8 research outputs found
Suramin Inhibits Hsp104 ATPase and Disaggregase Activity
<div><p>Hsp104 is a hexameric AAA+ protein that utilizes energy from ATP hydrolysis to dissolve disordered protein aggregates as well as amyloid fibers. Interestingly, Hsp104 orthologues are found in all kingdoms of life except animals. Thus, Hsp104 could represent an interesting drug target. Specific inhibition of Hsp104 activity might antagonize non-metazoan parasites that depend on a potent heat shock response, while producing little or no side effects to the host. However, no small molecule inhibitors of Hsp104 are known except guanidinium chloride. Here, we screen over 16,000 small molecules and identify 16 novel inhibitors of Hsp104 ATPase activity. Excluding compounds that inhibited Hsp104 activity by non-specific colloidal effects, we defined Suramin as an inhibitor of Hsp104 ATPase activity. Suramin is a polysulphonated naphthylurea and is used as an antiprotozoal drug for African Trypanosomiasis. Suramin also interfered with Hsp104 disaggregase, unfoldase, and translocase activities, and the inhibitory effect of Suramin was not rescued by Hsp70 and Hsp40. Suramin does not disrupt Hsp104 hexamers and does not effectively inhibit ClpB, the <i>E. coli</i> homolog of Hsp104, establishing yet another key difference between Hsp104 and ClpB behavior. Intriguingly, a potentiated Hsp104 variant, Hsp104<sup>A503V</sup>, is more sensitive to Suramin than wild-type Hsp104. By contrast, Hsp104 variants bearing inactivating sensor-1 mutations in nucleotide-binding domain (NBD) 1 or 2 are more resistant to Suramin. Thus, Suramin depends upon ATPase events at both NBDs to exert its maximal effect. Suramin could develop into an important mechanistic probe to study Hsp104 structure and function.</p></div
Suramin greatly inhibits Hsp104 refolding activity.
<p>(<b>A</b>) Urea-denatured firefly luciferase aggregates were incubated for 90 min at 25°C with Hsp104 (1 µM hexamer) plus 1∶1 mixtures of ATP and ATPγS and varying concentrations of GdmCl. Luciferase reactivation was then determined and converted to % WT disaggregase activity in the presence of ATPγS: ATP. Values represent mean ±S.D. (<i>n</i> = 3–6). (<b>B</b>) Reactions were performed as in (<b>A</b>) except varying concentrations of Suramin were used. Values represent mean ±S.D. (<i>n</i> = 6–10). (<b>C</b>) Half maximal inhibitory concentration (IC<sub>50</sub>) for Suramin-mediated inhibition of Hsp104. IC<sub>50</sub> was calculating by fitting luciferase-refolding data at different Suramin concentrations. (<b>D</b>) Reactions were performed as in (<b>A</b>) except that 1∶1 mixtures of ATP and ATPγS were replaced with Hsp70 (1 µM) and Hsp40 (1 µM) plus ATP. The Hsp104 alone condition (yellow bars) was carried out with 1∶1 mixtures of ATP and ATPγS as in (<b>A</b>). Each condition is normalized to the refolding activity in each corresponding condition in the absence of inhibitor.</p
Hsp104 variants respond differently to Suramin than Hsp104<sup>WT</sup>.
<p>(<b>A</b>) Hsp104 variants (0.25 µM monomer) were incubated for 10 min with ATP (1 mM) and varying concentrations of Suramin. The amount of Pi produced was measured by absorbance at 635 nm. Absorbance values were normalized to the absorbance produced by each Hsp104 variant in the absence of inhibitor. Values represent mean ±S.D. (<i>n</i> = 6). (<b>B</b>) Urea-denatured firefly luciferase aggregates were incubated for 90 min at 25°C with Hsp104 variants (1 µM hexamer) plus 1∶1 mixtures of ATP and ATPγS (or ATP alone) and varying concentrations of Suramin. Luciferase reactivation was then determined and normalized to untreated disaggregase activity for each variant in the presence of ATPγS: ATP (or ATP alone). 1∶1 mixtures of ATP and ATPγS were used for Hsp104<sup>WT</sup> and Hsp104<sup>T317A</sup>, while ATP alone was used for Hsp104<sup>A503V</sup> and Hsp104<sup>N728A</sup>. Values represent mean ±S.D. (<i>n</i> = 6–12).</p
Sixteen molecules inhibit hydrolysis of ATP by Hsp104.
