Metazoan Hsp70 machines use Hsp110 to power protein disaggregation

Abstract

Accumulation of aggregation-prone misfolded proteins disrupts normal cellular function and promotes ageing and disease. Bacteria, fungi and plants counteract this by solubilizing and refolding aggregated proteins via a powerful cytosolic ATP-dependent bichaperone system, comprising the AAA+ disaggregase Hsp100 and the Hsp70-Hsp40 system. Metazoa, however, lack Hsp100 disaggregases. We show that instead the Hsp110 member of the Hsp70 superfamily remodels the human Hsp70-Hsp40 system to efficiently disaggregate and refold aggregates of heat and chemically denatured proteins in vitro and in cell extracts. This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP-driven chaperoning is not required. Knock-down of nematode Caenorhabditis elegans Hsp110, but not an unrelated nucleotide exchange factor, compromises dissolution of heat-induced protein aggregates and severely shortens lifespan after heat shock. We conclude that in metazoa, Hsp70-Hsp40 powered by Hsp110 nucleotide exchange represents the crucial disaggregation machinery that reestablishes protein homeostasis to counteract protein unfolding stress.

Figure 1

Chemically aggregated luciferase is efficiently reactivated by the human Hsp70 system including Hsp110 as NEF. (A) Luciferase levels in supernatants and pellets of native or chemically aggregated luciferase samples. Data shown represent the average of at least three experiments±s.e. (B) Reactivation of 20 nM luciferase from urea-induced aggregates was monitored upon addition of the human Hsp70-Hsp40 (2 μM Hsc70, 1 μM Hdj1) or Hsp70-Hsp40-Hsp110 system (Hsc70, Hdj1 and 0.2–0.4 μM Hsp105, Apg2 or Apg1). (C) Reactivation of chemically aggregated luciferase by the yeast Hsp70-Hsp40-NEF system (2 μM Ssa1, 1 μM Ydj1±0.1 μM Sse1 or 2 μM Snl1ΔN) or bichaperone system including the disaggregase Hsp104 (Ssa1, Ydj1, 1 μM Hsp104±Sse1). Reactivation data shown represent the average of at least three experiments±s.e.

Figure 2

The disaggregation activity of the human Hsp70-Hsp40-Hsp110 extends to heat-aggregated luciferase as well as MDH. (A) Luciferase levels in supernatants and pellets of native or thermally aggregated luciferase samples. (B) Luciferase (2 μM) was heat aggregated in the presence of Hsp26, diluted to 20 nM luciferase and subsequently, reactivation was monitored upon addition of the human Hsp70 system±Hsp110 (2 μM Hsc70, 1 μM Hdj1 and 0.4 μM Apg2). (C) Reactivation of heat-aggregated luciferase treated as in (B) was monitored upon addition of the yeast Hsp70-Hsp40-NEF system (2 μM Ssa1, 1 μM Ydj1±0.1 μM Sse1 or 2 μM Snl1ΔN) or bichaperone system including the disaggregase Hsp104 (Ssa1, Ydj1, 1 μM Hsp104±Sse1). (D) Luciferase (20 nM) was heat aggregated in the presence of Hsp26 and reactivation was monitored upon addition of the human Hsp70 system (2 μM Hsc70, 1 μM Hdj1±0.2 μM Apg2). (E) Luciferase (20 nM) was heat aggregated in the presence of Hsp26 and reactivation was monitored upon addition of the yeast Hsp70 or bichaperone system (2 μM Ssa1, 1 μM Ydj1±0.2 μM Sse1, ±1 μM Hsp104). (F) MDH (0.5 μM) was thermally aggregated and, after four-fold dilution, reactivation was monitored upon addition of the human Hsp70-Hsp40-NEF system (4 μM Hsp70, 2 μM Hdj1±0.4 μM Apg2). Reactivation data shown represent the average of at least three experiments±s.e.

Figure 3

The human Hsp70-Hsp40-Hsp110 system resolubilizes aggregates from cell lysates containing EGFP-luciferase. (A) EGFP-luciferase levels in supernatants and pellets of native or heat-shocked cell lysates after incubation at 30°C in the absence of chaperones or in the presence of 2 μM Hsc70, 1 μM Hdj1 and 0.2 μM Apg2 or the E. coli bichaperone system consisting of Hsp70, Hsp40, NEF and Hsp100-type disaggregase (2 μM DnaK, 0.4 μM DnaJ, 0.2 μM GrpE (DnaKJE) and 1 μM ClpB). (B) Reactivation of heat-aggregated EGFP-luciferase after 4 h at 30°C in the absence or presence of the indicated chaperones. Concentrations were as indicated above, the yeast chaperones were added at the following concentrations: Ssa1 2 μM, Ydj1 1 μM, Sse1 0.2 μM, Hsp104 1 μM. Aside from the last combination for which one representative data set out of two is shown, reactivation data shown represent the average of at least four experiments±s.e.

