Journal: Redox Biology

Article Title: Deficient immunoproteasome assembly drives gain of α-synuclein pathology in Parkinson's disease

doi: 10.1016/j.redox.2021.102167

Figure Lengend Snippet: Immunoproteasome function is inhibited in α-Syn overexpressed SH-SY5Y cells and A53T α-Syn transgenic mice. (A) Cluster of gene expression profiles in WT α-Syn or A53T α-Syn overexpressed SH-SY5Y cells graphed as heatmap. (B) The constitution of 20S core particle of immunoproteasome and DEGs associated with immunoproteasome β1i (PSMB9) , β2i (PSMB10) , and β5i (PSMB8) were visualized using heatmap. The protein levels of immunoproteasome β1i, β2i and β5i subunits were determined by Western blot in SH-SY5Y cells (C, D) and in the SN of A53T α-Syn transgenic mice (G – J) . Chymotrypsin-like (E, K) and trypsin-like (F, L) activity were measured by fluorescence from the breakdown of Suc-LLVY-AMC and Z-ARR-AMC fluorogenic substrate, respectively. * P < 0.05, ** P < 0.01.

Article Snippet: To confirm the specificity of immunoproteasome activities, we analyzed proteolytic activities in the presence or absence of selective β1i inhibitor ML604440 (MCE, 500 nM) and β5i inhibitor ONX0914 (MCE, 500 nM), respectively.

Techniques: Transgenic Assay, Gene Expression, Western Blot, Activity Assay, Fluorescence

Journal: Redox Biology

Article Title: Deficient immunoproteasome assembly drives gain of α-synuclein pathology in Parkinson's disease

doi: 10.1016/j.redox.2021.102167

Figure Lengend Snippet: β5i interacts with PLK2 and participates in its degradation. (A, B) Endogenous interactions between β5i and PLK2 were determined in SH-SY5Y cells immunoprecipitated with anti-β5i or anti-PLK2 antibody and analyzed by Western blot to detect PLK2 and β5i protein levels. (C, D) SH-SY5Y cells were transfected with the indicated plasmids. The interactions between β5i and PLK2 were determined by immunoprecipitation with anti-HA or anti-Myc antibody and analyzed by Western blot with the indicated antibodies. (E, F) The mRNA levels of PLK2 in SH-SY5Y cells with β5i overexpression or β5i knockdown were determined by real-time PCR. (G, H) The ubiquitylation levels of PLK2 in SH-SY5Y cells with β5i overexpression or β5i knockdown were examined by in vitro ubiquitin conjugation assay. The protein levels of PLK2 in SH-SY5Y cells with β5i overexpression (I, J) or β5i knockdown (K, L) were determined by Western blot. The half-life of PLK2 was assesses in SH-SY5Y cells with β5i overexpression (M, N) or β5i knockdown (O, P) treated with 10 μg/mL CHX for the indicated time. * P < 0.05.

Article Snippet: To confirm the specificity of immunoproteasome activities, we analyzed proteolytic activities in the presence or absence of selective β1i inhibitor ML604440 (MCE, 500 nM) and β5i inhibitor ONX0914 (MCE, 500 nM), respectively.

Techniques: Immunoprecipitation, Western Blot, Transfection, Over Expression, Knockdown, Real-time Polymerase Chain Reaction, In Vitro, Ubiquitin Proteomics, Conjugation Assay

Journal: Redox Biology

Article Title: Deficient immunoproteasome assembly drives gain of α-synuclein pathology in Parkinson's disease

doi: 10.1016/j.redox.2021.102167

Figure Lengend Snippet: Immunoproteasome deficiency induces α-Syn phosphorylation and accumulation. (A) Immunostaining of pS129 α-Syn in WT α-Syn or A53T α-Syn transfected SH-SY5Y cells. The protein levels of PLK2, pS129 α-Syn in β5i overexpressed SH-SY5Y cells in the presence or absence of BI 2536 (B – D) and in the SN of 12-month A53T α-Syn transgenic mice with dopaminergic neurons-specific β5i overexpression (E – G) . (H) Immunofluorescence was applied to observe nigral pS129 α-Syn in 12-month A53T α-Syn transgenic mice with dopaminergic neurons-specific β5i overexpression. The soluble and insoluble α-Syn in β5i overexpressed SH-SY5Y cells in the presence or absence of BI 2536 (I, J) and in the SN of 12-month A53T α-Syn transgenic mice with dopaminergic neurons-specific β5i overexpression (K, L) . (M) The aggregation of α-Syn was detected by ThT immunofluorescence in the SN of 12-month A53T α-Syn transgenic mice with dopaminergic neurons-specific β5i overexpression. Representative immunofluorescent images of TH staining (N) and stereological quantification of TH-positive neurons (O) in the SN of 12-month A53T α-Syn transgenic mice with dopaminergic neurons-specific β5i overexpression. (P, Q) western blot was conducted to detect nigral TH protein levels in 12-month A53T α-Syn transgenic mice with dopaminergic neurons-specific β5i overexpression. Scale bar = 100 μm * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To confirm the specificity of immunoproteasome activities, we analyzed proteolytic activities in the presence or absence of selective β1i inhibitor ML604440 (MCE, 500 nM) and β5i inhibitor ONX0914 (MCE, 500 nM), respectively.

