Yu et al. 2021

A Chemoproteomics Approach to Profile Phospholipase D-Derived Phosphatidyl Alcohol Interactions

Published

December 15, 2021

Authors

Weizhi Yu, Zhi Lin, Christina M. Woo, Jeremy M. Baskin

Journal

ACS Chemical Biology

https://doi.org/10.1021/acschembio.1c00584

Abstract

Alcohol consumption leads to formation of phosphatidylethanol (PEth) via the transphosphatidylation activity of phospholipase D (PLD) enzymes. Though this non-natural phospholipid routinely serves as a biomarker of chronic alcoholism, its pathophysiological roles remain unknown. We use a minimalist diazirine alkyne alcohol as an ethanol surrogate to generate clickable, photoaffinity lipid reporters of PEth localization and lipid–protein interactions via PLD-mediated transphosphatidylation. We use these tools to visualize phosphatidyl alcohols in a manner compatible with standard permeabilization and immunofluorescence methods. We also use click chemistry tagging, enrichment, and proteomics analysis to define the phosphatidyl alcohol interactome. Our analysis reveals an enrichment of putative interactors at various membrane locations, and we validate one such interaction with the single-pass transmembrane protein basigin/CD147. This study provides a comprehensive view of the molecular interactions of phosphatidyl alcohols with the cellular proteome and points to future work to connect such interactions to potential pathophysiological roles of PEth.

Lipid probes utilized

Phosphatidyl Alcohol Phosphatidyl diazirine alkyne alcohol

Cell line analyzed

HeLa

Uncaging & Crosslinking timeline

Mass spectrometry quantification method

PSM

Data visualization

Volcano plots depict the relative enrichment of each protein versus non-crosslinked control (x-axis) and the statistical significance of each protein following a Student’s t-test (y-axis, -log10 transformed). Black proteins are designated as not statistically significant. Orange “enriched hit” denotes proteins with fold-changes > 1.5 (logFC > 0.5) pvalue < 0.05. Only proteins identified in all three replicates are displayed.

Data as reported in Yu et al., 2021, 2024. Black proteins are designated as not statistically significant. Orange “enriched hit” denotes proteins with fold-changes > 1.5 (logFC > 0.5) pvalue < 0.05. Only proteins identified in all three replicates are displayed.

Data as reported in Yu et al., 2021. Black proteins are designated as not statistically significant. Orange “enriched hit” denotes proteins with fold-changes > 1.5 (logFC > 0.5) pvalue < 0.05. Only proteins identified in all three replicates are displayed.

Gene Ontology Analysis

In beta: GO analysis still under development

No enriched molecular functions identified among enriched hits and candidates.

No enriched biological processes identified among enriched hits and candidates.

Figure 1: GO Dot plots display the enrichment of GO terms among the proteins enriched to the probe. For these analyses, only proteins categorized as “enriched candidates” and “enriched hits” were subject to GO analysis. The Cell Compartment analysis assesses whether the list of enriched proteins contains a statistically significant number of proteins in the same cellular region; appropriately, the Molecular Function and Biological Process analyses does the same for molecular function and biological process, respectively. Click here for more information about Gene Ontology Analysis.

Data exploration


Data download