Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction.

HSP-100 protein machines, such as ClpB, play an essential role in reactivating protein aggregates that can otherwise be lethal to cells. Although the players involved are known, including the DnaK/DnaJ/GrpE chaperone system in bacteria, details of the molecular interactions are not well understood. Using methyl-transverse relaxation-optimized nuclear magnetic resonance spectroscopy, we present an atomic-resolution model for the ClpB-DnaK complex, which we verified by mutagenesis and functional assays. ClpB and GrpE compete for binding to the DnaK nucleotide binding domain, with GrpE binding inhibiting disaggregation. DnaK, in turn, plays a dual role in both disaggregation and subsequent refolding of polypeptide chains as they emerge from the aggregate. On the basis of a combined structural-biochemical analysis, we propose a model for the mechanism of protein aggregate reactivation by ClpB.

A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI (+) ].

Most prions in yeast form amyloid fibrils that must be severed by the protein disaggregase Hsp104 to be propagated and transmitted efficiently to newly formed buds. Only one yeast prion, [PSI (+) ], is cured by Hsp104 overexpression. We investigated the interaction between Hsp104 and Sup35, the priongenic protein in yeast that forms the [PSI (+) ] prion.1 We found that a 20-amino acid segment within the highly-charged, unstructured middle domain of Sup35 contributes to the physical interaction between the middle domain and Hsp104. When this segment was deleted from Sup35, the efficiency of [PSI (+) ] severing was substantially reduced, resulting in larger Sup35 particles and weakening of the [PSI (+) ] phenotype. Furthermore, [PSI (+) ] in these cells was completely resistant to Hsp104 curing. The affinity of Hsp104 was considerably weaker than that of model Hsp104-binding proteins and peptides, implying that Sup35 prions are not ideal substrates for Hsp104-mediated remodeling. In light of this finding, we present a modified model of Hsp104-mediated [PSI (+) ] propagation and curing that requires only partial remodeling of Sup35 assembled into amyloid fibrils.

Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104).

Yeast prions are a powerful model for understanding the dynamics of protein aggregation associated with a number of human neurodegenerative disorders. The AAA+ protein disaggregase Hsp104 can sever the amyloid fibrils produced by yeast prions. This action results in the propagation of "seeds" that are transmitted to daughter cells during budding. Overexpression of Hsp104 eliminates the [PSI+] prion but not other prions. Using biochemical methods we identified Hsp104 binding sites in the highly charged middle domain of Sup35, the protein determinant of [PSI+]. Deletion of a short segment of the middle domain (amino acids 129-148) diminishes Hsp104 binding and strongly affects the ability of the middle domain to stimulate the ATPase activity of Hsp104. In yeast, [PSI+] maintained by Sup35 lacking this segment, like other prions, is propagated by Hsp104 but cannot be cured by Hsp104 overexpression. These results provide new insight into the enigmatic specificity of Hsp104-mediated curing of yeast prions and sheds light on the limitations of the ability of Hsp104 to eliminate aggregates produced by other aggregation-prone proteins.

A multistage system for the automated detection of epileptic seizures in neonatal electroencephalography.

This paper describes the design and test results of a three-stage automated system for neonatal EEG seizure detection. Stage I of the system is the initial detection stage and identifies overlapping 5-second segments of suspected seizure activity in each EEG channel. In stage II, the detected segments from stage I are spatiotemporally clustered to produce multichannel candidate seizures. In stage III, the candidate seizures are processed further using measures of quality and context-based rules to eliminate false candidates. False candidates because of artifacts and commonly occurring EEG background patterns such as bifrontal delta activity are also rejected. Seizures at least 10 seconds in duration are considered for reporting results. The testing data consisted of recordings of 28 seizure subjects (34 hours of data) and 48 nonseizure subjects (87 hours of data) obtained in the neonatal intensive care unit. The data were not edited to remove artifacts and were identical in every way to data normally processed visually. The system was able to detect seizures of widely varying morphology with an average detection sensitivity of almost 80% and a subject sensitivity of 96%, in comparison with a team of clinical neurophysiologists who had scored the same recordings. The average false detection rate obtained in nonseizure subjects was 0.74 per hour.

Remodeling of protein aggregates by Hsp104.

