytoscape.

(B) Western blot confirmatory analysis of myc-HPSE immunopurified from human corneal epithelial (HCE) cells and probed for binding to multiple sensors of double-stranded DNA breaks. Isotype indicates non-specific mouse IgG of the same immunoglobulin subtype as anti-myc IP antibody.

Nuclear preclusion of double-stranded DNA damage transducer ATM by HPSE upon genotoxic stress

Upon further analysis of the parallel HPSE-deficient mouse embryonic fibroblast (MEF) system, it became clear that cells respond more robustly to DNA damage in the absence of HPSE. Nuclear localization of phosphorylated ataxia telangiectasia mutated (p-ATM) and total cellular phosphorylated ATM substrates (noted by the specific p-S∗/T∗Q motif) were used as markers of activation of the DNA damage sensing system (Traven and Heierhorst, 2005). Likewise, HSV-1 and etoposide, a chemotherapeutic known for its ability to induce dsDNA breaks through the inhibition of topoisomerases, were used as inducers of DNA damage in cells containing and lacking HPSE. Phospho-ATM expression and nuclear localization and subsequent phosphorylation of ATM substrates are markedly increased in cells deficient in HPSE after induction of a DNA damage stimulus, whether through etoposide or viral infection (Figure 3A). Image analysis of multiple thresholded confocal micrographs using CellProfiler on an individual cell basis yielded highly significant increases in activation of the ATM system in the absence of HPSE (Figures 3B−3D). Etoposide was also found to preferentially induce IFN-β in the absence of HPSE, further supporting the notion that HPSE serves as an important link between DNA damage and IFN signaling (Figure 3E). Transcription of the downstream interferon stimulated gene 15 (ISG15) is dramatically elevated in HPSE-deficient cells, and is unchanged by HSV-1 or etoposide, suggesting that the interferon system is constitutively active in the absence of HPSE (Figure 3F).

Figure 3.

Figure 3

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Nuclear preclusion of DNA damage response transducer ATM by HPSE upon genotoxic stress

(A) Representative immunofluorescence micrographs displaying extent of phosphorylated ATM (green) and ATM substrate phosphorylation motif (p-S∗/T∗Q) (red) 8 h after exposure to HSV-1 (MOI = 1) or etoposide (50 μM) in Hpse +/+ and Hpse −/− MEFs. Scale bar, 20 μm.

(B) Demonstration of thresholding method performed with CellProfiler to quantify nuclear and total fluorescence intensity of individual cells.

(C and D) Quantification of fluorescence intensity of p-ATM present in individual nuclei (C) or ATM substrates present in whole cells (D). Significance determined by Wilcoxon signed-ranks test.

(E and F) Quantitative PCR measurement of IFN-β (E) and ISG15 (F) transcripts relative to β-actin after treatment of wild-type and HPSE-deficient MEFs with indicated agents at specific MOIs/concentrations for 24 h (HSV-1) or 8 h (etoposide).

Data are represented as mean ± SEM. Significance determined by Mann-Whitney test (n = 4). ∗∗∗p<0.001, ∗∗∗∗p < 0.0001.

HPSE interfaces with regulators of DNA damage response to influence cell fate

Large increases in cell death were also noted in cells without HPSE upon exposure to etoposide, based on propidium iodide uptake from culture media in cells with loss of membrane integrity (Figures 4A and 4B). Treatment with KU-55933, a specific commercial inhibitor of DNA damage sensor ATM, shows that this enhanced sensitivity to DNA damage is mediated through ATM in HPSE-deficient cells (Figures 4B and 4C). Propidium iodide index indicates the quotient of propidium iodide-stained events to Hoechst (cell-permeable nucleic acid stain)-positive events. Although etoposide was found to be a more effective driver of cell death than HSV-1 infection, treatment of HPSE-deficient cells with KU-55933 successfully prevented the induction of cell death in both cases (Figure 4C). Further investigation into the biochemical mediators of the DNA damage response in Hpse-deficient cells confirmed that ATM phosphorylation is elevated in the absence of HPSE (Figure 4D). Initial phosphorylation of ATM precedes phosphorylation of the downstream mediator checkpoint kinase 2 (CHK2) and phosphorylation of the histone H2Ax at serine 139 to produce γH2Ax, all indicators of activation of the DNA damage response. Western blot analysis of the ATM substrate motif p-S∗/T∗Q also indicates a differential pattern of downstream activation that remains to be explored. In the case of HSV-1 infection, which is a multifactorial set of processes involving a multitude of cellular factors in addition to DNA damage, this phosphorylation of ATM appears later in the time course (Figure 4E). Phosphorylated CHK2 was not detected in this system, although differential phosphorylation of γH2Ax was still apparent.

