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Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation

Nature volume 524, pages 247–251 (2015)Cite this article

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Abstract

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

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Figure 1: Simultaneous presence of class A and B J-proteins unleashes protein disaggregation activity and broadens target aggregate range of the HSP70 machinery.
Figure 2: Intermolecular JD–CTD interaction is required for mixed-class J-protein complex formation.
Figure 3: Conserved electrostatic potential distributions in A and B J-protein classes are complementary and direct mixed-class J-protein interactions for complex formation.
Figure 4: Model of individual versus complexed class A and class B J-protein function in protein disaggregation.

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Acknowledgements

We thank A. Mogk for critical reading of the manuscript and S. Ungelenk for Hsp26. This work was funded by the Deutsche Forschungsgemeinschaft (SFB1036, BU617/19-1 to B.B.; EXC257, SFB740 to J.K.), Alexander von Humboldt Foundation Postdoctoral Fellowships (to N.B.N. and A.Sz.), National Institutes of Health (the NIGMS, NIA, NIMS), Ellison Medical Foundation and Daniel F. and Ada L. Rice Foundation (to R.I.M.), German Federal Ministry of Education and Research (BMBF) Virtual Liver Network and EU FEP Flagship Programme Human Brain Project (0315749, 604102 to R.C.W.), Klaus Tschira Foundation (to M.B., A.St. and R.C.W.), Sir Henry Wellcome Postdoctoral Fellowship (to F.S.), ETH Zurich and ERC advanced grant Proteomics v3.0 (233226 to R.A.).

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Authors and Affiliations

  1. Center for Molecular Biology of the University of Heidelberg (ZMBH), German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, 69120, Germany

    Nadinath B. Nillegoda, Anna Szlachcic, Xuechao Gao, D. Lys Guilbride, Rebecca C. Wade, Matthias P. Mayer & Bernd Bukau

  2. Leibniz-Institute for Molecular Pharmacology (FMP), Berlin, 13125, Germany

    Janine Kirstein, Kristin Arnsburg & Annika Scior

  3. Heidelberg Institute for Theoretical Studies (HITS), Heidelberg, 69118, Germany

    Mykhaylo Berynskyy, Antonia Stank & Rebecca C. Wade

  4. Heidelberg Graduate School of Mathematical and Computational Methods for the Sciences, Heidelberg University, Heidelberg, 69120, Germany

    Antonia Stank

  5. Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, 8093, Switzerland

    Florian Stengel & Ruedi Aebersold

  6. Faculty of Science, University of Zurich, Zurich, 8057, Switzerland

    Ruedi Aebersold

  7. Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, Heidelberg, 69120, Germany

    Rebecca C. Wade

  8. Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, Evanston, 60208, Illinois, USA

    Richard I. Morimoto

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  1. Nadinath B. Nillegoda
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Contributions

N.B.N. and B.B. conceived the study. N.B.N., J.K., A.Sz., M.B., A.St., F.S., D.L.G., R.C.W., M.P.M. and B.B. designed the experiments. N.B.N., J.K., A.Sz., M.B., A.St., F.S., K.A., X.G. and A.Sc. performed the experiments. N.B.N., J.K., A.Sz., M.B., A.St., F.S., R.A., R.C.W., R.I.M., D.L.G., M.P.M. and B.B. analysed the data. N.B.N., D.L.G., M.P.M. and B.B. wrote the manuscript.

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Correspondence to Nadinath B. Nillegoda or Bernd Bukau.

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Extended data figures and tables

Extended Data Figure 1 Characterization of protein disaggregation/refolding and refolding-only reactions.