Hsp104 is molecular chaperone in the AAA+ family of ATPases that specializes in the resolubilization and refolding of thermally denatured proteins in yeast. In addition to providing high levels of thermotolerance, Hsp104 plays a pivotal role in the propagation of yeast prions, self-replicating, amyloid-like aggregates that are inherited during mitosis and meiosis. In this review, the structure and function of Hsp104 is discussed, its functional interaction with other molecular chaperones, and a model for disaggregation and refolding is proposed.

The SmpB-tmRNA tagging system plays important roles in Streptomyces coelicolor growth and development.

The ssrA gene encodes tmRNA that, together with a specialized tmRNA-binding protein, SmpB, forms part of a ribonucleoprotein complex, provides a template for the resumption of translation elongation, subsequent termination and recycling of stalled ribosomes. In addition, the mRNA-like domain of tmRNA encodes a peptide that tags polypeptides derived from stalled ribosomes for degradation. Streptomyces are unique bacteria that undergo a developmental cycle culminating at sporulation that is at least partly controlled at the level of translation elongation by the abundance of a rare tRNA that decodes UUA codons found in a relatively small number of open reading frames prompting us to examine the role of tmRNA in S. coelicolor. Using a temperature sensitive replicon, we found that the ssrA gene could be disrupted only in cells with an extra-copy wild type gene but not in wild type cells or cells with an extra-copy mutant tmRNA (tmRNA(DD)) encoding a degradation-resistant tag. A cosmid-based gene replacement method that does not include a high temperature step enabled us to disrupt both the ssrA and smpB genes separately and at the same time suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Indeed, mutant cells show growth and sporulation defects at high temperature and under optimal culture conditions. Interestingly, even though these defects can be completely restored by wild type genes, the DeltassrA strain was only partially corrected by tmRNA(DD). In addition, wildtype tmRNA can restore the hygromycin-resistance to DeltassrA cells while tmRNA(DD) failed to do so suggesting that degradation of aberrant peptides is important for antibiotic resistance. Overall, these results suggest that the tmRNA tagging system plays important roles during Streptomyces growth and sporulation under both normal and stress conditions.

Peptide and protein binding in the axial channel of Hsp104. Insights into the mechanism of protein unfolding.

The AAA+ molecular chaperone Hsp104 mediates the extraction of proteins from aggregates by unfolding and threading them through its axial channel in an ATP-driven process. An Hsp104-binding peptide selected from solid phase arrays enhanced the refolding of a firefly luciferase-peptide fusion protein. Analysis of peptide binding using tryptophan fluorescence revealed two distinct binding sites, one in each AAA+ module of Hsp104. As a further indication of the relevance of peptide binding to the Hsp104 mechanism, we found that it competes with the binding of a model unfolded protein, reduced carboxymethylated alpha-lactalbumin. Inactivation of the pore loops in either AAA+ module prevented stable peptide and protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of ATPase turnover in the second AAA+ module of this mutant was abolished. Drawing on these data, we propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module.

The C-terminal extension of Saccharomyces cerevisiae Hsp104 plays a role in oligomer assembly.

The Saccharomyces cerevisiae protein Hsp104, a member of the Hsp100/Clp AAA+ family of ATPases, and its orthologues in plants (Hsp101) and bacteria (ClpB) function to disaggregate and refold thermally denatured proteins following heat shock and play important roles in thermotolerance. The primary sequences of fungal Hsp104's contain a largely acidic C-terminal extension not present in bacterial ClpB's. In this work, deletion mutants were used to determine the role this extension plays in Hsp104 structure and function. Elimination of the C-terminal tetrapeptide DDLD diminishes binding of the tetratricopeptide repeat domain cochaperone Cpr7 but is dispensable for Hsp104-mediated thermotolerance. The acidic region of the extension is also dispensable for thermotolerance and for the stimulation of Hsp104 ATPase activity by poly-l-lysine, but its truncation results in an oligomerization defect and reduced ATPase activity in vitro. Finally, sequence alignments reveal that the C-terminal extension contains a sequence (VLPNH) that is conserved in fungal Hsp104's but not in other orthologues. Hsp104 lacking the entire C-terminal extension including the VLPNH region does not assemble and has very low ATPase activity. In the presence of a molecular crowding agent the ATPase activities of mutants with longer truncations are partially restored possibly through enhanced oligomer formation. However, elimination of the whole C-terminal extension results in an Hsp104 molecule which is unable to assemble and becomes aggregation prone at high temperature, highlighting a novel structural role for this region.

