Dataset Information

E-cadherin interactome complexity and robustness resolved by quantitative proteomics

ABSTRACT: E-cadherin-mediated cell-cell adhesion and signaling plays an essential role in development and maintenance of healthy epithelial tissues. Adhesiveness mediated by E-cadherin is conferred by its extracellular cadherin domains and is regulated by an assembly of adaptors and enzymes associated with its cytoplasmic tail. Here, we used proximity biotinylation and quantitative proteomics to identify 561 proteins in the vicinity of E-cadherin’s cytoplasmic tail. Isolation of E-cadherin-mediated adhesion plaques from a cell-glass interface added positional context to the proteomic results. Moreover, using expression of GFP-tagged fusion proteins, we determine the subcellular localization of 83 of the E-cadherin proximal proteins, and identify 24 novel adherens junction components. We employed a structure-informed database of protein-protein interactions to construct a comprehensive E-cadherin interactome, containing 82 known and 419 previously uncharacterized proteins. Finally, we found that calcium chelation did not disrupt most protein interactions with E-cadherin, suggesting that most E-cadherin interactions are independent of cell-cell adhesion.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sara Zanivan

LAB HEAD: Sara Zanivan

PROVIDER: PXD000448 | Pride | 2016-07-06

REPOSITORIES: Pride

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Publications

E-cadherin interactome complexity and robustness resolved by quantitative proteomics.

Guo Zhenhuan Z Neilson Lisa J LJ Zhong Hang H Murray Paul S PS Zanivan Sara S Zaidel-Bar Ronen R

Science signaling 20141202 354

E-cadherin-mediated cell-cell adhesion and signaling plays an essential role in development and maintenance of healthy epithelial tissues. Adhesiveness mediated by E-cadherin is conferred by its extracellular cadherin domains and is regulated by an assembly of intracellular adaptors and enzymes associated with its cytoplasmic tail. We used proximity biotinylation and quantitative proteomics to identify 561 proteins in the vicinity of the cytoplasmic tail of E-cadherin. In addition, we used prote ...[more]

PMID: 25468996

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Project description:Kallikrein-related peptidase 3 (KLK3, also known as prostate-specific antigen, PSA) is a chymotrypsin-like kallikrein that has been shown to have anti-angiogenic properties. KLK3 expression in prostate cancer is associated with low microvessel density. We have previously shown in a human umbilical vein endothelial cell (HUVEC) model that the anti-angiogenic effect of KLK3 is related to its enzymatic activity. However, the mechanism of this effect remains to be clarified. To this end we have used a DNA microarray to study the KLK3-induced changes in gene expression associated with reduction of HUVEC tube formation. Among the 41000 genes studied, 311 were differentially expressed in control and KLK3-treated cells. These changes were enriched in several pathways, including the pathways associated with proteasome, ubiquitin-mediated proteolysis, focal adhesion and regulation of actin cytoskeleton. Furthermore, the changes were opposite to those described previously to occur during tubulogenesis. In conclusion, our results show that KLK3 induces gene expression changes in HUVECs. While these changes might be relevant for the mechanism by which KLK3 exerts its anti-angiogenic activity, it cannot be judged by the present results whether the changes are secondary to morphogenic differentiation of the cells or the primary mechanism mediating the effect of KLK3. Keywords: KLK3 treatment, tube formation Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cord veins and grown in Endothelial Cell Basal Medium supplemented with an Endothelial Cell Growth Medium Supplement Pack (PromoCell). The cells were cultured on Matrigel basement membrane prepara-tion together with 500 nM enzymatically active KLK3 (PSA). Phosphate buffered saline (PBS) was used for control. HUVECs were grown on Matrigel for 18 h. RNA was isolated from KLK3-treated and control HUVECs from three different individuals. Cell Recovery Solution (BD Biosciences) was used to detach cells from the Matrigel. Total RNA was isolated with the RNeasy Mini Kit (Qiagen) according to manufacturerâ??s instructions. RNA con-centration and quality of the samples was determined with an Agilent 2100 bioanalyser (Agilent Technologies) by measuring the RNA integrity number (RIN). All samples contained high quality RNA (RIN 9.5-10). DNA microarray analysis was performed with the Agilent Whole Human Genome Oligo Microar-ray 4x44K (G4112F) (Agilent Technologies Inc.) following the manufacturerâ??s protocol. Total RNA (1 ï?g) was labeled using the RNA input fluorescent linear amplification kit according to the manufacturer's recommendations (Agilent Technologies Inc.). RNA was hybridized on Agilent's Human 4x44K Oligo Microarrays containing 44000 features (41000 unique genes and transcripts). Pre-processing of the data was performed by Feature Extraction software (v 7.5.1).

Dataset Information

E-cadherin interactome complexity and robustness resolved by quantitative proteomics

Publications

E-cadherin interactome complexity and robustness resolved by quantitative proteomics.

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