Upregulation of <i>Bcl-2</i> protects GD cells from apoptosis induced by proteostasis modulators.

<p>(<b>A</b>) Relative mRNA expression levels of Bcl-2 in cells treated with EerI (2 and 6 µM), MG-132 (0.6 µM), and fluvastatin (100 nM) for 24 hrs evaluated by quantitative RT-PCR and calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061418#pone-0061418-g004" target="_blank">Figure 4</a> (ANOVA, p<0.05). (<b>B</b>) PI binding population change (%) of cells treated with EerI (2 and 6 µM), MG-132 (0.6 µM), and fluvastatin (100 nM) for 16 hrs compared to untreated cells (p<0.01). The data is reported as mean ± SD. Number of total counted cells: 10,000. (<b>C</b>) L444P GC activities of GD fibroblasts treated with EerI (2 and 6 µM), MG-132 (0.6 µM), and fluvastatin (100 nM) for 48 hrs. Relative GC activities were evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061418#pone-0061418-g001" target="_blank">Figure 1B</a> (ANOVA, p<0.01). Experiments were repeated three times and data points are reported as mean ± SD. MG, MG-132; Flu, fluvastatin.</p

Silencing <i>Bcl-2</i> aggravates the apoptotic effect of proteostasis modulators.

<p>(<b>A</b>) Relative mRNA expression levels of Bcl-2 in GD fibroblasts incubated with siRNA for 48 hrs and treated with lacidipine (10 µM) and EerI (6 µM) for additional 24 hrs evaluated by quantitative RT-PCR and calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061418#pone-0061418-g004" target="_blank">Figure 4</a> (ANOVA, p<0.05). (<b>B</b>) Flow cytometry analysis of PI binding population change (%) of GD fibroblasts incubated with siRNA for 48 hrs followed by lacidipine (10 µM) and EerI (6 µM) treatment for 16 hrs (ANOVA, p<0.01). The change in PI binding population (%) was calculated by subtracting PI binding values of cells treated with small molecules to that of cells only incubated with control siRNA. The data is reported as mean ± SD. Number of total counted cells: 10,000. (<b>C</b>) Relative L444P GC activities in cells incubated with Bcl-2 or control siRNA for 48 hrs followed by lacidipine (10 µM) and EerI (6 µM) treatment for additional 48 hrs. Relative GC activities were evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061418#pone-0061418-g001" target="_blank">Figure 1B</a> (ANOVA, p<0.01). Experiments were repeated three times and data points are reported as mean ± SD. Lac, lacidipine.</p

Co-treatment with EerI and lacidipine enhances lysosomal trafficking and activity of L444P GC.

<p>(<b>A</b>) Lacidipine and EerI modulate distinct pathways of the proteostasis network that regulate the processing of GC. Lacidipine enhances ER folding by restoring Ca²⁺ homeostasis in GD cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061418#pone.0061418-Wang2" target="_blank">[14]</a>. Specifically, lacidipine inhibits extracellular Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels (LTCC) on the plasma membrane and blocks ER Ca²⁺ efflux through ryanodine receptors (RyRs) on the ER membrane, thus restoring the intracellular gradient of [Ca²⁺]. EerI treatment enhances retention of unstable proteins in the ER. Particularly, EerI inhibits p97 ATPase activity, which promotes retro-translocation of misfolded substrates from the ER to the cytoplasm for ER-associated degradation (ERAD). (<b>B</b>) L444P GC activities of GD cells treated with a range of concentrations of EerI and constant doses of lacidipine (5, 10, or 20 µM) for 48 hrs. Relative GC activities were evaluated by normalizing GC activities measured in treated cells to the activity in untreated cells (left y axis), (ANOVA, p<0.01 if not specified; *p<0.001). The corresponding fraction of WT GC activity is also reported (right y axis). Experiments were repeated three times and data points are reported as mean ± SD. Lac, lacidipine. (<b>C–D</b>) Immunofluorescence microscopy of GC and CNX (an ER marker), and GC and LAMP-1 (a lysosomal marker) in L444P GC fibroblasts. Cells were treated with EerI (6 µM), and lacidipine (10 µM) for 48 hrs. (<b>C</b>) Colocalization of CNX (grey, column 1) and GC (red, column 2) is shown in pink (column 3). (<b>D</b>) Colocalization of LAMP-1 (blue, column 1) and GC (red, column 2) is shown in purple (column 3). Heatmaps of co-localization images were obtained with NIH ImageJ analysis software (column 4). Hot colors represent positive correlation (co-localization), whereas cold colors represent negative correlation (exclusion).</p