Nucleocytoplasmic trafficking of the molecular chaperone Hsp104 in unstressed and heat-shocked cells.

Hsp104 is a molecular chaperone in yeast that restores solubility and activity to inactivated proteins after severe heat shock. We investigated the mechanisms that influence Hsp104 subcellular distribution in both unstressed and heat-shocked cells. In unstressed cells, Hsp104 and a green fluorescent protein-Hsp104 fusion protein were detected in both the nucleus and the cytoplasm. We demonstrate that a 17-amino-acid sequence of Hsp104 nuclear localization sequence 17 (NLS17) is sufficient to target a reporter molecule to the nucleus and is also necessary for normal Hsp104 subcellular distribution. The nuclear targeting function of NLS17 is genetically dependent on KAP95 and KAP121. In addition, wild-type Hsp104, but not an NLS17-mutated Hsp104 variant, accumulated in the nucleus of cells depleted for the general export factor Xpo1. Interestingly, severe, nonlethal heat shock enhances the nuclear levels of Hsp104 in an NLS17-independent manner. Under these conditions, we demonstrate that karyopherin-mediated nuclear transport is impaired, while the integrity of the nuclear-cytoplasmic barrier remains intact. Based on these observations, we propose that Hsp104 continues to access the nucleus during severe heat shock using a karyopherin-independent mechanism.

Detection of pseudosinusoidal epileptic seizure segments in the neonatal EEG by cascading a rule-based algorithm with a neural network.

This paper presents an approach to detect epileptic seizure segments in the neonatal electroencephalogram (EEG) by characterizing the spectral features of the EEG waveform using a rule-based algorithm cascaded with a neural network. A rule-based algorithm screens out short segments of pseudosinusoidal EEG patterns as epileptic based on features in the power spectrum. The output of the rule-based algorithm is used to train and compare the performance of conventional feedforward neural networks and quantum neural networks. The results indicate that the trained neural networks, cascaded with the rule-based algorithm, improved the performance of the rule-based algorithm acting by itself. The evaluation of the proposed cascaded scheme for the detection of pseudosinusoidal seizure segments reveals its potential as a building block of the automated seizure detection system under development.

BAG5 inhibits parkin and enhances dopaminergic neuron degeneration.

Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, are the major cause of early-onset Parkinson's disease (PD). Decreases in parkin activity may also contribute to neurodegeneration in sporadic forms of PD. Here, we show that bcl-2-associated athanogene 5 (BAG5), a BAG family member, directly interacts with parkin and the chaperone Hsp70. Within this complex, BAG5 inhibits both parkin E3 ubiquitin ligase activity and Hsp70-mediated refolding of misfolded proteins. BAG5 enhances parkin sequestration within protein aggregates and mitigates parkin-dependent preservation of proteasome function. Finally, BAG5 enhances dopamine neuron death in an in vivo model of PD, whereas a mutant that inhibits BAG5 activity attenuates dopaminergic neurodegeneration. This contrasts with the antideath functions ascribed to BAG family members and suggests a potential role for BAG5 in promoting neurodegeneration in sporadic PD through its functional interactions with parkin and Hsp70.

Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling.

Hsp104, the most potent thermotolerance factor in Saccharomyces cerevisiae, is an unusual molecular chaperone that is associated with the dispersal of aggregated, non-native proteins in vivo and in vitro. The close cooperation between Hsp100 oligomeric disaggregases and specific Hsp70 chaperone/cochaperone systems to refold and reactivate heat-damaged proteins has been dubbed a "bichaperone network". Interestingly, animal genomes do not encode a Hsp104 ortholog. To investigate the biochemical and biological consequences of introducing into human cells a stress tolerance factor that has protein refolding capabilities distinct from those already present, Hsp104 was expressed as a transgene in a human leukemic T-cell line (PEER). Hsp104 inhibited heat-shock-induced loss of viability in PEER cells, and this action correlated with reduced procaspase-3 cleavage but not with reduced c-Jun N-terminal kinase phosphorylation. Hsp104 cooperated with endogenous human Hsp70 and Hsc70 molecular chaperones and their J-domain-containing cochaperones Hdj1 and Hdj2 to produce a functional hybrid bichaperone network capable of refolding aggregated luciferase. We also established that Hsp104 shuttles across the nuclear envelope and enhances the chaperoning capacity of both the cytoplasm and nucleoplasm of intact cells. Our results establish the fundamental properties of protein disaggregase function in human cells with implications for the use of Hsp104 or related proteins as therapeutic agents in diseases associated with protein aggregation.

