Identification of proteins associated with Aha1 in HeLa cells by quantitative proteomics

• Interactions of 147 proteins with Aha1 were identified by LC-MS/MS.
• The interactions of 33 of the proteins with Aha1 were verified by Western blotting.
• The identified proteins are involved in many crucial and diverse cell processes.
• Aha1 participates in diverse biological pathways to modulate Hsp90 function.

The identification of the activator of heat shock protein 90 (Hsp90) ATPase’s (Aha1) protein-protein interaction (PPI) network will provide critical insights into the relationship of Aha1 with multi-molecular complexes and shed light onto Aha1’s interconnections with Hsp90-regulated biological functions. Flag-tagged Aha1 was over-expressed in HeLa cells and isolated by anti-Flag affinity pull downs, followed by trypsin digestion and identification co-adsorbing proteins by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). A probability-based identification of Aha1 PPIs was generated from the LC-MS/MS analysis by using a relative quantification strategy, spectral counting (SC). By comparing the SC-based protein levels between Aha1 pull-down samples and negative controls, 164 Aha1-interacting proteins were identified that were quantitatively enriched in the pull-down samples over the controls. The identified Aha1-interacting proteins are involved in a wide number of intracellular bioprocesses, including DNA maintenance, chromatin structure, RNA processing, translation, nucleocytoplasmic and vesicle transport, among others. The interactions of 33 of the identified proteins with Aha1 were further confirmed by Western blotting, demonstrating the reliability of our affinity-purification-coupled quantitative SC-MS strategy. Our proteomic data suggests that Aha1 may participate in diverse biological pathways to facilitate Hsp90 chaperone functions in response to stress.

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