Lacidipine reduces cytosolic [Ca²⁺] in GD fibroblasts treated with EerI.

<p>GD fibroblasts were cultured with lacidipine (10 µM) and EerI (6 µM) for 5, 10, 20 and 40 min, respectively. Cytosolic [Ca²⁺] level was evaluated by measuring excitation 340/380 ratio of fura-2 acetoxymethyl ester and normalized to that at time zero. Each data point was repeated three times and reported as mean ± SD.</p

Lacidipine attenuates induction of apoptosis in GD cells treated with EerI.

<p>(<b>A</b>) Flow cytometry histograms of Annexin V-FITC fluorescence intensities (x-axis, log scale) plotted against cell counts (y-axis, linear scale) obtained from the analysis of untreated cells and cells treated with lacidipine (10 µM) and EerI (6 µM). Three independent experiments were conducted and results of one representative experiment are reported. (<b>B</b>) PI binding population change (%) of cells treated with lacidipine (10 µM) and EerI (6 µM) for 16 hrs compared to untreated cells (ANOVA, p<0.01). Number of total cells counted: 10,000. The data is reported as mean ± SD.</p

Lacidipine remodels the UPR pathway in GD fibroblasts treated with EerI.

<p>Cells were treated with lacidipine (10 µM) and EerI (6 µM) for 24 hrs. (<b>A</b>) Xbp-1 mRNA splicing was determined by RT-PCR followed by gel electrophoresis. (<b>B</b>) Spliced Xbp-1 band intensities were quantified with NIH ImageJ analysis software. Relative mRNA expression levels of (<b>C</b>) ATF4, (<b>D</b>) CHOP, (<b>E</b>) Bcl-2, and (<b>F</b>) GC were obtained by quantitative RT-PCR, corrected by the expression of the housekeeping gene GAPDH, and normalized by that of untreated cells (ANOVA, p<0.05). The data is reported as mean ± SD. (<b>G</b>) Western blot analysis of cells treated with EerI (6 µM) and lacidipine (10 µM) for 48 hrs using GC specific antibody. GAPDH expression was used as a loading control. (<b>H</b>) Western blot band quantification. GC bands were quantified by NIH ImageJ analysis software and corrected by GAPDH band intensities.</p

Quantitatively Predictable Control of Cellular Protein Levels through Proteasomal Degradation

Protein function is typically studied and engineered by modulating protein levels within the complex cellular environment. To achieve fast, targeted, and predictable control of cellular protein levels without genetic manipulation of the target, we developed a technology for post-translational depletion based on a bifunctional molecule (NanoDeg) consisting of the antigen-binding fragment from the <i>Camelidae</i> species heavy-chain antibody (nanobody) fused to a degron signal that mediates degradation through the proteasome. We provide proof-of-principle demonstration of targeted degradation using a nanobody against the green fluorescent protein (GFP). Guided by predictive modeling, we show that customizing the NanoDeg rate of synthesis, rate of degradation, and mode of degradation enables quantitative and predictable control over the target’s levels. Integrating the GFP-specific NanoDeg within a genetic circuit based on stimulus-dependent GFP output results in enhanced dynamic range and resolution of the output signal. By providing predictable control over cellular proteins’ levels, the NanoDeg system could be readily used for a variety of systems-level analyses of cellular protein function

Genetic and Chemical Activation of TFEB Mediates Clearance of Aggregated α-Synuclein