Figure 4.

Figure 4

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HPSE interfaces with regulators of DNA damage response to influence cell fate

(A) Cell death upon induction of dsDNA breaks with etoposide, measured by uptake of propidium iodide (PI) stain from culture medium in Hpse +/+ and Hpse −/− MEFs. Hoechst nucleic acid stain is permeable to all cells, whereas PI only fluoresces upon loss of membrane integrity. Scale bar, 50 μm.

(B) Quantification of cell death in Hpse +/+ and Hpse −/− MEFs with varying doses of etoposide and ATM kinase inhibitor (ATMi, KU-55933 10 μM) at indicated times post treatment. Data are represented as mean ± SEM. Significance determined by Mann-Whitney test (n = 3).

(C) Representative flow cytometry analysis of PI uptake of cells pre-treated for 18 h with DMSO vehicle or ATMi 10 μM, followed by either HSV-1 for 24 h (left) or etoposide for 8 h (right). Mock (gray) curves denote cells that were pre-treated and then received no further stressor.

(D) Representative western blot analysis of Hpse +/+ and Hpse −/− MEFs treated with etoposide at 50 μM for specified times.

(E) Representative western blot analysis of Hpse +/+ and Hpse −/− MEFs infected with HSV-1 KOS at MOI = 0.1 for specified times.

∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

With an additional focus on the mechanism of etoposide-induced cell death occurring in the absence of HPSE, we observed that apoptosis is promoted at later time points. Drivers of apoptosis including caspase-3 and caspase-8 are cleaved after etoposide treatment of Hpse-deficient cells, whereas this is not observed in wild-type cells under these treatment conditions. On the other hand, cleavage of caspase-1 and gasdermin D, classically associated with activation of pyroptosis, is not seen. Collectively, these results suggest that HPSE acts to suppress ATM activation, potentially by serving as an insulator of signaling from the MRE11-RAD50-NBS1 complex to downstream mediators of the DNA damage response including CHK2.

Inhibitor modulation of cell death induced by etoposide

Chemical inhibitors of various forms of cell death and the DNA damage response were used to further dissect the involvement of HPSE in these processes induced by etoposide (Figures 5A−5C). Representative immunofluorescence images of cells treated in the presence of the dyes CellEvent Caspase-3/7 green reagent and propidium iodide indicate the extent of apoptosis and loss of membrane integrity, respectively. Nec-1, an inhibitor of RIPK1 and necroptosis, and belnacasan, an inhibitor of caspase-1, showed no effect on apoptosis and loss of membrane integrity compared with vehicle treatment. ZVAD, a potent inhibitor of all caspases, showed a marked decrease in apoptosis activation and some degree of reduction in propidium iodide cellular influx. KU-55933 (ATMi) treatment does display initial rescue of apoptosis until the final collection time, where prolonged cellular toxicity of this compound likely dominates. Interestingly, treatment with mirin, a chemical inhibitor of the MRN complex, protects cells from etoposide-induced apoptosis and eventual loss of membrane integrity, and exhibits a similar profile to ZVAD in these assays. Together these findings suggest again that HPSE plays an important role as a regulator of DNA damage response signals acting through the MRN complex.

Figure 5.

Figure 5

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Inhibitor modulation of cell death induced by DNA damage

(A) Representative immunofluorescence micrographs of Hpse −/− MEFs treated with etoposide (50 μM) for 12 h in the presence of specified inhibitors. Scale bar, 100 μm.

