Mechanistic Insights into Hsp104 Potentiation

Potentiated variants of Hsp104, a protein disaggregase from yeast, can dissolve protein aggregates connected to neurodegenerative diseases such as Parkinson disease and amyotrophic lateral sclerosis. However, the mechanisms underlying Hsp104 potentiation remain incompletely defined. Here, we establish that 2–3 subunits of the Hsp104 hexamer must bear an A503V potentiating mutation to elicit enhanced disaggregase activity in the absence of Hsp70. We also define the ATPase and substrate-binding modalities needed for potentiated Hsp104A503V activity in vitro and in vivo. Hsp104A503V disaggregase activity is strongly inhibited by the Y257A mutation that disrupts substrate binding to the nucleotide-binding domain 1 (NBD1) pore loop and is abolished by the Y662A mutation that disrupts substrate binding to the NBD2 pore loop. Intriguingly, Hsp104A503V disaggregase activity responds to mixtures of ATP and adenosine 5′-(γ-thio)-triphosphate (a slowly hydrolyzable ATP analogue) differently from Hsp104. Indeed, an altered pattern of ATP hydrolysis and altered allosteric signaling between NBD1 and NBD2 are likely critical for potentiation. Hsp104A503V variants bearing inactivating Walker A or Walker B mutations in both NBDs are inoperative. Unexpectedly, however, Hsp104A503V retains potentiated activity upon introduction of sensor-1 mutations that reduce ATP hydrolysis at NBD1 (T317A) or NBD2 (N728A). Hsp104T317A/A503V and Hsp104A503V/N728A rescue TDP-43 (TAR DNA-binding protein 43), FUS (fused in sarcoma), and α-synuclein toxicity in yeast. Thus, Hsp104A503V displays a more robust activity that is unperturbed by sensor-1 mutations that greatly reduce Hsp104 activity in vivo. Indeed, ATPase activity at NBD1 or NBD2 is sufficient for Hsp104 potentiation. Our findings will empower design of ameliorated therapeutic disaggregases for various neurodegenerative diseases

Changes in Histone H3 Acetylation on Lysine 9 Accompany Aβ 1-40 Overexpression in an Alzheimer’s Disease Yeast Model

Alzheimer’s Disease (AD), the most common type of dementia, is a neurodegenerative disease characterized by plaques of amyloid-beta (Aβ) peptides found in the cerebral cortex of the brain. The pathological mechanism by which Aβ aggregation leads to neurodegeneration remains unknown. Interestingly, genetic mutations do not explain most AD cases suggesting that other mechanisms are at play. Epigenetic mechanisms, such as histone post-translational modifications (PTMs), may provide insight into the development of AD. Here, we exploit a yeast Aβ overexpression model to map out the histone PTM landscape associated with AD. We find a modest decrease in the acetylation levels on lysine 9 of histone H3 in the context of Aβ 1-40 overexpression. This change is accompanied by a decrease in RNA levels. Our results support a potential role for H3K9ac in AD pathology and allude to the role of epigenetics in AD and other neurodegenerative diseases

Proteomic Interrogation of Human Chromatin

Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the “Chromatome”) is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes

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^A503V, 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 (IC₅₀) for Suramin-mediated inhibition of Hsp104. IC₅₀ 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

Suramin inhibits Hsp104-mediated substrate unfolding and translocation.

<p>(<b>A</b>) RepA_1–70-GFP was incubated with Hsp104^WT and GroEL_trap plus a 1∶1 mixture of ATP and ATPγS. GFP unfolding was measured by fluorescence. GroEL_trap 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_i 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

Hsp104 variants respond differently to Suramin than Hsp104^WT.

<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^WT and Hsp104^T317A, while ATP alone was used for Hsp104^A503V and Hsp104^N728A. 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_i 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