Semen harbors amyloid fibrils formed by proteolytic fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120) and semenogelins (SEM1 and SEM2) that potently enhance HIV infectivity. Amyloid but not soluble forms of these peptides enhance HIV infection. Thus, agents that remodel these amyloid fibrils could prevent HIV transmission. Here, we confirm that the green tea polyphenol, epigallocatechin-3-gallate (EGCG), slowly remodels fibrils formed by PAP248-286 termed SEVI (semen derived enhancer of viral infection) and also exerts a direct anti-viral effect. We elucidate for the first time that EGCG remodels PAP85-120, SEM1(45-107), and SEM2(49-107) fibrils more rapidly than SEVI fibrils. We establish EGCG as the first small molecule that can remodel all four classes of seminal amyloid. The combined anti-amyloid and anti-viral properties of EGCG could have utility in preventing HIV transmission.

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.

Despite its discovery over 30 years ago, human immunodeficiency virus (HIV) continues to threaten public health worldwide. Semen is the principal vehicle for the transmission of this retrovirus and several endogenous peptides in semen, including fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120) and semenogelins (SEM1 and SEM2), assemble into amyloid fibrils that promote HIV infection. For example, PAP248-286 fibrils, termed SEVI (Semen derived Enhancer of Viral Infection), potentiate HIV infection by up to 105-fold. Fibrils enhance infectivity by facilitating virion attachment and fusion to target cells, whereas soluble peptides have no effect. Importantly, the stimulatory effect is greatest at low viral titers, which mimics mucosal transmission of HIV, where relatively few virions traverse the mucosal barrier. Devising a method to rapidly reverse fibril formation (rather than simply inhibit it) would provide an innovative and urgently needed preventative strategy for reducing HIV infection via the sexual route. Targeting a host-encoded protein conformer represents a departure from traditional microbicidal approaches that target the viral machinery, and could synergize with direct antiviral approaches. Here, we review the identification of these amyloidogenic peptides, their mechanism of action, and various strategies for inhibiting their HIV-enhancing effects.

Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.

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.

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.

Hsp104 is an AAA+ protein disaggregase with powerful amyloid-remodeling activity. All nonmetazoan eukaryotes express Hsp104 while eubacteria express an Hsp104 ortholog, ClpB. However, most studies have focused on Hsp104 from Saccharomyces cerevisiae and ClpB orthologs from two eubacterial species. Thus, the natural spectrum of Hsp104/ClpB molecular architectures and protein-remodeling activities remains largely unexplored. Here, we report two structures of Hsp104 from the thermophilic fungus Calcarisporiella thermophila (CtHsp104), a 2.70Å crystal structure and 4.0Å cryo-electron microscopy structure. Both structures reveal left-handed, helical assemblies with all domains clearly resolved. We thus provide the highest resolution and most complete view of Hsp104 hexamers to date. We also establish that CtHsp104 antagonizes several toxic protein-misfolding events in vivo where S. cerevisiae Hsp104 is ineffective, including rescue of TDP-43, polyglutamine, and α-synuclein toxicity. We suggest that natural Hsp104 variation is an invaluable, untapped resource for illuminating therapeutic disaggregases for fatal neurodegenerative diseases.

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 in C. 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.