a, HSP70–J-protein–HSP110 (HSPA8–J-protein–HSPH2) functional cycle. Concomitant interaction of HSP70 with a J-protein and substrate results in allosteric stimulation of ATP hydrolysis; this traps the substrate in HSP70 (ref. 8). Subsequent NEF (for example, HSP110) promoted ADP dissociation from HSP70, then allows ATP rebinding, which triggers substrate release to complete the cycle54,55. b, Scheme for in vitro disaggregation/refolding and refolding-only reactions. The aggregates used in disaggregation/refolding assays are preformed by heating luciferase with yeast small heat-shock protein (sHSP) Hsp26 (ref. 4), which is known to co-aggregate with misfolded proteins in vivo27,28 (see Methods for detailed description). If HSP70, J-protein and HSP110 are instead heated together with substrate and Hsp26, luciferase is denatured into a more easily refoldable, inactive and largely monomeric substrate form used in refolding-only assays. c, SEC profiles of aggregated 3H-labelled luciferase (black; size range 200 kDa to ≥5,000 kDa representing ∼2 to >50 aggregated luciferase molecules) and monomeric native luciferase (red; size ∼63 kDa). Arrows indicate elution size (kDa). Inset, activity of loaded material. d, SEC profile of partially denatured and largely monomeric luciferase (starting material for refolding-only reactions). Inset, activity of loaded material. e, Chaperone nomenclature. f, Disaggregation and reactivation of preformed luciferase aggregates using human HSP70–HSP110 with human J-proteins JA2, green; JB1, blue; JA2+JB1, magenta or no J-protein, black. Under limiting chaperone (HSP70/HSP110) and increasing J-protein concentrations (A, solid or B, dashed) (n = 3). Data are mean ± s.e.m. Precise concentrations are shown in Extended Data Table 1.

Extended Data Figure 2 Effects of mixed-class J-proteins on disaggregation/refolding and refolding-only activity of the HSP70 system.

a, Disaggregation/refolding of aggregated luciferase compared for human class A (JA1 and JA2) and class B (JB1 and JB4) J-proteins (n = 3). b, Luciferase refolding-only compared for JA1, JA2, JB1 and JB4 (n = 3). c, Reactivation of heat-aggregated luciferase with nematode HSP70 machinery, using reduced substrate:HSP70 ratio of 1:20, containing DNJ-12 (A), DNJ-13 (B) or DNJ-12+DNJ-13 (A+B) (n = 2). d, Disaggregation/refolding of luciferase using human HSP70 and HSP110 combined with nematode J-proteins (n = 3). e, Reactivation of luciferase showing optimal JA2:JB1 ratio for disaggregation/refolding (n = 2). f, Initial disaggregation/refolding rates for e. g, Final yields of refolded luciferase (120 min) for e. Data are mean ± s.e.m. Precise concentrations are shown in Extended Data Table 1.

Extended Data Figure 3 Disaggregation synergy is independent of sHSP incorporation, NEF, substrate and aggregate character, and is not explained by sequential J-protein class activity.

a, Disaggregation/refolding reaction for luciferase aggregates without incorporating sHSP Hsp26 (n = 3). b, Reactivation without NEF (HSPH2) (n = 3). c, Reactivation of α-glucosidase aggregates (n = 3). d, Reactivation of preformed MDH aggregates in the presence of GroEL plus the GroES protein foldase system (GroELS) (n = 2). GroELS is required for efficient MDH refolding56. GroELS alone is in black. JB1:JA2 denotes the stoichiomety of each reaction. e, Disaggregation/refolding of stringent aggregates (≥5,000 kDa) formed using 2 μM luciferase (n = 3). f, Disaggregation/refolding of aggregated luciferase at reduced substrate:HSP70 ratio (luciferase:HSP70:J-protein:HSP110 = 1:7.5:3.8:0.4) (n = 3). The aggregated luciferase concentration is 100 nM. g, h, Holdase function of J-proteins (class A (g) and class B (h)) during luciferase aggregation at 42 °C, shown by decreased light scattering. Concentrations: 1× luciferase; 4× J-protein; 4× BSA (control) (n = 2). i, Reactivation with sequential JA2 and JB1 addition. J-protein added at t = 0 min (black graph legends); J-protein added after 30 or 60 min (red graph legends and arrows) (n = 2). Data are mean ± s.e.m. Precise concentrations are shown in Extended Data Table 1.