Techniques: Phospho-proteomics, Immunostaining, Transfection, Transgenic Assay, Over Expression, Immunofluorescence, Staining, Western Blot

Journal: Redox Biology

Article Title: Deficient immunoproteasome assembly drives gain of α-synuclein pathology in Parkinson's disease

doi: 10.1016/j.redox.2021.102167

Figure Lengend Snippet: Loss of POMP induced by α-Syn disturbs immunoproteasome β-ring assembly. POMP mRNA and protein levels in WT α-Syn or A53T α-Syn transfected SH-SY5Y cells (A – C) and in the SN of 12-month A53T α-Syn transgenic mice (D – F) . (G) The interaction between β1i, β5i precursors and POMP in SH-SY5Y cells was examined immunoprecipitated with anti-Myc antibody. The whole-cell lysate was subjected to immunoblot with anti-β1i, anti-β5i and anti-Myc antibody. (H) The interaction between β5i precursor and POMP was detected in WT α-Syn or A53T α-Syn transfected SH-SY5Y cells immunoprecipitated with anti-Myc antibody. The whole-cell lysate was subjected to immunoblot with anti-β5i, anti-Myc and anti-Flag antibody. (I–K) The protein levels of β1i and β5i were determined by Western blot in WT α-Syn or A53T α-Syn transfected SH-SY5Y cells with POMP knockdown. (L, M) Chymotrypsin-like and trypsin-like activity were measured in WT α-Syn or A53T α-Syn transfected SH-SY5Y cells with POMP knockdown. * P < 0.05.

Article Snippet: To confirm the specificity of immunoproteasome activities, we analyzed proteolytic activities in the presence or absence of selective β1i inhibitor ML604440 (MCE, 500 nM) and β5i inhibitor ONX0914 (MCE, 500 nM), respectively.

Techniques: Transfection, Transgenic Assay, Immunoprecipitation, Western Blot, Knockdown, Activity Assay

Journal: Redox Biology

Article Title: Deficient immunoproteasome assembly drives gain of α-synuclein pathology in Parkinson's disease

doi: 10.1016/j.redox.2021.102167

Figure Lengend Snippet: Dopaminergic neurons-specific overexpression of NRF2-POMP axis alleviates α-Syn pathology. (A) Homo sapiens POMP promoter contains NRF2 binding region (TGCTGTGTCAC) and ARE sequence (TGAGCGGCG). (B, C) Dual-luciferase reporter assay was applied to detect the interaction between POMP promoter and NRF2. (D – H) Western blot was applied to detect the nigral protein levels of β1i, β5i, PLK2, pS129 α-Syn in 12-month A53T α-Syn transgenic mice with dopaminergic neurons overexpressing NRF2 or POMP. (I, J) The soluble and insoluble α-Syn levels were detected in the SN of 12-month A53T α-Syn transgenic mice with dopaminergic neurons overexpressing NRF2 or POMP. (K) α-Syn aggregation was detected by ThT immunofluorescence in the SN of 12-month A53T α-Syn transgenic mice with dopaminergic neurons overexpressing NRF2 or POMP. (L, M) Western blot was applied to detect the nigral TH in 12-month A53T α-Syn transgenic mice with dopaminergic neurons overexpressing NRF2 or POMP. (N) Rotarod test was used to evaluate the motor ability in 12-month A53T transgenic mice with dopaminergic neurons-specific overexpression of NRF2-POMP axis. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To confirm the specificity of immunoproteasome activities, we analyzed proteolytic activities in the presence or absence of selective β1i inhibitor ML604440 (MCE, 500 nM) and β5i inhibitor ONX0914 (MCE, 500 nM), respectively.

Techniques: Over Expression, Binding Assay, Sequencing, Luciferase, Reporter Assay, Western Blot, Transgenic Assay, Immunofluorescence