scholarly journals Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein with Orthogonal Immunoprecipitation-Mass Spectrometry Assays

2020 ◽  
Author(s):  
Zhiqiang Fu ◽  
Yasmine Rais ◽  
X. Chris Le ◽  
Andrei P. Drabovich
Keyword(s):  
Prostate Cancer ◽  
Mass Spectrometry ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcript Level ◽  
Protein Isoforms ◽  
Protein Protein Interactions ◽  
Molecular Alteration ◽  
And Function ◽  
First Time

SummaryTMPRSS2-ERG gene fusion, a molecular alteration driving nearly a half of prostate cancer cases, has been intensively characterized at the transcript level, while limited studies explored the molecular identity and function of the endogenous fusion at the protein level. Here, we developed and applied immunoprecipitation-mass spectrometry (IP-MS) assays for the measurement of a low-abundance T1E4 TMPRSS2-ERG fusion protein, its isoforms and its interactome in VCaP prostate cancer cells. IP-MS assays quantified total ERG (∼27,000 copies/cell) and its four unique isoforms, and revealed that the T1E4-ERG isoform accounts for 71% of the total ERG protein in VCaP cells. For the first time, the N-terminal peptide (methionine-truncated and N-acetylated TASSSSDYGQTSK) unique for the T1/E4 fusion was identified and quantified. IP-MS with the C-terminal antibodies identified 29 proteins in the ERG interactome, including SWI/SNF chromatin remodeling complex subunits and numerous transcriptional co-regulators. Our data also suggested that TMPRSS2-ERG protein-protein interactions were exerted through at least two different regions. Knowledge on the distinct TMPRSS2-ERG protein isoforms and interactomes may facilitate development of more accurate diagnostics and targeted therapeutics of prostate cancer.

The Drosophila pipsqueak gene encodes a nuclear BTB-domain-containing protein required early in oogenesis

Development ◽  
1996 ◽  
Vol 122 (6) ◽  
pp. 1859-1871 ◽  
Author(s):  
H. Horowitz ◽  
C.A. Berg
Keyword(s):  
Fusion Protein ◽  
Protein Interactions ◽  
Protein Isoforms ◽  
Follicle Cells ◽  
Protein Protein Interactions ◽  
Gene Encoding ◽  
Btb Domain ◽  
Multiple Transcripts ◽  
C Terminus ◽  
Evolutionarily Conserved

Mutations at the pipsqueak locus affect early patterning in the Drosophila egg and embryo. We have cloned pipsqueak and found that it is a large and complex gene, encoding multiple transcripts and protein isoforms. One protein, PsqA, is absent in all of the mutants that we have examined. We show that PsqA is a nuclear protein present in the germ cells and somatically derived follicle cells throughout oogenesis and that it is required prior to stage one of oogenesis. PsqA contains a BTB (POZ) domain at its amino terminus; additionally, we have identified an evolutionarily conserved motif of unknown function present four times in tandem at the C terminus of the protein. PZ pipsqueak mutants produce a putative fusion protein containing the pipsqueak BTB domain fused to sequences resident on the PZ element (H. Horowitz and C. Berg, 1995 Genetics 139, 327–335). We demonstrate here that expression of this fusion protein in wild-type flies has a dominant effect, resulting in infertility and eggshell defects. These dominant phenotypes are discussed in light of current theories on the role of the BTB domain in protein-protein interactions.

scholarly journals Protein interaction landscapes revealed by advanced in vivo cross-linking–mass spectrometry

2021 ◽  
Vol 118 (32) ◽  
pp. e2023360118
Author(s):  
Andrew Wheat ◽  
Clinton Yu ◽  
Xiaorong Wang ◽  
Anthony M. Burke ◽  
Ilan E. Chemmama ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Cross Linking ◽  
Technological Advancement ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
293 Cells ◽  
And Function

Defining protein–protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking–mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry–based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine–lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

scholarly journals Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein

2021 ◽  
Vol 43 (2) ◽  
pp. 767-781
Author(s):  
Vanessa Pinatto Gaspar ◽  
Anelise Cardoso Ramos ◽  
Philippe Cloutier ◽  
José Renato Pattaro Junior ◽  
Francisco Ferreira Duarte Junior ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein Interactions ◽  
Ribosome Biogenesis ◽  
Cell Metabolism ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Cellular Mechanisms ◽  
Cancerous Cell ◽  
First Time

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein–protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.

scholarly journals Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions

2003 ◽  
Vol 17 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Raj Kumar ◽  
E. Brad Thompson
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Hormone Receptors ◽  
Nuclear Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Protein Interactions ◽  
Carboxy Terminal ◽  
And Function ◽  
Terminal Domains

Abstract The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.

scholarly journals PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

2016 ◽  
Vol 2016 ◽  
pp. 1-13
Author(s):  
Stefan Kalkhof ◽  
Stefan Schildbach ◽  
Conny Blumert ◽  
Friedemann Horn ◽  
Martin von Bergen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Isotope Labeling ◽  
Affinity Purification ◽  
Interaction Network ◽  
Mass Spectrometry Data ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Binding Behavior ◽  
Bona Fide

The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6.

scholarly journals Functional and Physical Interactions of the Herpes Simplex Virus Type 1 UL20 Membrane Protein with Glycoprotein K

2008 ◽  
Vol 82 (13) ◽  
pp. 6310-6323 ◽  
Author(s):  
Timothy P. Foster ◽  
Vladimir N. Chouljenko ◽  
K. G. Kousoulas
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Herpes Simplex Virus Type ◽  
Protein Protein Interactions ◽  
Glycoprotein K ◽  
Er Retention ◽  
Simplex Virus

ABSTRACT Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.

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