Proteomic Exploration of Membrane Curvature Sensors Using a Series of Spherical Supported Lipid Bilayers

Membrane
curvature-sensing (MCS) proteins recognize and regulate
the morphologies of biological membranes. As these proteins lack characteristic
sequence motifs in their primary structure, they are not instantly
recognizable by genomic databases. Overcoming this technological challenge
toward the agile identification of new proteins can promote the elucidation
of membrane morphological regulation. Here, for the selective identification
of MCS proteins, comparative proteomic analysis was performed using
different sizes of the spherical supported lipid bilayer (SSLB), which
consists of spherical SiO2 particles covered with a lipid
bilayer. Because of the presence of SiO2 core, the curvature
of the surrounding membrane is well-controlled and stable even on
a micron scale. To prove this concept, known membrane curvature-sensing
protein domains, Bin/Amphiphysin/Rvs (BAR) and Epsin N-terminal homology
(ENTH), were evaluated by performing a binding assay using SSLBs,
and the preferential binding to the highly curved membrane was confirmed.
Peripheral membrane proteins obtained from normal human dermal fibroblast
(NHDF) and human breast cancer (MDA-MB-231) cells were used in shotgun
proteomic analysis, and 786 and 949 proteins were identified from
SSLBs as lipid membrane binders, respectively. Statistical quantitative
analyses of proteins detected from each SSLB with a different size
revealed 118 candidate proteins, including 23 proteins unique to MDA-MB-231
cells, as membrane curvature sensors, including some previously reported
curvature sensors. Functional clustering analysis based on the KEGG
orthology database revealed that the protein-binding property to specific
high or low membrane curvature correlated with their functions. Further
investigation of candidate proteins will lead to the identification
of new MCS proteins as well as cancer biomarkers.