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The environmental species Pseudomonas aeruginosa thrives in a variety of habitats. Within the epidemic population structure of P. aeruginosa, occassionally highly successful clones that are equally capable to succeed in the environment and the human host arise. Framed by a highly conserved core genome, individual members of successful clones are characterized by a high variability in their accessory genome. The abundance of successful clones might be funded in specific features of the core genome or, although not mutually exclusive, in the variability of the accessory genome. In clone C, one of the most predominant clones, the plasmid pKLC102 and the PACGI-1 genomic island are two ubiquitous accessory genetic elements. The conserved transmissible locus of protein quality control (TLPQC) at the border of PACGI-1 is a unique horizontally transferred compository element, which codes predominantly for stress-related cargo gene products such as involved in protein homeostasis. As a hallmark, most TLPQC xenologues possess a core genome equivalent. With elevated temperature tolerance as a characteristic of clone C strains, the unique P. aeruginosa and clone C specific disaggregase ClpG is a major contributor to tolerance. As other successful clones, such as PA14, do not encode the TLPQC locus, ubiquitous denominators of success, if existing, need to be identified., Molecular epidemiology of Pseudomonas aeruginosa unraveled the occurence of few predominant clones that can thrive in various habitats with clone C one of the most abundant group of closely related strains that harbor common and strain specific entities which provide unique features such as xenologues of core genome genes involved in protein homeostasis to clone C strains.

Neurodegenerative diseases (NDs) are increasingly positioned as leading causes of global deaths. The accelerated aging of the population and its strong relationship with neurodegeneration forecast these pathologies as a huge global health problem in the upcoming years. In this scenario, there is an urgent need for understanding the basic molecular mechanisms associated with such diseases. A major molecular hallmark of most NDs is the accumulation of insoluble and toxic protein aggregates, known as amyloids, in extracellular or intracellular deposits. Here, we review the current knowledge on how molecular chaperones, and more specifically a ternary protein complex referred to as the human disaggregase, deals with amyloids. This machinery, composed of the constitutive Hsp70 (Hsc70), the class B J-protein DnaJB1 and the nucleotide exchange factor Apg2 (Hsp110), disassembles amyloids of α-synuclein implicated in Parkinson's disease as well as of other disease-associated proteins such as tau and huntingtin. We highlight recent studies that have led to the dissection of the mechanism used by this chaperone system to perform its disaggregase activity. We also discuss whether this chaperone-mediated disassembly mechanism could be used to solubilize other amyloidogenic substrates. Finally, we evaluate the implications of the chaperone system in amyloid clearance and associated toxicity, which could be critical for the development of new therapies., This research was funded by MCI/AEI/FEDER, UE (grant PID2019-111068GB-I00) and by the Basque Government (grant IT1201-19). L.V.-C. is the recipient of a predoctoral fellowship from the UPV/EHU and N.O. holds a contract funded by Fundacion Biofisika Bizkaia.

11 pags., 5 figs., α-synuclein aggregation is present in Parkinson’s disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially dis-aggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders., This work was supported by MCI/AEI/FEDER, UE (Grants PGC2018-101282-B-I00 to J.M.G.V, PGC2018-096335-B-100 to N.C., and PID2019-111068GB-I00 to A.M.), MINECO/FEDER, UE (Grants RYC-2012-12068 and BFU2015-64119-P to N.C.), and by the Basque Government (Grant IT1201-19 to A.M. and A.P.). A.C. also acknowledges funding from MCIU, PID2019-111096GA-I00; MCIU/AEI/FEDER MINECOG19/P66 , RYC2018-024686-I, and Basque Government T1270-19. L.S. acknowledges support from the University of California, Davis. A.F. thanks a predoctoral fellowship from the Basque Government. The technical and human support provided by the microscopy service of SGIker (UPV/EHU/ERDF, EU) is acknowledged. We thank J. M. Valpuesta and J. Cuellar for the visualization of α-syn oligomers by EM

Neurodegenerative diseases and other protein-misfolding disorders represent a longstanding biomedical challenge, and effective therapies remain largely elusive. This failure is due, in part, to the recalcitrant and diverse nature of misfolded protein conformers. Recent work has uncovered that many aggregation-prone proteins can also undergo liquid–liquid phase separation, a process by which macromolecules self-associate to form dense condensates with liquid properties that are compositionally distinct from the bulk cellular milieu. Efforts to combat diseases caused by toxic protein states focus on exploiting or enhancing the proteostasis machinery to prevent and reverse pathological protein conformations. Here, we discuss recent advances in elucidating and engineering therapeutic agents to combat the diverse aberrant protein states that underlie protein-misfolding disorders., Summary: This Special Article discusses cutting-edge approaches to counter aberrant phase transitions in disease using protein disaggregase- and RNA-based strategies.