Extended Data Figure 4 Stoichiometry of class A and B J-proteins determines the range of aggregate sizes resolved.

a, The GroEL trap (GroELD87K) facilitates the capture of 3H-luciferase monomers liberated by protein disaggregation before the refolding step. b, Refolding of disaggregated 3H-luciferase monomers (40 min) in the absence (solid bars) and presence of GroEL trap (open bars). c, SEC profile after disaggregation/refolding of aggregated tritiated α-glucosidase (60 min) with either J-protein class alone (green (A) or blue (B)) or J-proteins combined (magenta). Control reaction without chaperones (black). Elution fractions F1–F4 (red lines). Table shows size distribution of aggregates in each fraction; F1 luciferase aggregates ≥4,000 kDa; F2, aggregates ∼400–4,000 kDa; F3, aggregates ∼150–400 kDa, F4 disaggregated monomers (∼68 kDa). d, Quantification of SEC profile measuring disaggregation of tritiated α-glucosidase from aggregates (F1–F3) from c, also showing concomitant accumulation of disaggregated monomer (F4) from c (n = 3). e, ATP depletion by apyrase abrogates disaggregation. f, Quantification of SEC profile measuring disaggregation of tritiated luciferase from aggregates (F1–F3) with concomitant accumulation of disaggregated monomer (F4), using the HSP70–HSP110 system with JA2 or JB1 alone, or with JA2 plus JB1. Stoichiometry range used for JA2:JB1, 1:1 to 4:1 to 1:4. Specifically, 0.2 JB1:0.8 JA2 (orange); 0.2 JA2:0.8 JB1 (red). Solid colours denote 40-min reaction time; hash denotes 120 min. Control reaction without chaperones (black). Two-tailed t-test, *P < 0.05, **P < 0.01 (n = 3). Data are mean ± s.e.m. Precise concentrations are shown in Extended Data Table 1.

Extended Data Figure 5 JA2 and JB1 form homodimers and interact transiently.

a, Identified JA2 and JB1 inter-molecular cross-links; ‘Id’, amino acid sequence of peptides showing cross-linked lysines (K, orange). Protein 1 and 2 denote source proteins for cross-linked peptides; position 1 and 2 denote positions of cross-linked lysines within proteins; deltaS is the delta score for each crosslink; cut-off = 0.9. ld-Score is the linear discriminant score. b, Representative mass spectrometry spectra for inter-molecular JA2 and JB1 cross-links. Common peaks, green; cross-linked, red; matched peaks, diamonds (no peaks above 1,100 m/z detected). c, SEC profiles of 3H-labelled JA2 dimer (green cartoon) and 3H-labelled JB1 dimer (blue cartoon) mixed with unlabelled J-protein from the other class. Precise concentrations are shown in Extended Data Table 1.

Extended Data Figure 6 Electrostatic interactions between J-domain and CTD predominate in JA2 and JB1 complexes.

a, FRET efficiencies for JD–CTD and CTD–CTD interactions with 0–0.2% Tween20 titration. Percentage efficiency is relative to untreated (0% Tween20) samples. Donor quenching (black); acceptor fluorescence (red); below, fluorophore positions in J-protein protomers (JA2, green; JB1, blue). N-termini of JDJA2 and JDJB1 labelled with acceptor fluorophore ReAsH. CTDJA2 and CTDJB1 labelled with donor fluorophores FlAsH and Alexa Fluor 488 at residues 241 and 278, respectively. b, Disaggregation/refolding of preformed luciferase by JA2 and/or JB1 with increasing amounts of Tween20 (n = 2). c, FRET efficiencies for JA2 and JB1 interactions at increasing salt concentrations. d, Disaggregation/refolding of preformed luciferase aggregates by JA2 and JB1 with increasing salt concentrations; control, 50 mM salt, no chaperones (n = 2). e, Luciferase disaggregation/refolding in the presence of excess J-domain fragments carrying JD-QPNJA2/JB1 mutation of the HPD motif (n = 3). f, g, FRET between class A and B J-proteins. f, Competition with unlabelled full-length wild-type J-protein (FL); unlabelled competitor is 1–10× acceptor; (−), no competitor. g, Competition with unlabelled isolated JDJA2 and JDJB1. Data are mean ± s.e.m., average of at least two experiments for FRET experiments. Precise concentrations are shown in Extended Data Table 1.

