Protein Disaggregation Research Articles

Targeted Magnetic Nanoparticles for Remote Magnetothermal Disruption of Amyloid-β Aggregates.

Remotely triggered hysteretic heat dissipation by magnetic nanoparticles (MNPs) selectively attached to targeted proteins can be used to break up self-assembled aggregates. This magnetothermal approach is applied to the amyloid-β (Aβ) protein, which forms dense, insoluble plaques characteristic of Alzheimer's disease. Specific targeting of dilute MNPs to Aβ aggregates is confirmed via transmission electron microscopy (TEM) and is found to be consistent with a statistical model of MNP distribution on the Aβ substrates. MNP composition and size are selected to achieve efficient hysteretic power dissipation at physiologically safe alternating magnetic field (AMF) conditions. Dynamic light scattering, fluorescence spectroscopy, and TEM are used to characterize the morphology and size distribution of aggregates before and after exposure to AMF. A dramatic reduction in aggregate size from microns to tens of nanometers is observed, suggesting that exposure to an AMF effectively destabilizes Aβ deposits decorated with targeted MNPs. Experiments in primary hippocampal neuronal cultures indicate that the magnetothermal disruption of aggregates reduces Aβ cytotoxicity, which may enable future applications of this approach for studies of protein disaggregation in physiological environments.

Escherichia coli ClpB is a non-processive polypeptide translocase.

Escherichia coli caseinolytic protease (Clp)B is a hexameric AAA+ [expanded superfamily of AAA (ATPase associated with various cellular activities)] enzyme that has the unique ability to catalyse protein disaggregation. Such enzymes are essential for proteome maintenance. Based on structural comparisons to homologous enzymes involved in ATP-dependent proteolysis and clever protein engineering strategies, it has been reported that ClpB translocates polypeptide through its axial channel. Using single-turnover fluorescence and anisotropy experiments we show that ClpB is a non-processive polypeptide translocase that catalyses disaggregation by taking one or two translocation steps followed by rapid dissociation. Using single-turnover FRET experiments we show that ClpB containing the IGL loop from ClpA does not translocate substrate through its axial channel and into ClpP for proteolytic degradation. Rather, ClpB containing the IGL loop dysregulates ClpP leading to non-specific proteolysis reminiscent of ADEP (acyldepsipeptide) dysregulation. Our results support a molecular mechanism where ClpB catalyses protein disaggregation by tugging and releasing exposed tails or loops.

Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation.

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, 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 systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. 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.

Molecular biology: It takes two to untangle.

Yeast require the enzyme Hsp104 to untangle protein aggregates, which arise in stressed or aged cells. Animals lack Hsp104, but it emerges that proteins of the DNAJ family of molecular chaperones can fulfil this role. See Letter p.251 Microbes eliminate toxic intracellular protein aggregates thanks to HSP100 disaggregases, which are missing from animal cells. Now Bernd Bukau and colleagues have discovered an efficient protein disaggregation system in metazoan cells, which requires transient interactions between J-protein co-chaperones of classes A and B, which synergistically boost HSP70-dependent disaggregation activity. This system provides a flexible further level of regulation for metazoan protein quality control, with direct relevance to human diseases such as age-related neurodegeneration.

Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation.

Unicellular and sessile organisms are particularly exposed to environmental stress such as heat shock causing accumulation and aggregation of misfolded protein species. To counteract protein aggregation, bacteria, fungi, and plants encode a bi-chaperone system composed of ATP-dependent Hsp70 and hexameric Hsp100 (ClpB/Hsp104) chaperones, which rescue aggregated proteins and provide thermotolerance to cells. The partners act in a hierarchic manner with Hsp70 chaperones coating first the surface of protein aggregates and next recruiting Hsp100 through direct physical interaction. Hsp100 proteins bind to the ATPase domain of Hsp70 via their unique M-domain. This extra domain functions as a molecular toggle allosterically controlling ATPase and threading activities of Hsp100. Interactions between neighboring M-domains and the ATPase ring keep Hsp100 in a repressed state exhibiting low ATP turnover. Breakage of intermolecular M-domain interactions and dissociation of M-domains from the ATPase ring relieves repression and allows for Hsp70 interaction. Hsp70 binding in turn stabilizes Hsp100 in the activated state and primes Hsp100 ATPase domains for high activity upon substrate interaction. Hsp70 thereby couples Hsp100 substrate binding and motor activation. Hsp100 activation presumably relies on increased subunit cooperation leading to high ATP turnover and threading power. This Hsp70-mediated activity control of Hsp100 is crucial for cell viability as permanently activated Hsp100 variants are toxic. Hsp100 activation requires simultaneous binding of multiple Hsp70 partners, restricting high Hsp100 activity to the surface of protein aggregates and ensuring Hsp100 substrate specificity.

