De novo designed Hsp70 activator dissolves intracellular condensates

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.

Figure 1.. De novo design of Hsp70 binders/activators

(A) Experimental design. The driver of condensation (here, RIα) is fused with GFP. DnaJB1’s JD is fused to a GFP nanobody (GFPnb) and mCherry (mCh), and this tool is called condensate perturbator. (B) Expression of condensate perturbators using native JD partially dissolves GFP-tagged RIα puncta in HEK293T cells. Top: representative epifluorescence images of the various conditions tested. Scale bar, 10 μm. Bottom: quantification of number of RIα puncta per cell. Each point represents a single cell (n = 20 cells). Statistics are two-way Student’s t test. p values: *p < 0.05, **p < 0.01. (C) Structure models of native and designed Hsp70 complexes. Left: crystal structure of DnaK (Hsp70) with DnaJ (Hsp40) (PDB: 5NRO). Middle: AlphaFold structure prediction of DnaK with fully de novo protein designed to bind to the same region as DnaJ. Right: AlphaFold structure prediction of DnaK with partially redesigned DnaJ. (D) Design and biochemical characterization of fully de novo designed J-domain mimics (JDMs). Left: AlphaFold complex prediction of selected JDM with DnaK. Middle: representative trace of biolayer interferometry (BLI) measurements of JDM binding to either ATP or ADP-loaded Hsc70 (see STAR STAR Methods). Right: in vitro assays measuring ATP turnover by Hsc70 with either native DnaJA2 (Hsp40) or JDMs (see STAR Methods). Lines represent average of fluorescence quenching, which indicates ATP turnover (n = 3 experiments). Bottom: sequence alignment of the two JDMs presented here. (E) In vitro assays measuring the refolding of denatured luciferase where Hsc70 and either native DnaJA2 (Hsp40), E. coli DnaJ, or JDM37 fused to DnaJ without its native J-domain (JDM37-DnaJ^{no JD}) was added (see STAR Methods). Lines represent average luminescence recorded (n = 3 experiments). Dotted line represents the average luminescence level of native luciferase. Statistics are two-way ANOVA. p values: *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1–S6 and Table S1.

Figure 2.. De novo Hsp70 activator (JDM37) binds to and activates Hsp70 to dissolve intracellular condensates

(A) Experimental design. The driver of condensation (here, RIα) is fused with GFP. JDM37 is fused to a GFPnb and mCh (JDM37-GFPnb-mCh), and this tool is called condensate perturbator. (B) Expression of condensate perturbator using JDM37 dissolves GFP-tagged RIα puncta in HEK293T cells. Top: representative epifluorescence images of the various conditions tested in HEK293T cells transfected with the indicated constructs. 1 μM VER-155008 was added for 2 h prior to imaging. Scale bar, 10 μm. Bottom: quantification of number of RIα puncta per cell. Each point represents a single cell (n = 100 cells from 10 independent experiments). Statistics are one-way ANOVA. p values: ****p < 0.0001. (C) JDM recruits Hsc70 inside HEK293T cells. Left: representative epifluorescence images of the various conditions tested in HEK293T cells transfected with the indicated constructs. Scale bar, 10 μm. Right: quantitative analysis measuring the co-localization between RIα and Hsc70. Each point represents a separate biological replicate (n = 3 experiments, 51 cells in total for +JDM37-GFPnb, 47 cells in total for +JDM16-GFPnb, 45 cells in total for +GFPnb). Statistics are two-way Student’s t test. p values: *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Figure 3.. JDM37-mediated dissolution of endogenous condensates

(A) Experimental design. HEK293T cells with the 11^th β-strand of mNeonGreen inserted either at the 5′ or 3′ end of an endogenous gene locus and stably co-expressing remaining β-strands of mNeonGreen, thus allowing visualization of proteins expressed at their endogenous level. The proteins studied in this figure are proteins driving paraspeckle formation: NONO, PSPC1, SFPQ. The condensate perturbator here is JDM37-GFPnb-mCh. (B–D) Expression of JDM37-GFPnb-mCh dissolves GFP-tagged RIα puncta in HEK293T cells that enable visualization of endogenous NONO (293-NONO, B), PSPC1 (293-PSPC1, C), or SFPQ (293-SFPQ, D). Left: representative epifluorescence images of the various conditions tested. Scale bar, 10 μm. Right: quantification of number of paraspeckles per cell. Each point represents a single cell (n = 50 cells). Statistics are one-way ANOVA. p values: ****p < 0.0001. See also Figure S8.

Figure 4.. Dissolution of RIα condensates by de novo Hsp70 activator (JDM37) leads to global cAMP/PKA signaling increases

(A) Experimental design. The driver of condensation (here, RIα) is fused with mCherry. JDM37 is fused to an mCherry nanobody (mChnb) and mT-Sapphire (TSapphire), and this tool is called condensate perturbator. (B) Expression of engineered condensate perturbators dissolves mCherry-tagged RIα puncta in HEK293T cells (see STAR Methods for details). Top: representative epifluorescence images of the various conditions tested. Scale bar, 10 μm. Bottom: quantification of number of RIα puncta per cell. Each point represents a single cell (n = 100 cells from 10 independent experiments). For the quantification of number of puncta per cell, cells only with sufficient expression of the condensate perturbator were chosen for analysis (see STAR Methods for details). Statistics are one-way ANOVA. p values: *p < 0.05, ****p < 0.0001. (C and D) Time-course imaging of HEK293T cells expressing mCherry-tagged RIα, either cAMP sensor ICUE3 (C) or PKA sensor AKAR4 (D), and either JDM37-based or JDM16-based condensate perturbators. In each condition, 10 nM isoproterenol was added (n = at least 15 cells per curve). Statistics are two-way Student’s t test. p values: **p < 0.01. See also Figure S9.

Figure 5.. Dissolution of EML4-Alk condensates by JDM37 leads to decreased oncogenic signaling and cell growth

(A) Schematic of strategy to dissolve EML4-Alk oncogenic condensates. Protein that drives condensation (here, EML4-Alk) is tethered with mCherry. (B) Expression of engineered condensate perturbators dissolves mCherry-tagged EML4-Alk puncta in Beas2B cells (see STAR Methods for details). Top: representative epifluorescence images of the various conditions tested. Scale bar, 10 μm. Bottom: quantification of number of EML4-Alk puncta per cell. Each point represents a single cell (n = 100 cells from 27 independent experiments). For the quantification of number of puncta per cell, cells only with sufficient expression of the condensate perturbator were chosen for analysis (see STAR Methods for details). Statistics are one-way ANOVA. p values: ****p < 0.0001. (C) Raw FRET ratios of Beas2B cells expressing mCherry-tagged EML4-Alk, Ras sensor Ras-LOCKR-S, and either JDM37-based or JDM16-based condensate perturbators. Each point represents a single cell (n = 100 cells from 31 independent experiments). Statistics are two-way Student’s t test. p values: ****p < 0.0001. (D) Cell growth curves of Beas2B cells expressing mCherry-tagged EML4-Alk with or without condensate perturbators (n = 3 experiments). Line represents average from all 3 experiments. Statistics are two-way ANOVA. p values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S10.