Mass spectrometry techniques for studying the ubiquitin system

Heap, Rachel E.; Gant, M. S.; Lamoliatte, Frederic; Peltier, Julien; Trost, Matthias

doi:10.1042/bst20170091

Cited by 36 publications

(39 citation statements)

References 78 publications

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“…In order to identify the K63‐polyubiquitylated proteins, which bind the TAK1/MKK7/JNK complex on the phagosome of M2 macrophages, we enriched polyubiquitylated phagosomal proteins from M2 macrophages using tandem ubiquitin‐binding entities (TUBEs) of a repeat of the Npl4 Zinc Finger (NZF) domain of TAB2 tagged with Halo (termed here Halo‐TAB2). These constructs have been shown to bind specifically to K63‐polyubiquitin chains (Fig A; Hjerpe et al , ; Emmerich et al , ; Heap et al , ). Quantitative mass spectrometric analysis of these pull‐downs identified 538 phagosomal proteins that were reproducibly captured by Halo‐TAB2 compared to mutant, ubiquitin‐non‐binding Halo (T674A/F675A) TAB2 control beads (based on a twofold, P < 0.05 cut‐off; Dataset EV2).…”

Section: Resultsmentioning

confidence: 99%

Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages

et al. 2019

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Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL‐4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL‐4‐activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL‐4‐activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti‐inflammatory to a pro‐inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo . Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization.

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Section: Resultsmentioning

confidence: 99%

Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages

et al. 2019

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“…To confirm that the observed reduction in ubiquitin conjugates is related to polyubiquitin chains that are targeted to proteasomal degradation (Kulathu & Komander, ), we treated MCF7 cells with 1 μM palbociclib for 10 h in the presence or absence of the proteasome inhibitor bortezomib. We then enriched total polyubiquitin and analyzed the linkages by targeted parallel reaction monitoring (PRM) mass spectrometry (Heap et al , ). This analysis confirmed that amounts of K48 chains, as well as the K6, K29, and K63 chains, were significantly reduced upon palbociclib treatment, and this reduction was dependent on proteasomal activity (Fig ).…”

Section: Resultsmentioning

confidence: 99%

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK 4/6 inhibitor palbociclib

et al. 2018

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Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor‐positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib‐induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib‐induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence‐like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse‐free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.

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“…However, in most cases, they are due to changes in their activities, which is more challenging to monitor, in particular in patient samples. Mass spectrometry has been widely used to analyze, at the proteome scale, cell protein SUMOylome and Ubiquitylome (Heap et al, 2017;Hendriks & Vertegaal, 2016).…”

Section: Dysregulations Of the Ubiquitin And Sumo Conjugated Proteomementioning

confidence: 99%

Ubiquitin and SUMO conjugation as biomarkers of Acute Myeloid Leukemias response to chemotherapies

Gâtel

Brockly

Reynès

et al. 2019

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AbstractUbiquitin and the ubiquitin-like SUMO are covalently conjugated to thousands of proteins to modulate their function and fate. Many of the enzymes involved in their conjugation are dysregulated in cancers and involved in cancer cells response to therapies. We describe here the identification of biomarkers of the activity of these enzymes and their use to predict Acute Myeloid Leukemias (AML) response to standard chemotherapy (daunorubicine-DNR and cytarabine-Ara-C). We compared the ability of extracts from chemosensitive and chemoresistant AML cells to conjugate ubiquitin or SUMO-1 on 9000 proteins spotted on protein-arrays. We identified 122 proteins whose conjugation by these post-translational modifiers marks AML resistance to DNR and/or Ara-C. Based on this modifomic signature, we defined a statistical score able to predict AML patient response to standard chemotherapy. We finally developed a miniaturized assay to easily assess the modification level of the selected biomarkers and validated it in patient cell extracts. Thus, our work identifies a new type of ubiquitin-based biomarkers that could be used to predict cancer patients response to treatments.Summary blurbThis study describes the identification of a new class of biomarkers of cancer response to therapies based on protein modification by Ubiquitin and SUMO and provides the tools to analyze them in Acute Myeloid Leukemia patient samples.

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Mass spectrometry techniques for studying the ubiquitin system

Cited by 36 publications

References 78 publications

Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages

Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK 4/6 inhibitor palbociclib

Ubiquitin and SUMO conjugation as biomarkers of Acute Myeloid Leukemias response to chemotherapies

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