50 Interaction and collaboration between DnaK and ClpB during protein disaggregation

50 Interaction and collaboration between DnaK and ClpB during protein disaggregation

Study on microwave‐accelerated casein protein grafted with glucose and β‐cyclodextrin to improve the gel properties

SummaryThe effect of glycosylation treatment coupled with microwave heating (MH) on gel properties and structure change of casein were investigated. Conjugation was confirmed by electrophoresis. Protein disulphide bonds were broken by microwave irradiation to create an increase first and a decrease later in free sulfhydryl contents with time, thereby inducing subunit disaggregation. Also, the exposure of hydrophobic core residues was increased in MH‐treated casein, which brings about protein disaggregation and unfolding. It showed that graft reactions by MH had much faster than those by water bath heating (WH). The content of disulphide bonds is 7.2% and 6.0% in the casein–glucose and casein–β‐cyclodextrin, respectively, treated by MH which was reduced significantly than that of treated by WH. Moreover, MH can increase the hardness of the gel. Consequently, MH could improve the efficiency of the reaction and gel properties.

Role of Cyclophilin on Amyloid Formation and Protein Disaggregation

Oligomers and fibrils of protein and peptides are implicated in memory storage, neurotransmission and formation of amyloid diseases. It is believed that partially misfolded/folded states are trapped in a certain conformation and it eventually leads to formation of larger and more stable fibrillar aggregates. Recently, our laboratory demonstrated that AdK-aggregate could be disaggregated by cyclophilin CyP (LdCyP) in an isomerase- independent fashion, resulting in reactivation [Ref. 1]. We also observed the formation of elongated fibrils when lysozyme was co-incubated with cyclophilin [Ref. 2]. Very recently we tested the self aggregation process of insulin in the presence and absence of cyclophilin. The formation of insulin fiber was confirmed by ThT fluorescence assay when insulin was incubated in acidic condition at 65oC. However, co-incubation with cyclophilin, hinder the formation of amyloid fiber. Cyclophilin also showed potential to disaggregate amyloid aggregate of insulin. Cyclophilin itself was not formed amyloid fibril upon incubation for 50 days. TEM images, however, indicated formations of amorphous type of aggregates upon prolong incubation for several days at 65 oC. In our study, possibly, cyclophilin, helped to preserve the insulin native structure intact even at high temperature and did not allow it to partially unfold that may initiate the amyloid formation. Ref. 1. Mukherjee, D ; Patra, H ; Laskar, A ; Dasgupta, A ; Maiti, NC and Datta, AK, Archives Biochem. Biophys. 2013, 537, 82-90 Ref. 2. Das S., Pal U., Das S. and Maiti N.C. , Journal of Proteins and Proteomics, 4(2), 129-137, 2013.

Analysis of the Cooperative ATPase Cycle of the AAA+ Chaperone ClpB from Thermus thermophilus by Using Ordered Heterohexamers with an Alternating Subunit Arrangement

The ClpB/Hsp104 chaperone solubilizes and reactivates protein aggregates in cooperation with DnaK/Hsp70 and its cofactors. The ClpB/Hsp104 protomer has two AAA+ modules, AAA-1 and AAA-2, and forms a homohexamer. In the hexamer, these modules form a two-tiered ring in which each tier consists of homotypic AAA+ modules. By ATP binding and its hydrolysis at these AAA+ modules, ClpB/Hsp104 exerts the mechanical power required for protein disaggregation. Although ATPase cycle of this chaperone has been studied by several groups, an integrated understanding of this cycle has not been obtained because of the complexity of the mechanism and differences between species. To improve our understanding of the ATPase cycle, we prepared many ordered heterohexamers of ClpB from Thermus thermophilus, in which two subunits having different mutations were cross-linked to each other and arranged alternately and measured their nucleotide binding, ATP hydrolysis, and disaggregation abilities. The results indicated that the ATPase cycle of ClpB proceeded as follows: (i) the 12 AAA+ modules randomly bound ATP, (ii) the binding of four or more ATP to one AAA+ ring was sensed by a conserved Arg residue and converted another AAA+ ring into the ATPase-active form, and (iii) ATP hydrolysis occurred cooperatively in each ring. We also found that cooperative ATP hydrolysis in at least one ring was needed for the disaggregation activity of ClpB.

Common functionally important motions of the nucleotide-binding domain of Hsp70.

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate-binding domain (SBD) that binds client substrates, and the nucleotide-binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure-function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (PDB 3C7N:B) by all-atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP- and ATP-unique classes, which reflect conformational trends that are unique to either the ADP- or ATP-bound states, respectively. "Mutual" class motions generally describe "in-plane" and/or "out-of-plane" (scissor-like) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The "unique" class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the "unique" type, regions of enhanced mobility can be identified; these are termed "hot spots," and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide-binding pocket was also found to influence the dynamics of the NBD significantly.