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration inC. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.

Aggregates of α-synuclein contribute to the etiology of Parkinson's Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson's Disease. The protective role of PDI in Parkinson's Disease and other neurodegenerative disorders is linked to its chaperone function, yet the mechanism of neuroprotection remains unclear. Using Thioflavin-T fluorescence and transmission electron microscopy, we show here for the first time that PDI can break down nascent fibrils of α-synuclein. Mature fibrils were not affected by PDI. Another PDI family member, ERp57, could prevent but not reverse α-synuclein aggregation. The disaggregase activity of PDI was effective at a 1:50 molar ratio of PDI:α-synuclein and was blocked by S-nitrosylation. PDI could not reverse the aggregation of malate dehydrogenase, which indicated its disaggregase activity does not operate on all substrates. These findings establish a previously unrecognized disaggregase property of PDI that could underlie its neuroprotective function.

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SUMMARY Hsp104 is an AAA+ protein disaggregase, which can be potentiated via diverse mutations in its autoregulatory middle domain (MD) to mitigate toxic misfolding of TDP-43, FUS, and α-synuclein implicated in fatal neurodegenerative disorders. Problematically, potentiated MD variants can exhibit off-target toxicity. Here, we mine disaggregase sequence space to safely enhance Hsp104 activity via single mutations in nucleotide-binding domain 1 (NBD1) or NBD2. Like MD variants, NBD variants counter TDP-43, FUS, and α-synuclein toxicity and exhibit elevated ATPase and disaggregase activity. Unlike MD variants, non-toxic NBD1 and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity. Potentiating substitutions alter NBD1 residues that contact ATP, ATP-binding residues, or the MD. Mutating the NBD2 protomer interface can also safely ameliorate Hsp104. Thus, we disambiguate allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity., In Brief Tariq et al. disambiguate the allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity. Non-toxic nucleotide-binding domain 1 (NBD1) and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity connected to neurodegenerative disease., Graphical Abstract

Amyloid fibrils are protein homopolymers that adopt diverse cross-β conformations. Some amyloid fibrils are associated with the pathogenesis of devastating neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Conversely, functional amyloids play beneficial roles in melanosome biogenesis, long-term memory formation and release of peptide hormones. Here, we showcase advances in our understanding of amyloid assembly and structure, and how distinct amyloid strains formed by the same protein can cause distinct neurodegenerative diseases. We discuss how mutant steric zippers promote deleterious amyloidogenesis and aberrant liquid-to-gel phase transitions. We also highlight effective strategies to combat amyloidogenesis and related toxicity, including: (1) small-molecule drugs (e.g. tafamidis) to inhibit amyloid formation or (2) stimulate amyloid degradation by the proteasome and autophagy, and (3) protein disaggregases that disassemble toxic amyloid and soluble oligomers. We anticipate that these advances will inspire therapeutics for several fatal neurodegenerative diseases., Summary: This Review showcases important advances in our understanding of amyloid structure, assembly and disassembly, which are inspiring novel therapeutic strategies for amyloid disorders.

ATPases associated with diverse cellular activities (AAA+) form a superfamily of proteins involved in a variety of functions and are characterized by the presence of an ATPase module containing two conserved motifs known as Walker A and Walker B. ClpB and Hsp104, chaperones that have disaggregase activities, are members of a subset of this superfamily, known as the AAA family, and are characterized by the presence of a second highly conserved motif, known as the second region of homology (SRH). Hsp104 and its homolog Hsp78 (78 kDa heat shock protein) are representatives of the Clp family in yeast. The structure and function of Hsp78 is reviewed and the possible existence of other homologs in metazoans is discussed.