Extended Data Figure 7 In silico prediction of JD–CTD interactions between class A and B J-proteins and in vitro evidence that physical interactions between J-proteins do not overlap J-protein substrate binding sites.

a, Preferred positions of the centres of geometry (CoG) of J-domains (y axis, JA1, JA2, JB1 and JB4) around CTD dimers (x axis, class A, green, class B, blue) obtained from molecular docking simulations. JDJA1/JB1, wireframe meshes; JDJA2/JB4, brown contours, each contoured at the isovalue given in the top left of each image. The higher scores for class A CTDs indicate greater specificity of the complexes formed with J-domains; the lower scores for class B CTDs indicate much less specific interactions. Lysines in inter- and intra-J-protein JA2–JB1 cross-links, orange spheres. b, Properties of the docking arrangements obtained after clustering. Total number of clusters per simulation, denominator; number of selected clusters (corresponding to 90% of all docked complexes), numerator, bold. In parentheses, the range of average energy values (in units of kT) for the selected clusters. Lower energy values indicate more favourable binding; fewer clusters indicate a more defined binding mode (see Methods). JDJA2 docking to CTDJB1 is much weaker and less specific than JDJB1 docking to CTDJA1, but docking arrangements compatible with cross-linking results still obtain (Fig. 2d). c, Competition of isolated JDJA2 fragments against JA2 holdase function in luciferase aggregation at 42 °C (n = 2). d, Competition of isolated JDJB1 fragments against JA2 holdase function (n = 2). Luciferase, 1×; JA2, 4×; isolated J-domain fragments, 20× (red; 5-fold excess over JA2), or 40× (orange; 10-fold excess over JA2). Light scattering measured at 600 nm. Precise concentrations are shown in Extended Data Table 1.

Extended Data Figure 8 Possible configurations of the JA2–JB1 mixed-class complex.

a, Compact configuration. b, Open configuration. Configurations were derived from computational docking, using constraints from experimental FRET and cross-linking data (Fig. 2a–d and Extended Data Fig. 5a). Each configuration is shown from two views (left and right) rotated by 135 degrees with respect to each other and in ribbon (top) and molecular surface (bottom) representations. In both cases J-domains of JA2 dock onto the CTD dimer of JB1, and similarly J-domains of JB1 dock to the CTD dimer of JA2. Both CTDJB1 protomers are within cross-linking distance of CTDJA2. Unstructured glycine/phenylalanine (G/F)-rich flexible regions connecting J-domains and CTDs shown by dark blue (JB1) or green (JA2) dashed lines. Residues at FRET fluorophore sites are shown in space-filling representation (red on JA2, magenta on JB1). Inter-molecular crosslinking lysine pairs (gold and cyan, space-filling) are connected by dotted lines. Bottom left within a: molecular surface representation of compact configuration of the JA2–JB1 complex, showing substrate binding sites from crystallographic24 (yellow) and biochemical57 (orange, cyan) data. HPD motif, red. Residues implicated in JD–HSP70 interactions19,58,59 (dark teal and dark green on JDJA2; purple and dark blue on JDJB1). Bottom right within a: rotated image. Table shows fluorophore separation distances; calculated percentage FRET efficiencies in parentheses. a, Both CTDJB1 protomers are within cross-linking distance of CTDJA2. b, As in a, but with only a single CTDJB1 protomer within cross-linking distance to CTDJA2; one JDJA2 docks onto CTDJB1, the other JDJA2 is free. Similarly, one JDJB1 docks onto CTDJA2, the other JDJB1 docks onto its own CTD, consistent with SAXS-determination of class B J-proteins16,17. Model of JB1 (blue) based on the crystal structure of CTD and NMR structure of J-domain. Homology model of JA2 (green) based on the crystal structure of Ydj1 (see Methods).

Extended Data Table 1 Tabulated reaction conditions
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Nillegoda, N., Kirstein, J., Szlachcic, A. et al. Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature 524, 247–251 (2015). https://doi.org/10.1038/nature14884

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