Thermal proteome profiling monitors ligand interactions with cellular membrane proteins

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Thermal proteome profiling (TPP) is a method to measure changes in protein thermal stability.
• TPP has been primarily used to study soluble proteins.
• Detecting interactions with membrane proteins presents unique challenges.

Purpose of the Study:

• To adapt and apply thermal proteome profiling for the detection of transmembrane protein-small molecule interactions.
• To investigate the effects of detergents on ATP-binding profiles of transmembrane proteins.
• To analyze cellular thermal shifts in response to drug treatments affecting membrane proteins.

Main Methods:

• Utilized thermal proteome profiling in cultured human cells.
• Assessed changes in protein denaturation temperatures upon small molecule treatment.
• Investigated ATP-binding profiles and cellular thermal shifts in response to specific signaling pathways and drugs.

Main Results:

• Observed shifts in denaturation temperatures for ATP-binding transmembrane proteins when assessing detergent effects.
• Detected cellular thermal shifts in T cell-receptor signaling, identifying the membrane target CD45 and downstream pathway components.
• Identified interactions with transmembrane transporters ATP1A1 and MDR1 upon drug treatment.

Conclusions:

• Thermal proteome profiling can be effectively extended to study transmembrane protein-small molecule interactions.
• The method allows for the delineation of membrane targets and associated signaling pathways.
• This approach provides insights into drug mechanisms of action on membrane proteins.