<div><p>Aggregation of α-synuclein (α-syn) is associated with the development of a number of neurodegenerative diseases, including Parkinson’s disease (PD). The formation of α-syn aggregates results from aberrant accumulation of misfolded α-syn and insufficient or impaired activity of the two main intracellular protein degradation systems, namely the ubiquitin-proteasome system and the autophagy-lysosomal pathway. In this study, we investigated the role of transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in preventing the accumulation of α-syn aggregates in human neuroglioma cells. We found that TFEB overexpression reduces the accumulation of aggregated α-syn by inducing autophagic clearance of α-syn. Furthermore, we showed that pharmacological activation of TFEB using 2-hydroxypropyl-β-cyclodextrin promotes autophagic clearance of aggregated α-syn. In summary, our findings demonstrate that TFEB modulates autophagic clearance of α-syn and suggest that pharmacological activation of TFEB is a promising strategy to enhance the degradation of α-syn aggregates.</p></div

HPβCD treatment induces activation of TFEB in H4/α-syn-GFP cells.

<p><b>a-b)</b> Immunofluorescence microscopy analysis of TFEB subcellular localization in H4/α-syn-GFP cells treated with HPβCD (1 mM). TFEB nuclear localization was monitored using a TFEB-specific antibody and DAPI nuclear stain. Colocalization of DAPI (blue, row 1) and TFEB (red, row 2) is shown in purple (row 3). Scale bar represents 10 μm. <b>c)</b> Percentage of HPβCD-treated cells presenting TFEB nuclear localization. Representative fields containing 50–100 cells were analyzed (p < 0.05). <b>d)</b> Relative mRNA expression levels of representative CLEAR network genes in H4/α-syn-GFP cells treated with HPβCD (1 mM) for 24 h. <i>GBA</i>, <i>HEXA</i>, and <i>LAMP1</i> mRNA expression levels were obtained by qRT-PCR and calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120819#pone.0120819.g002" target="_blank">Fig. 2C</a>. Data are reported as mean ± SD (n ≥ 3; p < 0.01).</p

TFEB overexpression results in reduced accumulation of α-syn aggregates.

<p><b>a)</b> Fluorescence microscopy analyses of H4/α-syn-GFP cells transduced to express TFEB-3xFLAG or S142A TFEB-3xFLAG. Images of α-syn-GFP fluorescence (green, column 1) and aggregates, detected using the ProteoStat dye (red, column 2), were merged (column 3) and analyzed using NIH ImageJ software. Representative images are reported. Scale bar represents 20 μm. <b>b)</b> Total protein aggregation in H4/α-syn-GFP cells transduced to express TFEB-3xFLAG or S142A TFEB-3xFLAG. Total protein aggregation was quantified by measuring binding of the ProteoStat dye by flow cytometry. The aggregation propensity factor (APF) was calculated as described in Methods and normalized to TFEB mRNA expression. Data are reported as mean ± SD (n ≥ 3; p < 0.01). <b>c)</b> Fluorescence microscopy analyses of H4/α-syn-GFP cells treated with control siRNA or <i>TFEB</i> siRNA. Images were analyzed as described in (a). Representative images are reported. Scale bar represents 20 μm. <b>d)</b> Total protein aggregation in H4/α-syn-GFP treated with control siRNA or <i>TFEB</i> siRNA. Total protein aggregation was quantified as described in (b). Data are reported as mean ± SD (n ≥ 3; p < 0.01). e) Immunofluorescence microscopy analyses of TFEB subcellular localization in H4/α-syn-GFP cells transduced to express TFEB-3xFLAG or S142A TFEB-3xFLAG. TFEB nuclear localization was monitored using a FLAG-specific antibody and DAPI nuclear stain. Colocalization of DAPI (blue, column 1) and TFEB-3xFLAG (red, column 2) is shown in purple (column 3). Representative images are reported. Scale bar represents 10 μm. <b>f)</b> Percentage of cells transduced as described in (e) presenting TFEB nuclear localization. Representative fields containing 50–100 cells were analyzed (p < 0.05).</p