<p>Hsp104 (0.25 µM monomer) was incubated for 60 min with ATP (1 mM) plus 16,850 small-molecule drugs (10 µM). The amount of P<sub>i</sub> produced was measured by absorbance at 635 nm. Absorbance values were normalized to the absorbance produced by Hsp104 in the absence of the small molecules. Sixteen molecules were found to reproducibly interfere with Hsp104 ATPase activity. Inhibition values represent mean ±S.D. (<i>n</i> = 2).</p><p>Sixteen molecules inhibit hydrolysis of ATP by Hsp104.</p
Suramin, but not Cisplatin, greatly inhibits Hsp104 ATPase activity.
<p>(<b>A</b>) Hsp104 (0.25 µM monomer) was incubated for 60 min with ATP (1 mM) and varying concentrations of Suramin, Cisplatin and Guanidinium Chloride (GdmCl). The amount of P<sub>i</sub> produced was measured by absorbance at 635 nm. Raw absorbance values were normalized to the average absorbance yielded by Hsp104<sup>WT</sup> in the absence of inhibitor. Values represent mean ±S.D. (<i>n</i> = 6). (<b>B</b>) Hsp104 (0.25 µM monomer) was incubated for 10 min with ATP (1 mM) and varying concentrations of Suramin. Raw absorbance values were normalized to the average absorbance yielded by Hsp104<sup>WT</sup> in the absence of inhibitor. Values represent mean ±S.D. (<i>n</i> = 6).</p
Suramin inhibits Hsp104-mediated substrate unfolding and translocation.
<p>(<b>A</b>) RepA<sub>1–70</sub>-GFP was incubated with Hsp104<sup>WT</sup> and GroEL<sub>trap</sub> plus a 1∶1 mixture of ATP and ATPγS. GFP unfolding was measured by fluorescence. GroEL<sub>trap</sub> alone is shown as a negative control (red line). Fluorescence values were normalized to initial raw fluorescence for each sample. Values represent mean ±S.D. (<i>n</i> = 3). (<b>B</b>) FITC-casein (0.1 µM) was incubated at 25°C with HAP (1 µM hexamer) and ClpP (21 µM monomer) plus ATP (5 mM) and an ATP regeneration system. Degradation of FITC-casein was monitored by fluorescence. Initial fluorescence was subtracted from raw fluorescence values for each sample. HAP alone is shown as a negative control (red line). Values represent mean ±S.D. (<i>n</i> = 5).</p
ClpB displays resistance to Suramin.
<p>(<b>A</b>) ClpB (0.25 µM monomer) was incubated for 10 min with ATP (1 mM) and varying concentrations of Suramin. The amount of P<sub>i</sub> produced was measured by absorbance at 635 nm. Absorbance values were normalized to the absorbance produced by ClpB in the absence of inhibitor. Values represent mean ±S.D. (<i>n</i> = 6). (<b>B</b>) Urea-denatured firefly luciferase aggregates were incubated for 90 min at 25°C with ClpB (1 µM hexamer) in the presence of 1∶1 mixtures of ATP and ATPγS and varying concentrations of Suramin. Reactivation of luciferase was then determined by luminescence and converted to % WT ClpB activity (activity of 1 µM WT ClpB in the presence of ATP and ATPγS). Values represent mean ±S.D. (<i>n = </i>6–12).</p
Neurodegenerative Disease Proteinopathies Are Connected to Distinct Histone Post-translational Modification Landscapes
Amyotrophic
lateral sclerosis (ALS) and Parkinson’s disease
(PD) are devastating neurodegenerative diseases involving the progressive
degeneration of neurons. No cure is available for patients diagnosed
with these diseases. A prominent feature of both ALS and PD is the
accumulation of protein inclusions in the cytoplasm of degenerating
neurons; however, the particular proteins constituting these inclusions
vary: the RNA-binding proteins TDP-43 and FUS are most notable in
ALS, while α-synuclein aggregates into Lewy bodies in PD. In
both diseases, genetic causes fail to explain the occurrence of a
large proportion of cases, and thus, both are considered mostly sporadic.
Despite mounting evidence for a possible role of epigenetics in the
occurrence and progression of ALS and PD, epigenetic mechanisms in
the context of these diseases remain mostly unexplored. Here we comprehensively
delineate histone post-translational modification (PTM) profiles in
ALS and PD yeast proteinopathy models. Remarkably, we find distinct
changes in histone modification profiles for each. We detect the most
striking changes in the context of FUS aggregation: changes in several
histone marks support a global decrease in gene transcription. We
also detect more modest changes in histone modifications in cells
overexpressing TDP-43 or α-synuclein. Our results highlight
a great need for the inclusion of epigenetic mechanisms in the study
of neurodegeneration. We hope our work will pave the way for the discovery
of more effective therapies to treat patients suffering from ALS,
PD, and other neurodegenerative diseases