Metazoan Hsp70 machines use Hsp110 to power protein disaggregation

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.     

Cellular strategies for controlling protein aggregation

The aggregation of misfolded proteins is associated with the perturbation of cellular function, ageing and various human disorders. Mounting evidence suggests that protein aggregation is often part of the cellular response to an imbalanced protein homeostasis rather than an unspecific and uncontrolled dead-end pathway. It is a regulated process in cells from bacteria to humans, leading to the deposition of aggregates at specific sites. The sequestration of misfolded proteins in such a way is protective for cell function as it allows for their efficient solubilization and refolding or degradation by components of the protein quality-control network. The organized aggregation of misfolded proteins might also allow their asymmetric distribution to daughter cells during cell division.     

Cytosolic aggregates perturb the degradation of nontranslocated secretory and membrane proteins

A wide range of diseases are associated with the accumulation of cytosolic protein aggregates. The effects of these aggregates on various aspects of normal cellular protein homeostasis remain to be determined. Here we find that cytosolic aggregates, without necessarily disrupting proteasome function, can markedly delay the normally rapid degradation of nontranslocated secretory and membrane protein precursors. In the case of mammalian prion protein (PrP), the nontranslocated fraction is recruited into preexisting aggregates before its triage for degradation. This recruitment permits the growth and persistence of cytosolic PrP aggregates, explaining their apparent "self-conversion" seen in earlier studies of transient proteasome inhibition. For other proteins, the aggregate-mediated delay in precursor degradation led to aggregation and/or soluble residence in the cytosol, often causing aberrant cellular morphology. Remarkably, improving signal sequence efficiency mitigated these effects of aggregates. These observations identify a previously unappreciated consequence of cytosolic aggregates for nontranslocated secretory and membrane proteins, a minor but potentially disruptive population the rapid disposal of which is critical to maintaining cellular homeostasis.     

Cytosolic aggregates in presence of non‐translocated proteins perturb endoplasmic reticulum structure and dynamics

Presence of cytosolic protein aggregates and membrane damage are two common attributes of neurodegenerative diseases. These aggregates delay degradation of non‐translocated protein precursors leading to their persistence and accumulation in the cytosol. Here, we find that cells with intracellular protein aggregates (of cytosolic prion protein or huntingtin) destabilize the endoplasmic reticulum (ER) morphology and dynamics when non‐translocated protein load is high. This affects trafficking of proteins out from the ER, relative distribution of the rough and smooth ER and three‐way junctions that are essential for the structural integrity of the membrane network. The changes in ER membranes may be due to high aggregation tendency of the ER structural proteins—reticulons, and altered distribution of those associated with the three‐way ER junctions—Lunapark. Reticulon4 is seen to be enriched in the aggregate fractions in presence of non‐translocated protein precursors. This could be mitigated by improving signal sequence efficiencies of the proteins targeted to the ER. These were observed using PrP variants and the seven‐pass transmembrane protein (CRFR1) with different signal sequences that led to diverse translocation efficiencies. This identifies a previously unappreciated consequence of cytosolic aggregates on non‐translocated precursor proteins—their persistent presence affects ER morphology and dynamics. This may be one of the ways in which cytosolic aggregates can affect endomembranes during neurodegenerative disease.     

Substrate relay in an Hsp70‐cochaperone cascade safeguards tail‐anchored membrane protein targeting

Membrane proteins are aggregation‐prone in aqueous environments, and their biogenesis poses acute challenges to cellular protein homeostasis. How the chaperone network effectively protects integral membrane proteins during their post‐translational targeting is not well understood. Here, biochemical reconstitutions showed that the yeast cytosolic Hsp70 is responsible for capturing newly synthesized tail‐anchored membrane proteins (TAs) in the soluble form. Moreover, direct interaction of Hsp70 with the cochaperone Sgt2 initiates a sequential series of TA relays to the dedicated TA targeting factor Get3. In contrast to direct loading of TAs to downstream chaperones, stepwise substrate loading via Hsp70 maintains the solubility and targeting competence of TAs, ensuring their efficient delivery to the endoplasmic reticulum (ER). Inactivation of cytosolic Hsp70 severely impairs TA translocation in vivo. Our results demonstrate a new role of cytosolic Hsp70 in directly assisting the targeting of an essential class of integral membrane proteins and provide a paradigm for how “substrate funneling” through a chaperone cascade preserves the conformational quality of nascent membrane proteins during their biogenesis.     

Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double‐membraned chloroplasts through binary fission. A key mediator of division‐complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.     

Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

Neurofilament protein aggregation in a cell line model system

Protein aggregates are associated with many diseases and even aggregates of proteins that have no role in disease are inherently toxic to both neuronal and non-neuronal cells. We have developed a model system to explore the mechanism of protein aggregation using a mouse muscle cell line expressing chimeric neurofilament (NF) proteins, a constituent of the protein aggregates in ALS, Lewy body dementia, and Charcot-Marie-Tooth disease. Formation of protein aggregates in these cells leads to reduced cell viability and activated caspases. Aggregates contained both chimeric NF proteins and ubiquitin by immunolocalization and were predominately cytosolic when proteins were expressed at low levels or for shorter periods of time but were present in the nucleus when expression levels increased. This system represents a flexible, new tool to decipher the molecular mechanism of protein aggregation and the contributions of aggregation to cell toxicity.     

