Tumor protein D54 binds intracellular nanovesicles via an extended amphipathic region

Area of Science:

• Cell biology
• Protein biochemistry
• Biophysics

Background:

• Tumor protein D54 (TPD54) is a cytosolic protein overexpressed in cancers.
• TPD54 interacts with small (30 nm) cytosolic vesicles, distinct from classical transport vesicles.
• The precise function of TPD54 and its interaction mechanism with vesicles remain largely unknown.

Purpose of the Study:

• To elucidate the mechanism by which TPD54 captures intracellular nanovesicles.
• To investigate the structural changes in TPD54 upon membrane binding.
• To identify the specific regions of TPD54 responsible for nanovesicle interaction.

Main Methods:

• Bioinformatical analysis of TPD54 structure.
• Limited proteolysis, CD spectroscopy, and tryptophan fluorescence assays.
• Cysteine mutagenesis and membrane-sensitive probe binding experiments.
• Site-directed mutagenesis and liposome binding assays.

Main Results:

• TPD54 undergoes significant structural alterations in its amphipathic helices upon binding to small liposomes.
• Amphipathic helices AH2 and AH3 are crucial for TPD54 membrane binding in vitro and in cells.
• TPD54's AH3 motif functions as an amphipathic lipid packing sensor (ALPS), sensitive to membrane curvature and lipid unsaturation.
• TPD54 binding to liposomes demonstrates high sensitivity to membrane curvature and lipid unsaturation.

Conclusions:

• TPD54 utilizes both ALPS-dependent and ALPS-independent mechanisms for nanovesicle recognition.
• The ALPS motif in AH3 plays a key role in sensing and binding to specific nanovesicle characteristics.
• TPD54's interaction with nanovesicles is a complex process involving structural flexibility and specific membrane sensing capabilities.