Identification of Helicase Proteins as Clients for HSP90

The
90-kDa heat shock protein (HSP90) is a molecular chaperone
that maintains the proper folding of its client proteins including
protein kinases and steroid hormone receptors. Helicases are a group
of nucleic acid-binding ATPases that can unwind DNA and/or RNA and
function in almost every aspect of nucleic acid metabolism. Not much,
however, is known about the interactions between HSP90 and helicase
proteins. Herein, we developed a parallel-reaction monitoring (PRM)-based
targeted proteomic method that allows for quantifying >80% of the
human helicase proteome. By employing this method, we demonstrated
that a large number of helicase proteins exhibited diminished expression
in cultured human cells upon treatment with two small-molecule inhibitors
of HSP90. We further introduced a tandem affinity tag to the C-terminus
of endogenous HSP90β protein by using the CRISPR-Cas9 genome
editing method. Affinity purification followed by LC-PRM analysis
revealed an enrichment of 40 out of the 66 quantified helicases from
the lysate of cells expressing tagged HSP90β. Together, we developed
a high-throughput targeted proteomic method for assessing quantitatively
the human helicase proteome, and our results support that helicases
may constitute an important group of client proteins for HSP90.