ClpB of E. coli and yeast Hsp104 are homologous molecular chaperones and members of the AAA+ (ATPases Associated with various cellular Activities) superfamily of ATPases. They are required for thermotolerance and function in disaggregation and reactivation of aggregated proteins that form during severe stress conditions. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro. In yeast, the propagation of prions depends upon Hsp104. Since protein aggregation and amyloid formation are associated with many diseases, including neurodegenerative diseases and cancer, understanding how disaggregases function is important. In this study, we have explored the innate substrate preferences of ClpB and Hsp104 in the absence of the DnaK and Hsp70 chaperone system. The results suggest that substrate specificity is determined by nucleotide binding domain-1.□

Protein misfolding is implicated in numerous lethal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD). There are no therapies that reverse these protein-misfolding events. We aim to apply Hsp104, a hexameric AAA+ protein from yeast, to target misfolded conformers for reactivation. Hsp104 solubilizes disordered aggregates and amyloid, but has limited activity against human neurodegenerative disease proteins. Thus, we have previously engineered potentiated Hsp104 variants that suppress aggregation, proteotoxicity and restore proper protein localization of ALS and PD proteins in Saccharomyces cerevisiae, and mitigate neurodegeneration in an animal PD model. Here, we establish that potentiated Hsp104 variants possess broad substrate specificity and, in yeast, suppress toxicity and aggregation induced by wild-type TDP-43, FUS and α-synuclein, as well as missense mutant versions of these proteins that cause neurodegenerative disease. Potentiated Hsp104 variants also rescue toxicity and aggregation of TAF15 but not EWSR1, two RNA-binding proteins with a prion-like domain that are connected with development of ALS and frontotemporal dementia. Thus, potentiated Hsp104 variants are not entirely non-specific. Indeed, they do not unfold just any natively folded protein. Rather, potentiated Hsp104 variants are finely tuned to unfold proteins bearing short unstructured tracts that are not recognized by wild-type Hsp104. Our studies establish the broad utility of potentiated Hsp104 variants.

Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector.

The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of protein aggregates, an insoluble association of polypeptides that harm cell physiology, either by themselves or in the process of formation. Several biological processes have evolved to prevent and eliminate the existence of non-functional and amyloidogenic aggregates, as they are associated with several human pathologies. Molecular chaperones and heat shock proteins are specialized in controlling the quality of the proteins in the cell, specifically by aiding proper folding, and dissolution and clearance of already formed protein aggregates. The latter is a function of disaggregases, mainly represented by the ClpB/Hsp104 subfamily of molecular chaperones, that are ubiquitous in all organisms but, surprisingly, have no orthologs in the cytosol of metazoan cells. This review aims to describe the characteristics of disaggregases and to discuss the function of yeast Hsp104, a disaggregase that is also involved in prion propagation and inheritance. O processo de enovelamento é um evento importante para o tempo de vida de uma proteína, uma vez que é essencial para a função adequada da proteína e, por conseguinte, para a fisiologia celular. Enovelamento inadequado, ou mau enovelamento, pode não só conduzir à perda de função, mas também para a formação de agregados de proteína, uma associação insolúvel de polipeptídos que prejudicam a fisiologia celular, quer por si próprios ou no processo de formação. Vários processos biológicos têm evoluído para prevenir e eliminar a existência de agregados não funcionais e amiloidogênicos, uma vez que estão associados com várias patologias humanas. Chaperonas moleculares e proteínas de choque térmico são especializadas no controle de qualidade de proteínas na célula, especificamente, auxiliando o enovelamento correto, dissolução e remoção de agregados de proteína já formados. Esta última é uma função das 'disagregases', representadas principalmente pela subfamília ClpB/ Hsp104 de chaperonas moleculares, que são comuns em todos os organismos, mas, surpreendentemente, não possuem ortólogos no citosol de células de metazoários. Esta revisão tem como objetivo descrever as características das disagregases e discutir a função da Hsp104 de levedura, uma disagregase que também está envolvida na propagação e herança de príons.

By virtue of their general ability to bind (hold) translocating or unfolding polypeptides otherwise doomed to aggregate molecular chaperones are commonly dubbed "holdases". Yet chaperones also carry physiological functions that do not necessitate prevention of aggregation such as altering the native states of proteins as in the disassembly of SNARE complexes and clathrin coats. To carry such physiological functions major members of the Hsp70 Hsp110 Hsp100 and Hsp60/CCT chaperone families act as catalytic unfolding enzymes or unfoldases that drive iterative cycles of protein binding unfolding/pulling and release. One unfoldase chaperone may thus successively convert many misfolded or alternatively folded polypeptide substrates into transiently unfolded intermediates which once released can spontaneously refold into low affinity native products. Whereas during stress a large excess of non catalytic chaperones in holding mode may optimally prevent protein aggregation after the stress catalytic disaggregases and unfoldases may act as nanomachines that use the energy of ATP hydrolysis to repair proteins with compromised conformations. Thus holding and catalytic unfolding chaperones can act as primary cellular defenses against the formation of early misfolded and aggregated proteotoxic conformers in order to avert or retard the onset of degenerative protein conformational diseases. © 2014 The Author(s).