Figure 4

(A) The Apg2 mutant Apg2-N619Y/E622A is NEF deficient. Dissociation rates of MABA-ADP from 0.5 μM Hsc70 were determined upon addition of 1 μM Apg2 or Apg2-N619Y/E622A. Data represent the average of six experiments±s.d. (B) The Apg2 mutant Apg2-N619Y/E622A is deficient in complex formation with Hsc70. Interaction between mutant or WT Apg2 and His₆-Smt3-Hsc70 was tested by a pulldown on Co-IDA, followed by elution with Ulp1 protease. (C) The Apg2 variant Apg2-N619Y/E622A does not support efficient disaggregation of chemically aggregated luciferase. Reactivation of 20 nM luciferase from urea-induced aggregates was monitored upon addition of 2 μM Hsc70 and 1 μM Hdj1 in the presence or absence of 0.2 μM Apg2 or Apg2-N619Y/E622A. Reactivation data represent the average of at least four experiments±s.e. (D) The stimulatory effect of Apg2 on disaggregation is strongly concentration dependent. Reactivation of 20 nM luciferase from heat-induced aggregates obtained at a concentration of 2 μM luciferase was monitored upon addition of 2 μM Hsc70, 1 μM Hdj1 and varying concentrations of Apg2. The values shown represent the final yield of reactivated luciferase after 120 min. Reactivation data represent the average of three experiments±s.d. (E) The ATPase-deficient Apg2 mutant Apg2-D7S supports luciferase disaggregation equally well as Apg2. Reactivation of 20 nM luciferase from heat-induced aggregates obtained at a concentration of 2 μM luciferase was monitored upon addition of 2 μM Hsc70 and 1 μM Hdj1 in the presence or absence of 0.2μM Apg2 or Apg2-D7S. Reactivation data represent the average of at least three experiments±s.e. (F) Sse1 as well as the ATPase-deficient mutant Sse1-K69M support disaggregation together with Hsc70 and Hdj1. Reactivation of luciferase from heat-induced aggregates obtained at a concentration of 2 μM luciferase was monitored upon addition of 2 μM Hsc70, 1 μM Hdj1 and 0.2 μM Apg2 or Sse1 or Sse1-K69M. Reactivation data represent the average of six experiments±s.e.

Figure 5

Efficient disaggregation depends on all components of the Hsp70 system. (A) Reactivation of 20 nM luciferase from urea-induced aggregates was monitored upon addition of the human Hsp70 system (2 μM Hsc70, 1 μM Hdj1±0.4 μM Apg2 or 0.4 μM Bag-1 or 2 μM Snl1ΔN). (B) Luciferase (2 μM) was heat aggregated in the presence of Hsp26, diluted to 20 nM luciferase and subsequently, reactivation was monitored upon addition of the human Hsp70 system (2 μM Hsc70, 1 μM Hdj1±0.4 μM Apg2 or 0.4 μM Bag-1). (C) MDH (0.5 μM) was thermally aggregated and, after four-fold dilution, reactivation was monitored upon addition of the human Hsp70 system (4 μM Hsp70, 2 μM Hdj1±0.4 μM Apg2 or 0.4 μM Bag-1). For Bag-1, one representative data set out of two is shown. (D) Luciferase (2 μM) was heat aggregated in the presence of Hsp26, diluted to 20 nM luciferase and subsequently, reactivation was monitored upon addition of the human Hsp70 system (2 μM Hsc70, 1 μM DNAJA2 and 0.2 μM Apg2 or 0.8 μM Bag-1 or 1 μM Snl1ΔN). One representative data set out of two is shown. (E) Luciferase (20 nM) was heat aggregated in the presence of Hsp26 and reactivation was monitored upon addition of the human Hsp70 system (2 μM Hsc70, 1 μM DNAJA2 and 0.2 μM Apg2 or 0.4 μM Bag-1 or 1 μM Snl1ΔN). For Snl1ΔN, one representative data set out of two is shown. Reactivation data shown in this figure represent the average of at least three experiments±s.e. except where stated otherwise.

Figure 6

Knock-down of Hsp110 results in the persistence of heat shock-induced luciferase. (A) Luciferase-YFP expressing nematodes were heat-shocked at 35°C for 1 h and then transferred onto RNAi plates (C30C11.4 (Hsp110), bag-1 or empty vector (L4440) as control) and returned to 20°C. The aggregation propensity of luciferase-YFP was monitored during the indicated recovery time 12 h (left) and 24 h (right) post heat shock. For each condition, 20 nematodes were analysed. Representative images of the foci (white arrows) of the head region are shown. The two different time points depict different animals. The aggregation propensity however was indistinguishable between animals treated with the same RNAi and heat-shock conditions. The scale bars are 25 μM. (B) FRAP analysis demonstrates that foci of the Hsp110 knock-down are immobile aggregates (red), whereas the luciferase-YFP of the control (blue) or upon knock-down of bag-1 is soluble (green). FRAP was performed on three different animals of each genetic background. The graphs represent the average recovery of fluorescence and the variations are depicted by the error bars. (C) Quantification of luciferase-YFP foci of (A) of the whole nematode 12 and 24 h post heat shock.

Figure 7

Knock-down of Hsp110 decreases lifespan particularly upon heat shock. Left panel: Lifespan analysis of C. elegans upon knock-down of C30C11.4 (Hsp110), hsp-1 (Hsp70) and double knock-down of Hsp110/Hsp70 by RNAi at 20°C. Knock-down of Hsp110 or hsp-1 slightly decreases lifespan at 20°C. The double knock-down of Hsp110 and hsp-1 however reduces lifespan by ∼3 days. Right panel: Lifespan analysis of C. elegans upon knock-down of Hsp110, Hsp70 and double knock-down of Hsp110/Hsp70 by RNAi after heat shock at 35°C for 1 h on day 1 show an aggravated shortening of the lifespan. Knock-down of Hsp110 shortens lifespan by ∼4.5 days compared to the control and is further exacerbated upon additional knock-down of Hsp70. Data represent the average of 100 animals each.