Proximity‐dependent labeling methods for proteomic profiling in living cells: An update

Bosch, Justin A.; Chen, Chiao-Lin; Perrimon, Norbert

doi:10.1002/wdev.392

Cited by 78 publications

(67 citation statements)

References 154 publications

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“…There are many examples of non-catalytic accessory functions of epigenetic enzymes regulating both histone 76,77 and DNA modifications [78][79][80] . Future studies interrogating the replication fork-associated proteome 81 in cells with and without mutant DNMT3A in addition to differential recruitment of DNA repair proteins to chromatin and proximity-labeling proteomics approaches 82,83 will be necessary to clarify its role in preserving DNA integrity. This emerging knowledge will be instructive to develop further therapeutic combination strategies.…”

Section: Discussionmentioning

confidence: 99%

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

Venugopal

Nowialis

Feng

et al. 2021

Preprint

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Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

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Section: Discussionmentioning

confidence: 99%

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

Venugopal

Nowialis

Feng

et al. 2021

Preprint

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“…Besides the utilities described in this paper, HA Frankenbody could be applied to proximity labeling of HA-tagged POI by HA Frankenbody-APEX2 or BioID (Bosch et al, 2021), visualization of HA-POI in the extracellular space in vivo by a secreted form of HA Frankenbody, and so on. Our study provides a new platform for the functional analysis of HA-tagged proteins in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Multiple transgenic strains with a protein of interest (POI) harboring different tags are needed for in vivo functional analysis (Kanca et al, 2017). For example, a transgenic line with an epitope tag-fused construct for immunostaining or immunoprecipitation (Vandemoortele et al, 2019), a GFP or RFP-fused construct for live imaging (Dunst and Tomancak, 2019), a biotin ligase-fused construct for proximity labeling (Bosch et al, 2020), or an RFP-GFP tandem fluorescent-fused construct to monitor autophagic degradation in lysosomes (Kimura et al, 2007). Transgenesis methods introducing a tagged construct at a secondary site are well established in Drosophila (Venken and Bellen, 2014).…”

Section: Introductionmentioning

confidence: 99%

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

Murakawa

Nakamura

Kawaguchi

et al. 2021

Preprint

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For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: HA Frankenbody. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

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“…It allows most of the study of protein complexes in situ and in vivo TPCA profiling can be rapidly deployed to unravel the assembly state of protein complexes across cellular state, cell type, tissue and physiological conditions to provide insight into their functions in normal and diseased cells kinetics and over 30-fold increased signal-to-noise ratio compared to BirA* [18]. Horseradish peroxidase (HRP) can also convert a substrate into free radicals in the presence of H 2 O 2, thus covalently label neighbouring proteins on electron-rich amino acids [19]. However, HRP is mainly used for proximity labelling in oxidizing environments, such as the extracellular surface, due to its low reactivity in the reducing environment [20][21][22].…”

Section: Prosmentioning

confidence: 99%

“…The stability and activity of APEX2 were further improved by Huang et al by introducing a version of cysteine-free APEX2 with C32S mutation [27]. The directed evolution of proximity labelling components is discussed in detail by Bosch et al [19].…”

Section: Prosmentioning

confidence: 99%

Recent progress in mass spectrometry-based strategies for elucidating protein–protein interactions

Low

Syafruddin

Mohtar

et al. 2021

Cell. Mol. Life Sci.

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Protein-protein interactions are fundamental to various aspects of cell biology with many protein complexes participating in numerous fundamental biological processes such as transcription, translation and cell cycle. MS-based proteomics techniques are routinely applied for characterising the interactome, such as affinity purification coupled to mass spectrometry that has been used to selectively enrich and identify interacting partners of a bait protein. In recent years, many orthogonal MS-based techniques and approaches have surfaced including proximity-dependent labelling of neighbouring proteins, chemical cross-linking of two interacting proteins, as well as inferring PPIs from the co-behaviour of proteins such as the co-fractionating profiles and the thermal solubility profiles of proteins. This review discusses the underlying principles, advantages, limitations and experimental considerations of these emerging techniques. In addition, a brief account on how MS-based techniques are used to investigate the structural and functional properties of protein complexes, including their topology, stoichiometry, copy number and dynamics, are discussed. Keywords Affinity purification coupled to mass spectrometry (AP-MS) • Proximity-dependent biotinylation coupled to MS (PDB-MS) • Cross-linking mass spectrometry (XL-MS) • Co-fractionation mass spectrometry (coFrac-MS) • Thermal proximity coaggregation (TPCA)

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Proximity‐dependent labeling methods for proteomic profiling in living cells: An update

Cited by 78 publications

References 154 publications

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

Recent progress in mass spectrometry-based strategies for elucidating protein–protein interactions

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