(B) Thresholded image intensity quantification analysis of CellEvent caspase-3/7 green detection reagent in Hpse +/+ and Hpse −/− MEFs treated with etoposide and specified inhibitors at corresponding times performed with CellProfiler, as introduced in Figure 3. Boxplots indicate medians and interquartile ranges of 250 cells for each of the 4 images for a total of 1,000 cells analyzed per condition. Experiment was replicated two times with similar results.

(C) Propidium iodide uptake quantified as proportions of live and dead cells in Hpse +/+ and Hpse −/− MEFs treated with etoposide and specified inhibitors at corresponding times. Nuclei thresholded in CellProfiler with a propidium iodide intensity of greater than 0.2 were considered dead.

Discussion

With this exploration of various cellular responses to genotoxic stress, we demonstrate that HPSE serves as a key intersection between the detection and effector phases of signal transduction in the regulation of cell cycle and DNA damage responses. HPSE non-enzymatic activity in particular has been implicated in an array of cellular pathways, yet the mechanism of these roles remains unclear (Coombe and Gandhi, 2019; Jayatilleke and Hulett, 2020). Our recent study established that HPSE displays an inhibitory role over type I interferon and downstream responses (Agelidis et al., 2021). Based on results of the current work, HPSE non-enzymatic binding of proteins involved in DNA damage response, particularly MRE11, RAD50, and NBS1, is likely key in the dampening of interferon responses observed in our studies. Model agents of DNA damage used in this study were etoposide and HSV-1 KOS, which is a strain commonly used by laboratories studying HSV-1 infection worldwide. Other more virulent strains, including McKrae and 17, are likely to be more effective at inducing DNA damage, the details of which will be analyzed in future studies. At multiple doses of etoposide and with a low MOI of 0.1, cells without HPSE are more sensitive to induction of IFN-β transcription. One explanation for the lack of significant difference in IFN-β expression at MOI of 1 is that with the higher MOI, a substantial number of HPSE-deficient cells are in the process of cell death and the initial exaggerated IFN induction has likely already occurred by the time this qPCR snapshot was collected. Likewise, transcription of the downstream ISG15 is dramatically elevated in HPSE-deficient cells and is unchanged by HSV-1 or etoposide, suggesting that the interferon system is constitutively enhanced in the absence of HPSE.

Here and in prior work, we show that HPSE restricts multiple essential cellular defense responses previously linked to one another but not through one factor: type I interferon, cell death, DNA damage, and regulation of the cell cycle (Agelidis et al., 2021). With these related cellular processes in mind, we looked to the multifunctional transcription factor ATM for its potential involvement in the differential regulation by HPSE. Sensing of DNA damage through p-ATM has been shown to be a potent inducer of interferon production and eventual cell death in numerous studies (Dunphy et al., 2018; Hartlova et al., 2015; Kondo et al., 2013; Yu et al., 2015), yet the precise interactions and kinetics of signaling intermediaries including γH2Ax, p-CHK2, and the MRN complex remain unclear (Collins et al., 2020). Double-stranded DNA breaks, such as those caused by ionizing radiation or topoisomerase inhibition, result in the activation of serine/threonine kinases ATM and DNA-PK and subsequent phosphorylation of several hundred protein targets, with γH2Ax serving in many studies as a marker of DNA damage response activation (Jackson and Bartek, 2009; Natale et al., 2017). Likewise, the checkpoint kinase CHK2 is one of the most studied ATM targets, which functions in cell-cycle arrest and control of DNA repair (Shiloh, 2003). Our observation of increased γH2Ax and p-CHK2 levels in HPSE-deficient cells upon both etoposide and HSV-1 treatment thus suggests an inhibitory role of HPSE in the regulation of DNA damage response. Furthermore, our finding in HPSE-deficient cells that mirin reduces DNA damage-driven apoptosis indicates that HPSE interaction with the MRN complex is likely instrumental in dictating downstream responses and cell fate.

