RESEARCH
Understanding the Hsp70 chaperone code
Molecular chaperones keep the proteome folded and functional, but they do not act alone. Chaperones like Hsp70 are themselves heavily regulated, and that regulation reaches into almost every part of cell biology. Our lab studies a fundamental question: how are chaperone interactions changed by post-translational modifications? We work across purified proteins, budding yeast, and human cancer cells, using genetics, biochemistry, structural proteomics, and mass spectrometry.
Over the past decade, my laboratory has helped establish the concept of the Hsp70 chaperone code, demonstrating that site-specific post-translational modifications (PTMs) can reprogram chaperone activity, protein interactions, and cellular outcomes. Our early work revealed that phosphorylation of a single conserved residue on Hsp70 is sufficient to alter global chaperone function and cell cycle progression. Since that time, the number of identified PTMs on Hsp70 has expanded dramatically, yet our understanding of how these modifications are integrated into cellular signaling remains limited.
The central direction of our research program is to define how the Hsp70 chaperone code functions as a regulatory system that integrates diverse cellular signals to control proteostasis and signaling networks. Moving forward, we will expand beyond individual PTM-focused studies to develop a systems-level understanding of how combinations of modifications regulate Hsp70 activity across physiological contexts. In parallel, we are beginning to explore how PTMs on client proteins (“client code”) influence chaperone engagement, opening new directions at the interface of proteostasis and signaling.
Understanding the fundamental role of chaperone phosphorylation using chaperone code arrays
Our previous studies began with a top-down approach, observing the modulation of Hsp70 phosphorylation in response to unique stresses. While these studies have provided fascinating insights into individual stress responses, there remains a vast number of phosphorylation sites on Hsp70 with no known function. To clarify the role of these sites, we have developed a collection of 146 yeast strains containing single mutations in all 73 known phosphorylation sites of Ssa1 to either phospho-mutant (alanine) or phospho-mimic status (aspartic acid or glutamic acid). In this strain, Ssa1 is the sole Hsp70, providing cell viability.
We are currently creating a phenotypic fingerprint for each of the 146 chaperone code mutants. Our goal is to generate fitness profiles for chaperone code mutants across these stressors and cluster mutants based on the similarity of their phenotypic fingerprint. We have now developed equivalent arrays for other chaperones and chaperones including Kar2, Ydj1, Sis1 and Cdc37.
Understanding the role of Hsp70 phosphorylation in ALS
Phase transition and aggregation of the TAR DNA-binding protein (TDP-43) is associated with neurodegenerative disorders such as frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS) As such, TDP-43 represents a promising target for novel FTD and ALS therapeutics. However, key elements of TDP-43 regulation remain poorly understood, including how cells maintain TDP-43 in a soluble and functional state. Recent studies have implicated molecular chaperones such as Hsp70 in regulating the aggregation of TDP-43, but the exact mechanism and signaling behind this is not understood. We are currently using our chaperone code array technologies to identify chaperone PTMs that promote the destruction of TDP-43.
Understanding the role of chaperones in ribonucleotide reductase function in cancer
Ribonucleotide reductase (RNR) makes the dNTPs that cells need to replicate and repair DNA. It is also the target of widely used chemotherapies such as hydroxyurea and gemcitabine. Using proteomics to follow chaperone interactions after DNA damage, we discovered that RNR is a chaperone client. We later showed that the Hsp70 co-chaperone Ydj1, the yeast counterpart of human DNAJA1, is needed for RNR stability and activity. We also found that different Hsp70 paralogs regulate RNR through their C-terminal domains.
These findings have a direct clinical angle. A chemogenomic screen identified DNAJA1 as a hub for anticancer drug resistance: removing DNAJA1 makes cancer cells far more sensitive to a broad range of drugs. We are now using structural proteomics, biophysics, and cell biology to work out exactly how DNAJA1 engages and matures the RNR small subunit. We also want to learn whether targeting this interaction could make existing chemotherapies more effective.
Understanding the client code
Our work suggests that chaperone interactions are regulated not only by modifications on the chaperones themselves, but also by post-translational modifications on their client proteins. Our 2023 study published in PLOS Biology identified over a hundred direct interactions of Hsp70 that contained a PTM at the interaction interface.
We refer to this complementary regulatory system as the “Client Code.” We are investigating how phosphorylation and other client modifications control recognition by Hsp70 and Hsp90, and how these signals determine whether a client is folded, activated, localized or degraded. Together, the Chaperone Code and Client Code may provide a broader framework for understanding how cells dynamically control protein fate.