Examination of polypeptide substrate specificity for Escherichia coli ClpB.

Escherichia coli ClpB is a molecular chaperone that belongs to the Clp/Hsp100 family of AAA+ proteins. ClpB is able to form a hexameric ring structure to catalyze protein disaggregation with the assistance of the DnaK chaperone system. Our knowledge of the mechanism of how ClpB recognizes its substrates is still limited. In this study, we have quantitatively investigated ClpB binding to a number of unstructured polypeptides using steady-state anisotropy titrations. To precisely determine the binding affinity for the interaction between ClpB hexamers and polypeptide substrates the titration data were subjected to global non-linear least squares analysis incorporating the dynamic equilibrium of ClpB assembly. Our results show that ClpB hexamers bind tightly to unstructured polypeptides with binding affinities in the range of ∼3-16 nM. ClpB exhibits a modest preference of binding to Peptide B1 with a binding affinity of (1.7 ± 0.2) nM. Interestingly, we found that ClpB binds to an unstructured polypeptide substrate of 40 and 50 amino acids containing the SsrA sequence at the C-terminus with an affinity of (12 ± 3) nM and (4 ± 2) nM, respectively. Whereas, ClpB binds the 11-amino acid SsrA sequence with an affinity of (140 ± 20) nM, which is significantly weaker than other polypeptide substrates that we tested here. We hypothesize that ClpB, like ClpA, requires substrates with a minimum length for optimal binding. Finally, we present evidence showing that multiple ClpB hexamers are involved in binding to polypeptides ≥152 amino acids.

Interplay between E. coli DnaK, ClpB and GrpE during Protein Disaggregation

The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: Escherichia coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the middle domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomains IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK–ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.

Expression of factors associated with apoptosis in the porcine corpus luteum throughout the luteal phase of the estrous cycle and early pregnancy: Their possible involvement in acquisition of luteolytic sensitivity

The studies on the acquisition of luteolytic sensitivity have been focused mainly on molecular changes induced in the luteal tissue after treatment with exogenous PGF2α or on physiological changes occurring during the estrous cycle. The comparison of changes leading to the acquisition of luteolytic sensitivity after Day 12 of the estrous cycle and corresponding days of pregnancy has not been investigated in the pig. The present study was undertaken to evaluate (1) apoptosis measured as the proportions of early apoptotic, late apoptotic, and viable cells; (2) expression of factors involved in the extrinsic (TNFA/TNFα, TNFRSF1A/TNFR1, TNFRSF1B/TNFR2, FAS/Fas, and FASLG/FasL) and intrinsic (CASP3/Casp3, TP53/p-53, BAX/Bax, and BCL2/Bcl-2) apoptotic pathways, with two components of the activating protein-1 complex, i.e., FOS/Fos and JUN/Jun and IFNG/IFNγ; and (3) concentrations of luteal and blood plasma progesterone (P4) throughout the luteal phase of the estrous cycle and early pregnancy. Corpora lutea (CL) were collected postmortem on Days 8, 10, 12, and 14 of the estrous cycle and the corresponding days of pregnancy. The luteal tissue was subjected to RNA and/or protein isolation and disaggregation of CL cells followed by flow cytometry analysis aimed to determine apoptotic changes. Luteal and blood plasma P4 concentrations decreased on Day 14 of the estrous cycle versus pregnancy (P < 0.05 and P < 0.001, respectively). A significant increase in the number of early apoptotic cells and a decrease in the number of viable cells were observed on Day 14 of the estrous cycle (P < 0.001 and P < 0.05, respectively). Increase (P < 0.05) of TNFA messenger RNA (mRNA) level coincided with that of IFNG on Day 12 of the estrous cycle but not on the corresponding day of pregnancy. The content of FAS mRNA and protein increased on Day 14 of the estrous cycle versus pregnancy (P < 0.05). The mRNA expression of CASP3, BCL-2 and BAX was unchanged in cyclic and pregnant CL, while level of TP53 increased (P < 0.05) on Day 12 of the estrous cycle versus Day 8. The level of FOS and JUN mRNA increased (P < 0.05) on Day 14 of the estrous cycle versus the remaining days. The level of FOS and JUN mRNA was significantly higher (P < 0.001 and P < 0.05, respectively) on Day 14 of the estrous cycle than that on the corresponding day of pregnancy. In summary, the simultaneous increase of TNFA and IFNG transcript in cyclic CL suggests the crucial role of both cytokines in sensitization of porcine CL to further luteolytic action of PGF2α. The upregulated expression of FAS, FOS, and JUN mRNA in the late luteal phase in cyclic CL can indicate their involvement in structural luteolysis. The increased viability of luteal cells and elevated P4 concentrations in pregnant CL confirm the protective role of luteal P4 against apoptosis.

