Summary There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid, but has no metazoan homologue 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 Hsp 104 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.

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.DOI: http://dx.doi.org/10.7554/eLife.05397.001

Clinical and preclinical studies suggest that nicotinic acetylcholine receptors are involved in affective disorders; therefore, the potential therapeutic value of nicotinic partial agonists as treatments of these disorders is of growing interest. This study evaluated the effects of acute and chronic administration of nicotine and the ␣4␤2 nicotinic partial agonists varenicline and sazetidine-A in mouse models of anxiety and depression. Acutely, only nicotine and varenicline had anxiolytic effects in the marble-burying test and in the novelty-induced hypophagia (NIH) test. In contrast, in animal models of antidepressant efficacy, such as the forced swim and the tail suspension test, only acute sazetidine-A had significant antidepressant-like effects. The NIH test provides an anxiety-related measure that is sensitive to the effects of chronic but not acute antidepressant treatment. Chronic nicotine and chronic sazetidine-A treatment were effective in this paradigm, but varenicline was ineffective. These results suggest that the partial agonists varenicline and sazetidine-A may have diverse therapeutic benefits in affective disorders.

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.

16Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve 17 critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding 18 domains (NBDs) power polypeptide translocation through a central channel comprised of a 19 hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. To 20 elucidate the translocation mechanism, we determined the cryo-EM structure of a hyperactive 21 ClpB variant to 2.9 Å resolution bound to the model substrate, casein in the presence of slowly 22 hydrolysable ATPγS. Distinct substrate-gripping mechanisms are identified for NBD1 and NBD2 23 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide 24 substrate adjacent the topmost NBD1 contact. NBD conformations at the spiral seam reveal how 25 2 ATP hydrolysis and substrate engagement or disengagement are precisely tuned to drive a 26 stepwise translocation cycle. 27 28 effectively dissolve self-templating fibrils formed by human neurodegenerative disease proteins 52 including α-synuclein, TDP43, FUS, and TAF15 25,28-30 . In addition, amino-terminal domains 53 (NTDs) are connected to the NBD1 by a flexible linker 7 and form an additional ring in the 54 hexamer 31 that may facilitate substrate transfer to the AAA+ domains. Substrate interaction sites 55 have been identifed 32 and the NTDs are critical for the dissolution of prions by Hsp104 33 , 56 supporting plasticity in the mechanism, however, specific functions during translocation are 57 unknown. 58 Recent cryo-EM structures of substrate-bound Hsp104 6 and ClpB 5 complexes identify an 59 asymmetric architecture of the hexamer and a remarkable spiral of pore loop-substrate contacts 60by both NBD1 and NBD2. Five protomers contact an 80 Å-long unfolded polypeptide strand via 61 the conserved Tyr residues with an approximate 6.5 Å, dipeptide spacing while a sixth protomer 62 at the spiral seam is unbound to substrate and flexible 6 . Pore loop-substrate interactions are 63 similarly spaced for both NBD1 and NBD2 rings in Hsp104 6 , but are different for ClpB, with NBD1 64 adopting a more planar arrangement compared to NBD2 5 . The architecture and substrate 65 interactions are similar to other recent structures of single and double-ring AAA+ complexes 34-36 , 66 supporting a conserved substrate interaction mechanism. However, for the double-ring 67 translocases, higher resolution structures are needed to further define substrate interactions and 68 identify how coordination between domains drives translocation. In the cryo-EM dataset of 69Hsp104 we identified an additional conformation in which the seam protomer rearranges to 70 contact the substrate at the next position, resulting in a complete spiral of contacts by the 71 hexamer, and, together, supports a two amino acid-step rotary translocation mechanism 6 . This 72 extended state was not identified in ClpB. Additionally, substrate-free structures of Hsp104 6,20 with 73 ADP and the nonhydrolyzab...