Gorenberg, E. L. | bioRxiv Science

bioRxiv2024

Defining a small-molecule stimulator of the human Hsp70-disaggregase system with selectivity for DnaJB proteins

Hsp70, Hsp40, and Hsp110 form a human protein-disaggregase system that solubilizes and reactivates proteins trapped in aggregated states. However, this system fails to maintain proteostasis in fatal neurodegenerative diseases. Here, we potentiate the human Hsp70-disaggregase system pharmacologically. By scouring a collection of dihydropyrimidines, we disambiguate a small molecule that specifically stimulates the Hsp70-disaggregase system against disordered aggregates and -synuclein fibrils. The newly identified lead compound stimulates the disaggregase activity of multiple active human Hsp70, Hsp40, Hsp110 chaperone sets, with selectivity for combinations that include DnaJB1 or DnaJB4 as the Hsp40. We find that the relative stoichiometry of Hsp70, Hsp40, and Hsp110 dictates disaggregase activity. Remarkably, our lead compound shifts the composition of active chaperone stoichiometries by preferentially activating combinations with lower DnaJB1 concentrations. Our findings unveil a small molecule that stimulates the Hsp70-disaggregase system, even at suboptimal chaperone stoichiometries, which could be developed for the treatment of neurodegenerative diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/575109v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f1b574org.highwire.dtl.DTLVardef@1bfd3aborg.highwire.dtl.DTLVardef@e191e3org.highwire.dtl.DTLVardef@130d19c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Identification of palmitoyl protein thioesterase 1 substrates defines roles for synaptic depalmitoylation

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause Neuronal Ceroid Lipofuscinosis type 1 (CLN1), a devastating neurodegenerative disease. Here, we provide a resource identifying PPT1 substrates. We utilized Acyl Resin-Assisted Capture and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated >100 novel PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation regulates disulfide bond formation in a tertiary screen analyzing post-translational modifications. Collectively, the diverse PPT1 substrates highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of CLN1, and advance our basic understanding of the purpose of depalmitoylation. HighlightsO_LI[~]10% of palmitoylated proteins are palmitoyl protein thioesterase 1 (PPT1) substrates C_LIO_LIUnbiased proteomic approaches identify 9 distinct classes of PPT1 substrates, including synaptic adhesion molecules and endocytic proteins C_LIO_LIProtein degradation does not require depalmitoylation by PPT1 C_LIO_LIDepalmitoylation mediates disulfide bond formation in transmembrane PPT1 substrates C_LI

neuroscience↗