Given the extensive list of interacting proteins identified here by IP-MS and previously by other investigators, HPSE may be capable of this type of regulatory activity in a variety of cellular settings and pathways. Recent work has shown that HPSE binds to DNA with unknown consequences (He et al., 2012; Nobuhisa et al., 2007; Schubert et al., 2004; Yang et al., 2015). Future experiments including nuclease treatment of immunopurified HPSE may show whether any of the associations identified are due to bridging interactions with DNA. Further biochemical analysis of MRN complex binding to ATM in the context of HPSE will clarify the nature of this regulatory system. Interestingly, multiple forms of cancer display increased expression of HPSE, understood to drive late-stage metastatic disease (Purushothaman et al., 2011; Putz et al., 2017; Vlodavsky and Friedmann, 2001; Vlodavsky et al., 2018). These newly observed functions of HPSE can help explain the cell cycle dysregulation and loss of sensitivity to DNA damage observed in malignancy. In parallel, multiple viruses including HSV-1 are known to manipulate DNA damage responses and other cellular stress responses to promote their own replication and spread (Lilley et al., 2005; Luftig, 2014; Moretti and Blander, 2017; Turnell and Grand, 2012). Likewise, HPSE upregulation may provide a survival advantage to dysplastic or infected tissue by enabling cells to avoid detection of insults to DNA common to the processes of microbial infection and tumorigenesis.

Limitations of the study

Although widely used to study the impact of a particular gene, murine embryonic knockout cells present potential limitations, including the possibility of compensatory mechanisms that act to minimize the cellular effect of the genetic defect. It remains to be fully understood whether our findings are the direct result of the presence or absence of HPSE, or related to compensatory alterations that occurred during the knockout process. Our approach from multiple experimental angles including IP-MS of human HPSE and functional analysis of HPSE-deficient MEFs represents an introductory understanding of the role of HPSE and its interactome in the regulation of the DNA damage response. Further demonstration in models such as HPSE complementation of knockout cells and Crispr-Cas9 knockout of human cells will provide additional clarifying details of these findings.

Resource availability

Lead contact

Further information and requests for resources may be addressed to the lead contact, Deepak Shukla, PhD at dshukla@uic.edu.

Materials availability

This study did not generate new unique reagents.

Data and code availability

Raw data for IP-MS proteomics experiment can be found on ProteomeXchange under the identifier PXD014183.

Methods

All methods can be found in the accompanying Transparent methods supplemental file.

Acknowledgments

This work was supported by NIH grant R01EY029426 (DS), NIH fellowship F30EY025981 (AA), and departmental core grant P30EY001792 (DS).

Author contributions

Conceptualization, A.A. and D.S.; Methodology, A.A., R.K.S., C.D.P., A.C., D.J.G., and D.S.; Software, A.A., A.C.; Validation, A.A., R.K.S., C.D.P., and A.C.; Formal analysis, A.A. and A.C.; Investigation, A.A., R.K.S., C.D.P., and A.C.; Resources, A.A., A.C., D.J.G., and D.S.; Data curation, A.A. and A.C.; Writing – original draft, A.A.; Writing – review and editing, A.A., R.K.S., C.D.P., A.C., D.J.G., and D.S.; Visualization, A.A.; Supervision, A.A., D.J.G, and D.S.; Funding acquisition, A.A. and D.S.

Declaration of interests

The authors declare no competing interests.

Published: March 19, 2021

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2021.102242.

Supplemental information

Document S1. Transparent methods and Figures S1 and S2
mmc1.pdf (1.9MB, pdf)
Table S1. Immunopurification-mass spectrometry quantitative proteomics, Related to Figures 1 and 2
mmc2.xlsx (214.3KB, xlsx)
Table S2. Results of immunopurification-mass spectrometry quantitative proteomics ClueGO Gene Ontology analysis, Related to Figures 1 and 2
mmc3.xlsx (74.4KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent methods and Figures S1 and S2
mmc1.pdf (1.9MB, pdf)
Table S1. Immunopurification-mass spectrometry quantitative proteomics, Related to Figures 1 and 2
mmc2.xlsx (214.3KB, xlsx)
Table S2. Results of immunopurification-mass spectrometry quantitative proteomics ClueGO Gene Ontology analysis, Related to Figures 1 and 2
mmc3.xlsx (74.4KB, xlsx)

Data Availability Statement

Raw data for IP-MS proteomics experiment can be found on ProteomeXchange under the identifier PXD014183.

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