Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

The clinical trial landscape in amyotrophic lateral sclerosis-Past, present, and future.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease marked by progressive loss of muscle function. It is the most common adult-onset form of motor neuron disease, affecting about 16 000 people in the United States alone. The average survival is about 3 years. Only two interventional drugs, the antiglutamatergic small-molecule riluzole and the more recent antioxidant edaravone, have been approved for the treatment of ALS to date. Therapeutic strategies under investigation in clinical trials cover a range of different modalities and targets, and more than 70 different drugs have been tested in the clinic to date. Here, we summarize and classify interventional therapeutic strategies based on their molecular targets and phenotypic effects. We also discuss possible reasons for the failure of clinical trials in ALS and highlight emerging preclinical strategies that could provide a breakthrough in the battle against this relentless disease.

AAA+ Protein-Based Technologies to Counter Neurodegenerative Disease.

Protein misfolding and overloaded proteostasis networks underlie a range of neurodegenerative diseases. No cures exist for these diseases, but developing effective therapeutic agents targeting the toxic, misfolded protein species in disease is one promising strategy. AAA+ (ATPases associated with diverse cellular activities) protein translocases, which naturally unfold and translocate substrate proteins, could be potent therapeutic agents to disassemble toxic protein conformers in neurodegenerative disease. Here, we discuss repurposing AAA+ protein translocases Hsp104 and proteasome-activating nucleotidase (PAN) to alleviate the toxicity from protein misfolding in neurodegenerative disease. Hsp104 effectively protects various animal models from neurodegeneration underpinned by protein misfolding, and enhanced Hsp104 variants strongly counter neurodegenerative disease-associated protein misfolding toxicity in yeast, Caenorhabditis elegans, and mammalian cells. Similarly, a recently engineered PAN variant (PANet) mitigates photoreceptor degeneration instigated by protein misfolding in a mouse model of retinopathy. Further study and engineering of AAA+ translocases like Hsp104 and PAN will reveal promising agents to combat protein misfolding toxicity in neurodegenerative disease.

Hsp104 and Potentiated Variants Can Operate as Distinct Nonprocessive Translocases.

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104A503S, a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104A503S can operate as nonprocessive translocases for soluble substrates, indicating a "partial threading" model of translocation. Remarkably, Hsp104A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104A503S.

Potentiating Hsp104 activity via phosphomimetic mutations in the middle domain.

Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase separation by TDP-43, FUS and α-synuclein connected to devastating neurodegenerative disorders. Subtle missense mutations in the Hsp104 MD can enhance activity, indicating that post-translational modification of specific MD residues might also potentiate Hsp104. Indeed, several serine and threonine residues throughout Hsp104 can be phosphorylated in vivo. Here, we introduce phosphomimetic aspartate or glutamate residues at these positions and assess Hsp104 activity. Remarkably, phosphomimetic T499D/E and S535D/E mutations in the MD enable Hsp104 to counter TDP-43, FUS and α-synuclein aggregation and toxicity in yeast, whereas T499A/V/I and S535A do not. Moreover, Hsp104T499E and Hsp104S535E exhibit enhanced ATPase activity and Hsp70-independent disaggregase activity in vitro. We suggest that phosphorylation of T499 or S535 may elicit enhanced Hsp104 disaggregase activity in a reversible and regulated manner.

Avidity for Polypeptide Binding by Nucleotide-Bound Hsp104 Structures.

Recent Hsp104 structural studies have reported both planar and helical models of the hexameric structure. The conformation of Hsp104 monomers within the hexamer is affected by nucleotide ligation. After nucleotide-driven hexamer formation, Hsp104-catalyzed disruption of protein aggregates requires binding to the peptide substrate. Here, we examine the oligomeric state of Hsp104 and its peptide binding competency in the absence of nucleotide and in the presence of ADP, ATPγS, AMPPNP, or AMPPCP. Surprisingly, we found that only ATPγS facilitates avid peptide binding by Hsp104. We propose that the modulation between high- and low-peptide affinity states observed with these ATP analogues is an important component of the disaggregation mechanism of Hsp104.