Growth‐driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus

All living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein is collected at the cell poles and is retained throughout consecutive cell divisions only in old pole‐inheriting daughter cells, resulting in aggregation‐free progeny within a few generations. In this study, we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium, protein aggregates form as multiple distributed foci located throughout the cell volume. Time‐lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short‐lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long‐lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.     

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high‐energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA. However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La‐related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI‐Larp complex promotes the synthesis of a subset of nuclear‐encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI‐Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron‐transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI‐Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis.     

Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies

The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-like structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone beta-galactosidase fusion protein are clearly toxic for mammalian cells but the beta-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is the key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death.     

Role of sHsps in organizing cytosolic protein aggregation and disaggregation

Small heat shock proteins (sHsps) exhibit an ATP-independent chaperone activity to prevent the aggregation of misfolded proteins in vitro. The seemingly conflicting presence of sHsps in insoluble protein aggregates in cells obstructs a precise definition of sHsp function in proteostasis networks. Recent findings specify sHsp activities in protein quality control systems. The sHsps of yeast, Hsp42 and Hsp26, interact with early unfolding intermediates of substrates, keeping them in a ready-to-refold conformation close to the native state. This activity facilitates substrate refolding by ATP-dependent Hsp70-Hsp100 disaggregating chaperones. Hsp42 can actively sequester misfolded proteins and promote their deposition at specific cellular sites. This aggregase activity represents a cytoprotective protein quality control strategy. The aggregase function of Hsp42 controls the formation of cytosolic aggregates (CytoQs) under diverse stress regimes and can be reconstituted in vitro, demonstrating that Hsp42 is necessary and sufficient to promote protein aggregation. Substrates sequestered at CytoQs can be dissociated by Hsp70-Hsp100 disaggregases for subsequent triage between refolding and degradation pathways or are targeted for destruction by selective autophagy termed proteophagy.     

Motility and segregation of Hsp104-associated protein aggregates in budding yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.     

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat‐stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat‐induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat‐induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered.     

Structural basis for interaction of DivIVA/GpsB proteins with their ligands

DivIVA proteins and their GpsB homologues are late cell division proteins found in Gram‐positive bacteria. DivIVA/GpsB proteins associate with the inner leaflet of the cytosolic membrane and act as scaffolds for other proteins required for cell growth and division. DivIVA/GpsB proteins comprise an N‐terminal lipid‐binding domain for membrane association fused to C‐terminal domains supporting oligomerization. Despite sharing the same domain organization, DivIVA and GpsB serve different cellular functions: DivIVA plays diverse roles in division site selection, chromosome segregation and controlling peptidoglycan homeostasis, whereas GpsB contributes to the spatiotemporal control of penicillin‐binding protein activity. The crystal structures of the lipid‐binding domains of DivIVA from Bacillus subtilis and GpsB from several species share a fold unique to this group of proteins, whereas the C‐terminal domains of DivIVA and GpsB are radically different. A number of pivotal features identified from the crystal structures explain the functional differences between the proteins. Herein we discuss these structural and functional relationships and recent advances in our understanding of how DivIVA/GpsB proteins bind and recruit their interaction partners, knowledge that might be useful for future structure‐based DivIVA/GpsB inhibitor design.     

The nascent polypeptide‐associated complex is a key regulator of proteostasis

The adaptation of protein synthesis to environmental and physiological challenges is essential for cell viability. Here, we show that translation is tightly linked to the protein‐folding environment of the cell through the functional properties of the ribosome bound chaperone NAC (nascent polypeptide‐associated complex). Under non‐stress conditions, NAC associates with ribosomes to promote translation and protein folding. When proteostasis is imbalanced, NAC relocalizes from a ribosome‐associated state to protein aggregates in its role as a chaperone. This results in a functional depletion of NAC from the ribosome that diminishes translational capacity and the flux of nascent proteins. Depletion of NAC from polysomes and re‐localisation to protein aggregates is observed during ageing, in response to heat shock and upon expression of the highly aggregation‐prone polyglutamine‐expansion proteins and Aβ‐peptide. These results demonstrate that NAC has a central role as a proteostasis sensor to provide the cell with a regulatory feedback mechanism in which translational activity is also controlled by the folding state of the cellular proteome and the cellular response to stress.     

Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls

Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease‐specific ‘seed’ proteins; however, roles of other co‐aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control‐subject pools using magnetic‐bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high‐resolution proteomics. Although total detergent‐insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or Aβ_1–42 pulldown, the protein constituents of Alzheimer‐derived aggregates were more abundant, diverse, and post‐translationally modified than those from controls. Tau‐ and Aβ‐containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer‐specific protein enrichment in tau‐containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co‐aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co‐localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer‐specific enrichment in tau‐containing aggregates, were assessed for aggregation‐promoting roles in C. elegans by RNA‐interference ‘knockdown’. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ_1–42. These observations imply shared mechanisms driving both types of aggregation, and/or aggregate‐mediated cross‐talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.