Structural mechanisms of chaperone mediated protein disaggregation.

The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism—entropic pulling—may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context.

Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation.

The hexameric AAA+ chaperone ClpB reactivates aggregated proteins in cooperation with the Hsp70 system. Essential for disaggregation, the ClpB middle domain (MD) is a coiled-coil propeller that binds Hsp70. Although the ClpB subunit structure is known, positioning of the MD in the hexamer and its mechanism of action are unclear. We obtained electron microscopy (EM) structures of the BAP variant of ClpB that binds the protease ClpP, clearly revealing MD density on the surface of the ClpB ring. Mutant analysis and asymmetric reconstructions show that MDs adopt diverse positions in a single ClpB hexamer. Adjacent, horizontally oriented MDs form head-to-tail contacts and repress ClpB activity by preventing Hsp70 interaction. Tilting of the MD breaks this contact, allowing Hsp70 binding, and releasing the contact in adjacent subunits. Our data suggest a wavelike activation of ClpB subunits around the ring.DOI: http://dx.doi.org/10.7554/eLife.02481.001.

Surface-Mediated Protein Disaggregation

Preventing protein aggregation is of both biological and industrial importance. Interprotein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In this article, we show how surface chemistry and curvature can be tuned to mitigate these interprotein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that interprotein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose interprotein contacts to gain surface contacts, and thus the surface helps to reduce the interprotein interactions. Furthermore, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient at both reducing interprotein contacts and simultaneously retaining most of the native contacts probably as a result of confinement-induced stabilization.

A Thermal-Cycling Method for Disaggregating Monoclonal Antibody Oligomers

Non-native oligomeric forms of biopharmaceutical proteins are therapeutically inactive, and potentially toxic and immunogenic, and therefore undesirable in pharmaceutical formulations. Immunoglobulin G class of antibodies are known to form stable nonnative oligomers through Fab-Fab interactions. In this paper, we investigate thermal-cycling as a technique for disaggregating antibody oligomers. Aggregate containing monoclonal antibody (mAb) samples were exposed to rapid heating and cooling cycles in a thermal-cycler. The heating phase of the thermal-cycle resulted in partial unfolding of the Fab domain, leading to the release of monomer from the oligomer complexes, whereas the rapid cooling that followed led to refolding and minimized the probability of protein reaggregation. The extent of mAb oligomer disaggregation was determined by size-exclusion chromatography and hydrophobic interaction membrane chromatography, whereas protein refolding was assessed by circular dichroism spectroscopy. The thermal-cycling technique in addition to being suitable for disaggregating protein oligomer samples could also potentially be useful for studying the mechanisms of protein aggregation and disaggregation.

Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation

The homologous hexameric AAA(+) proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.

Coordination of Translational Control and Protein Homeostasis during Severe Heat Stress

Exposure of cells to severe heat stress causes not only misfolding and aggregation of proteins but also inhibition of translation and storage of mRNA in cytosolic heat stress granules (heat-SGs), limiting newly synthesized protein influx into overloaded proteome repair systems. How these two heat stress responses connect is unclear. Here, we show that both S.cerevisiae and D.melanogaster heat-SGs contain mRNA, translation machinery components (excluding ribosomes), and molecular chaperones and that heat-SGs coassemble with aggregates of misfolded, heat-labile proteins. Components in these mixed assemblies exhibit distinct molecular motilities reflecting differential trapping. Wedemonstrate that heat-SG disassembly and restoration of translation activity during heat stress recovery is intimately linked to disaggregation of damaged proteins present in the mixed assemblies and requires Hsp104 and Hsp70 activity. Chaperone-driven protein disaggregation directly coordinates timing of translation reinitiation with protein folding capacity during cellular protein quality surveillance, enabling efficient protein homeostasis.

Enhancing protein disaggregation restores proteasome activity in aged cells

The activity of the ubiquitin-proteasome system, UPS, declines during aging in several multicellular organisms. The reason behind this decline remains elusive. Here, using yeast as a model system, we show that while the level and potential capacity of the 26S proteasome is maintained in replicatively aged cells, the UPS is not functioning properlyin vivo. As a consequence cytosolic UPS substrates, such as ΔssCPY* are stabilized, accumulate, and form inclusions. By integrating a pGPD-HSP104 recombinant gene into the genome, we were able to constitutively elevate protein disaggregase activity, which diminished the accumulation of protein inclusions during aging. Remarkably, this elevated disaggregation restored degradation of a 26S proteasome substrate in aged cells without elevating proteasome levels, demonstrating that age-associated aggregation obstructs UPS function. The data supports the existence of a negative feedback loop that accelerates aging by exacerbating proteostatic decline once misfolded and aggregation-prone proteins reach a critical level.