De novo designed Hsp70 activator dissolves intracellular condensates

Jason Z Zhang; Nathan Greenwood; Jason Hernandez; Josh T Cuperus; Buwei Huang; Bryan D Ryder; Christine Queitsch; Jason E Gestwicki; David Baker

doi:10.1016/j.chembiol.2025.01.006

De novo designed Hsp70 activator dissolves intracellular condensates

Cell Chem Biol. 2025 Mar 20;32(3):463-473.e6. doi: 10.1016/j.chembiol.2025.01.006. Epub 2025 Feb 7.

Authors

Jason Z Zhang¹, Nathan Greenwood², Jason Hernandez³, Josh T Cuperus⁴, Buwei Huang², Bryan D Ryder³, Christine Queitsch⁵, Jason E Gestwicki³, David Baker⁶

Affiliations

¹ Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. Electronic address: jzz0428@uw.edu.
² Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA.
³ Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA.
⁴ Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
⁵ Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA; Brotman Baty Institute for Precision Medicine, Seattle, WA 98195, USA.
⁶ Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. Electronic address: dabaker@uw.edu.

Abstract

Protein quality control (PQC) is carried out in part by the chaperone Hsp70 in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of Hsp70 binding proteins that either inhibit or stimulate Hsp70 ATPase activity. An ATPase stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution and revealed roles as cell growth promoting signaling hubs. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to regulate condensates and other cellular targets.

Keywords: Hsp70; chaperones; condensates; protein design.

MeSH terms

Adenosine Triphosphatases / metabolism
HSP40 Heat-Shock Proteins* / chemistry
HSP40 Heat-Shock Proteins* / metabolism
HSP70 Heat-Shock Proteins* / chemistry
HSP70 Heat-Shock Proteins* / metabolism
Humans

Substances

HSP70 Heat-Shock Proteins
Adenosine Triphosphatases
HSP40 Heat-Shock Proteins

Abstract

MeSH terms

Substances

Grants and funding