Amino acid substitutions in the C-terminal AAA+ module of Hsp104 prevent substrate recognition by disrupting oligomerization and cause high temperature inactivation.

Hsp104 is an important determinant of thermotolerance in yeast and is an unusual molecular chaperone that specializes in the remodeling of aggregated proteins. The structural requirements for Hsp104-substrate interactions remain unclear. Upon mild heat shock Hsp104 formed cytosolic foci in live cells that indicated co-localization of the chaperone with aggregates of thermally denatured proteins. We generated random amino acid substitutions in the C-terminal 199 amino acid residues of a GFP-Hsp104 fusion protein, and we used a visual screen to identify mutants that remained diffusely distributed immediately after heat shock. Multiple amino acid substitutions were required for loss of heat-inducible redistribution, and this correlated with complete loss of nucleotide-dependent oligomerization. Based on the multiply substituted proteins, several single amino acid substitutions were generated by site-directed mutagenesis. The singly substituted proteins retained the ability to oligomerize and detect substrates. Intriguingly, some derivatives of Hsp104 functioned well in prion propagation and multiple stress tolerance but failed to protect yeast from extreme thermal stress. We demonstrate that these proteins co-aggregate in the presence of other thermolabile proteins during heat treatment both in vitro and in vivo suggesting a novel mechanism for uncoupling the function of Hsp104 in acute severe heat shock from its functions at moderate temperatures.

Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104.

Saccharomyces cerevisiae Hsp104, a hexameric member of the Hsp100/Clp subfamily of AAA+ ATPases with two nucleotide binding domains (NBD1 and 2), refolds aggregated proteins in conjunction with Hsp70 molecular chaperones. Hsp104 may act as a "molecular crowbar" to pry aggregates apart and/or may extract proteins from aggregates by unfolding and threading them through the axial channel of the Hsp104 hexamer. Targeting Tyr-662, located in a Gly-Tyr-Val-Gly motif that forms part of the axial channel loop in NBD2, we created conservative (Phe and Trp) and non-conservative (Ala and Lys) amino acid substitutions. Each of these Hsp104 derivatives was comparable to the wild type protein in their ability to hydrolyze ATP, assemble into hexamers, and associate with heat-shock-induced aggregates in living cells. However, only those with conservative substitutions complemented the thermotolerance defect of a Deltahsp104 yeast strain and promoted refolding of aggregated protein in vitro. Monitoring fluorescence from Trp-662 showed that titration of fully assembled molecules with either ATP or ADP progressively quenches fluorescence, suggesting that nucleotide binding determines the position of the loop within the axial channel. A Glu to Lys substitution at residue 645 in the NBD2 axial channel strongly alters the nucleotide-induced change in fluorescence of Trp-662 and specifically impairs in protein refolding. These data establish that the structural integrity of the axial channel through NBD2 is required for Hsp104 function and support the proposal that Hsp104 and ClpB use analogous unfolding/threading mechanisms to promote disaggregation and refolding that other Hsp100s use to promote protein degradation.

Enhanced spatio-temporal clustering in the detection of neonatal seizures using context-based rules.

This work describes the clustering stage of a three-stage automated neonatal seizure detection system. This stage clusters spatio-temporally the short candidate seizure segments detected in prior stages, and then applies a variety of context-based rules to eliminate false detections and determine the final detected seizures. The work discusses important considerations in the implementation of rules and presents preliminary results.

Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein.

AAA proteins remodel other proteins to affect a multitude of biological processes. Their power to remodel substrates must lie in their capacity to couple substrate binding to conformational changes via cycles of nucleotide binding and hydrolysis, but these relationships have not yet been deciphered for any member. We report that when one AAA protein, Hsp104, engages polypeptide at the C-terminal peptide-binding region, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2) drives a conformational change in the middle region. This, in turn, drives ATP hydrolysis in the N-terminal ATPase domain (NBD1). This interdomain communication pathway can be blocked by mutation in the middle region or bypassed by antibodies that bind there, demonstrating the crucial role this region plays in transducing signals from one end of the molecule to the other.