The APE2 Exonuclease Is a Client of the Hsp70–Hsp90 Axis in Yeast and Mammalian Cells

Abstract

Molecular chaperones such as Hsp70 and Hsp90 help fold and activate proteins in important signal transduction pathways that include DNA damage response (DDR). Previous studies have suggested that the levels of the mammalian APE2 exonuclease, a protein critical for DNA repair, may be dependent on chaperone activity. In this study, we demonstrate that the budding yeast Apn2 exonuclease interacts with molecular chaperones Ssa1 and Hsp82 and the co-chaperone Ydj1. Although Apn2 does not display a binding preference for any specific cytosolic Hsp70 or Hsp90 paralog, Ssa1 is unable to support Apn2 stability when present as the sole Ssa in the cell. Demonstrating conservation of this mechanism, the exonuclease APE2 also binds to Hsp70 and Hsp90 in mammalian cells. Inhibition of chaperone function via specific small molecule inhibitors results in a rapid loss of APE2 in a range of cancer cell lines. Taken together, these data identify APE2 and Apn2 as clients of the chaperone system in yeast and mammalian cells and suggest that chaperone inhibition may form the basis of novel anticancer therapies that target APE2-mediated processes.

A high-throughput exonuclease assay based on the fluorescent base analog 2-aminopurine

“…More than ten exo- and endonucleases are active in different forms of mammalian DNA repair (reviewed in 38 ). Furthermore, upregulation of EXO1 is associated with increased malignancies and correlated with a poor prognosis 39,40 , whereas the inhibition of APE2 has shown to reduce growth and malignancies 41,42 . Finally, also ExoN, the replicative exonuclease of the SARS-CoV-2 virus is the target of novel promising antiviral drugs 43 .…”

High-Throughput Exonuclease Assay Based on the Fluorescent Base Analogue 2-Aminopurine

“…More than 10 exo- and endonucleases are active in different forms of mammalian DNA repair (reviewed in ref ). Furthermore, upregulation of EXO1 is associated with increased malignancies and correlated with a poor prognosis, , whereas the inhibition of APE2 has shown to reduce growth and malignancies. , Finally, ExoN, the replicative exonuclease of the SARS-CoV-2 virus, is also the target of novel promising antiviral drugs …”

“…Furthermore, upregulation of EXO1 is associated with increased malignancies and correlated with a poor prognosis, 44,45 whereas the inhibition of APE2 has shown to reduce growth and malignancies. 46,47 Finally, ExoN, the replicative exonuclease of the SARS-CoV-2 virus, is also the target of novel promising antiviral drugs. 48 To aid in the search for novel exonuclease inhibitors, we have developed a simple and versatile exonuclease assay that can be used for both 3′−5′ and 5′−3′ exonucleases, as well as bacterial and human exonucleases.…”

Insights into Hsp90 mechanism and in vivo functions learned from studies in the yeast, Saccharomyces cerevisiae

“…Hsp90 interacts with multiple proteins involved in rRNA processing and ribosomal biogenesis and function ( Franzosa et al, 2011 ; Tenge et al, 2014 ; Kolhe et al, 2023 ). Hsp90 also has many roles in the nucleus, including transcription by RNA polymerase II, chromatin remodeling and organization, and DNA repair ( Flom et al, 2005 ; Gribun et al, 2008 ; Zhao et al, 2008 ; DeZwaan et al, 2009 ; Truman et al, 2015 ; Echtenkamp et al, 2016 ; Gvozdenov et al, 2019 ; Wang et al, 2020 ; Omkar et al, 2022 ). Hsp90 also has demonstrated roles in the cell cycle and the secretory pathway ( McClellan et al, 2007 ).…”