Proteomic analysis identifies ZMYM2 as endogenous binding partner of TBX18 protein in 293 and A549 cells

The TBX18 transcription factor regulates patterning and differentiation programs in the primordia of many organs yet the molecular complexes in which TBX18 resides to exert its crucial transcriptional function in these embryonic contexts have remained elusive. Here, we used 293 and A549 cells as an accessible cell source to search for endogenous protein interaction partners of TBX18 by an unbiased proteomic approach. We tagged endogenous TBX18 by CRISPR/Cas9 targeted genome editing with a triple FLAG peptide, and identified by anti-FLAG affinity purification and subsequent LC-MS analysis the ZMYM2 protein to be statistically enriched together with TBX18 in both 293 and A549 nuclear extracts. Using a variety of assays, we confirmed binding of TBX18 to ZMYM2, a component of the CoREST transcriptional corepressor complex. Tbx18 is coexpressed with Zmym2 in the mesenchymal compartment of the developing ureter of the mouse, and mutations in TBX18and in ZMYM2 were recently linked to congenital anomalies in the kidney and urinary tract (CAKUT) in line with a possible in vivo relevance of TBX18-ZMYM2 protein interaction in ureter development.

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Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo

PROTOPLASMA ◽

10.1007/s00709-016-1042-3 ◽

2016 ◽

Vol 254 (4) ◽

pp. 1547-1562 ◽

Cited By ~ 7

Author(s):

Jana Majerská ◽

Petra Procházková Schrumpfová ◽

Ladislav Dokládal ◽

Šárka Schořová ◽

Karel Stejskal ◽

...

Keyword(s):

Affinity Purification ◽

Tandem Affinity Purification ◽

Interaction Partners

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RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting

10.1101/2020.02.27.968297 ◽

2020 ◽

Cited By ~ 2

Author(s):

Shuo Han ◽

Boxuan Simen Zhao ◽

Samuel A. Myers ◽

Steven A. Carr ◽

Chuan He ◽

...

Keyword(s):

Protein Interaction ◽

Protein Interactions ◽

High Specificity ◽

Living Cells ◽

Cellular Processes ◽

Endogenous Protein ◽

Wide Range ◽

Interaction Partners ◽

Unbiased Manner ◽

Human Telomerase

AbstractRNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. Capitalizing on the ability of the engineered peroxidase APEX2 to identify protein interaction partners via proximity-dependent biotinylation, we used two approaches to target APEX2 to specific cellular RNAs. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were able to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured endogenous protein interaction partners of hTR, including more than a dozen proteins not previously linked to hTR. We validated the unexpected interaction between hTR and the N6-methyladenosine (m6A) demethylase ALKBH5. Further investigation showed that endogenous hTR is modified by m6A, which can be erased by ALKBH5, and that ALKBH5 influences both telomerase complex assembly and activity. These results highlight the ability of MS2- and Cas13-targeted APEX2 to identify novel RNA-protein interactions in living cells.

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Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

eLife ◽

10.7554/elife.58662 ◽

2020 ◽

Vol 9 ◽

Author(s):

Chanjae Lee ◽

Rachael M Cox ◽

Ophelia Papoulas ◽

Amjad Horani ◽

Kevin Drew ◽

...

Keyword(s):

Affinity Purification ◽

Human Genetic Disease ◽

Interaction Partners ◽

A Cell ◽

Cell Type Specific ◽

Axonemal Dynein ◽

And Function ◽

Affinity Purification Mass Spectrometry ◽

Functional Partitioning

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly-acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging in Xenopus to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity- purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

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Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

The Journal of Cell Biology ◽

10.1083/jcb.200805092 ◽

2008 ◽

Vol 183 (2) ◽

pp. 223-239 ◽

Cited By ~ 318

Author(s):

Laura Trinkle-Mulcahy ◽

Séverine Boulon ◽

Yun Wah Lam ◽

Roby Urcia ◽

François-Michel Boisvert ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Interaction ◽

Cell Biology ◽

Protein Complexes ◽

Affinity Purification ◽

Specific Protein ◽

Protein Interaction Data ◽

Quantitative Mass Spectrometry ◽

Cell Extracts ◽

Interaction Partners

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

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Protein-Protein Interaction Detection: Methods and Analysis

International Journal of Proteomics ◽

10.1155/2014/147648 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 180

Author(s):

V. Srinivasa Rao ◽

K. Srinivas ◽

G. N. Sujini ◽

G. N. Sunand Kumar

Keyword(s):

Protein Interaction ◽

Protein Function ◽

In Silico ◽

Affinity Purification ◽

Phylogenetic Profile ◽

Detection Methods ◽

Protein Protein Interaction ◽

Interaction Detection

Protein-protein interaction plays key role in predicting the protein function of target protein and drug ability of molecules. The majority of genes and proteins realize resulting phenotype functions as a set of interactions. The in vitro and in vivo methods like affinity purification, Y2H (yeast 2 hybrid), TAP (tandem affinity purification), and so forth have their own limitations like cost, time, and so forth, and the resultant data sets are noisy and have more false positives to annotate the function of drug molecules. Thus, in silico methods which include sequence-based approaches, structure-based approaches, chromosome proximity, gene fusion, in silico 2 hybrid, phylogenetic tree, phylogenetic profile, and gene expression-based approaches were developed. Elucidation of protein interaction networks also contributes greatly to the analysis of signal transduction pathways. Recent developments have also led to the construction of networks having all the protein-protein interactions using computational methods for signaling pathways and protein complex identification in specific diseases.

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Membrane-SPINE: An improved method to identify protein-protein interaction partners of membrane proteins in vivo

PROTEOMICS ◽

10.1002/pmic.201000558 ◽

2011 ◽

Vol 11 (10) ◽

pp. 2124-2128 ◽

Cited By ~ 13

Author(s):

Volker S. Müller ◽

Peter R. Jungblut ◽

Thomas F. Meyer ◽

Sabine Hunke

Keyword(s):

Membrane Proteins ◽

Protein Interaction ◽

Improved Method ◽

Protein Protein Interaction ◽

Interaction Partners

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Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

10.1101/2020.04.21.052837 ◽

2020 ◽

Cited By ~ 1

Author(s):

Chanjae Lee ◽

Rachael M. Cox ◽

Ophelia Papoulas ◽

Amjad Horani ◽

Kevin Drew ◽

...

Keyword(s):

Affinity Purification ◽

Human Genetic Disease ◽

Interaction Partners ◽

A Cell ◽

Cell Type Specific ◽

Axonemal Dynein ◽

And Function ◽

Affinity Purification Mass Spectrometry ◽

Functional Partitioning

AbstractCiliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

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