New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

Reprogramming EF-hands for design of catalytically amplified lanthanide sensors.

We recently reported that a computationally designed catalyst nicknamed AlleyCat facilitates C-H proton abstraction in Kemp elimination at neutral pH in a selective and calcium-dependent fashion by a factor of approximately 100,000 (Korendovych et al. in Proc. Natl. Acad. Sci. USA 108:6823, 2011). Kemp elimination produced a colored product that can be easily read out, thus making AlleyCat a catalytically amplified metal sensor for calcium. Here we report that metal-binding EF-hand motifs in AlleyCat could be redesigned to incorporate trivalent metal ions without significant loss of catalytic activity. Mutation of a single neutral residue at position 9 of each of the EF-hands to glutamate results in almost a two orders of magnitude improvement of selectivity for trivalent metal ions over calcium. Development of this new lanthanide-dependent switchable Kemp eliminase, named CuSeCat EE, provides the foundation for further selectivity improvement and broadening the scope of the repertoire of metals for sensing. A concerted effort in the design of switchable enzymes has many environmental, sensing, and metal ion tracking applications.

TRIM11 Prevents and Reverses Protein Aggregation and Rescues a Mouse Model of Parkinson's Disease.

Neurodegenerative diseases are characterized by the formation and propagation of protein aggregates, especially amyloid fibrils. However, what normally suppresses protein misfolding and aggregation in metazoan cells remains incompletely understood. Here, we show that TRIM11, a member of the metazoan tripartite motif (TRIM) family, both prevents the formation of protein aggregates and dissolves pre-existing protein deposits, including amyloid fibrils. These molecular chaperone and disaggregase activities are ATP independent. They enhance folding and solubility of normal proteins and cooperate with TRIM11 SUMO ligase activity to degrade aberrant proteins. TRIM11 abrogates α-synuclein fibrillization and restores viability in cell models of Parkinson's disease (PD). Intracranial adeno-associated viral delivery of TRIM11 mitigates α-synuclein-mediated pathology, neurodegeneration, and motor impairments in a PD mouse model. Other TRIMs can also function as ATP-independent molecular chaperones and disaggregases. Thus, we define TRIMs as a potent and multifunctional protein quality-control system in metazoa, which might be applied to treat neurodegenerative diseases.

Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and α-Synuclein Proteotoxicity.

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.

Ratchet-like polypeptide translocation mechanism of the AAA+ disaggregase Hsp104.

Hsp100 polypeptide translocases are conserved members of the AAA+ family (adenosine triphosphatases associated with diverse cellular activities) that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from Saccharomyces cerevisiae solubilizes stress-induced amorphous aggregates and amyloids. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo-electron microscopy structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions are mediated by conserved, pore-loop tyrosines that contact an 80-angstrom-long unfolded polypeptide along the axial channel. Two protomers undergo a ratchet-like conformational change that advances pore loop-substrate interactions by two amino acids. These changes are coupled to activation of specific nucleotide hydrolysis sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and substrate-responsive flexibility during Hsp104-catalyzed disaggregation.

Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity.

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.

Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation.

Hsp104, a conserved AAA+ protein disaggregase, promotes survival during cellular stress. Hsp104 remodels amyloids, thereby supporting prion propagation, and disassembles toxic oligomers associated with neurodegenerative diseases. However, a definitive structural mechanism for its disaggregase activity has remained elusive. We determined the cryo-EM structure of wild-type Saccharomyces cerevisiae Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the 12 AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, thus explaining roles in ATP hydrolysis and protein disaggregation. Remarkably, substrate-binding pore loops line the channel in a spiral arrangement optimized for substrate transfer across the AAA+ domains, thereby establishing a continuous path for polypeptide translocation.

Design of an allosterically regulated retroaldolase.

We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca(2+) ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue.