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Summary Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson’s disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.

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.

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 þ 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.

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Nedd8 is a small ubiquitin-like protein that can be conjugated to substrate-proteins in a process known as neddylation. Although neddylation plays a critical regulatory role in cell proliferation and development, the spectrum of Nedd8 substrates and its interaction network remain poorly understood. To explore the neddylation pathway at the proteome level, we have affinity purified Nedd8 modified and associated proteins from HEK293 cells stably expressing GST-Nedd8 and employed LC-MS/ MS for subsequent protein identification. A total of 496 GST-Nedd8 modified and associated proteins have been identified, including all of the eight cullin family members (i.e., Cul-1, -2, -3, -4A, -4B, -5, -7, and Parc) that are involved in the neddylation and ubiquitin-proteasome degradation pathway. In addition, a group of proteins involved in transcription, DNA repair and replication, cell cycle regulation and chromatin organization, and remodeling have been copurified and identified. Apart from protein identification, the neddylation sites of cullins were determined by MS/MS analysis, which agree well with previous mutagenesis studies. Furthermore, MS analyses revealed that Nedd8 K11, K22, K48, and K60 can form chains in vivo, whereas Nedd8 K22 and K48 can be neddylated in vitro. These results present the first molecular evidence for in vitro and in vivo polyneddylation, suggesting that chain formation of ubiquitin and ubiquitin-like proteins may be a general phenomenon for these modifications. Although much remains to be explored for the biological significance of the observations, this work provides critically important information regarding Nedd8 chain assembly and its interaction network. The vast amount of proteomic information obtained here

Ubr1 and Ubr2 ubiquitin ligases are shown to promote degradation of misfolded cytosolic polypeptides in vivo and in a purified system in association with Hsp70.