p sqstm1  (Cell Signaling Technology Inc)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Chchd10: A Novel Metabolic Sensor Modulating Adipose Tissue Homeostasis.

doi: 10.1002/advs.202408763

Figure Lengend Snippet: Figure 8. Chchd10 deficiency promotes TDP43-mediated Raptor mRNA stabilization thus promoting the mTORC1-mediated p62/Keap1/NRF2 activa- tion. A) Representative immunoblots of the protein abundance of Raptor, mTOR, Chchd10, and GAPDH in 4-day-differentiated WT and Chchd10 KO adipocytes. The right panel is the quantification of Raptor and mTOR band intensity normalized with GAPDH (n = 4). B) The relative mRNA abundance of Raptor in 4-day-differentiated WT and Chchd10 KO adipocytes treated with actinomycin D for 0, 2, and 4 h (n = 3). C) Representative immunoblots of the protein abundance of TDP43, p62, Chchd10, and HSP90 in eWAT of WT mice fed with STC or HFD for 3 days. The right panel is the quantification of TDP43 and p62 band intensity normalized with HSP90 (n = 6). D,E) 2-day-differentiated WT and Chchd10 KO adipocytes were treated with TDP43 siRNA (siTDP43) or controlled scramble siRNA (siCTR) for 48 h. D) Representative immunoblots of the protein abundance of TDP43, Raptor, p-p62 (S351), p62, Chchd10, and GAPDH. The right panel is the quantification of band intensity of TDP43, Raptor, p-p62 (S351), and p62 normalized with GAPDH (n = 4). E) Representative immunoblots of the protein abundance of the Keap1 and GAPDH in the cytoplasmic protein lysate and NRF2 and NPM1 in the nuclear protein lysate, respectively. The right panel is the quantification of Keap1 band intensity normalized with GAPDH and NRF2 band intensity normalized with NPM1, respectively (n = 4). F) Schematic diagram showing the mechanism by which Chchd10 reduction upon excess energy intake promotes TDP43 mediated Raptor stabilization and the subsequent upregulation of mTORC1 activity and p62/Keap1/NRF2 axis. Data are presented as mean ± SD. Statistical significance was analyzed by Mann-Whitney U test A, B and C) or two-way ANOVA D and E).

Article Snippet: The membranes were blocked with TBST containing 10% bovine serum albumin (BSA) and probed with primary antibodies against Chchd10 (25671-1-AP) and GAPDH (60004-1-Ig) from Proteintech; HSP90 (#4874), PPARγ (#2443), CEBPα (#2295), p62 (#5114), Keap1 (#8047), Lamin B1 (#13 435), and p-p62 (S349, #16 177) from Cell Signaling Technology; NRF2 (ab31163) and nucleophosmin 1 (NPM1, ab15440) from Abcam, FAS (sc-48357), Raptor (sc-81537), and TDP43 (sc376311) from Santa Cruz; GSTA4 (#PA5-106329) from Invitrogen, and pp62 (S351, MBS9212103) from MyBioSource at dilutions recommended by the suppliers.

Techniques: Western Blot, Quantitative Proteomics, Activity Assay, MANN-WHITNEY

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Chchd10: A Novel Metabolic Sensor Modulating Adipose Tissue Homeostasis.

doi: 10.1002/advs.202408763

Figure Lengend Snippet: Figure 9. Summary of the effect of Chchd10 reduction in adipocytes in response to excess energy intake. In response to excess energy intake with high glucose and fatty acid levels, Chchd10 expression is rapidly and persistently reduced in white adipocytes. In short term, reduction of Chchd10 enhances adipogenesis predominantly in subcutaneous adipose tissue (SAT) to potentiate lipid storage, thus accommodating the excess energy intake. In the long term, Chchd10 reduction helps maintain the normal function of adipocytes, mainly in visceral adipose tissue (VAT), by promoting 4-HNE clearance. Mechanistically, reduction of Chchd10 promotes TDP43 expression, stabilizing Raptor mRNA thus promoting Raptor protein synthesis. Increased Raptor promotes the localization of the mTORC1 complex on the lysosome and enhances the mTORC1-mediated phosphorylation of p62. p-p62 potentiates Keap1 degradation and enhances the dissociation of the Keap1-NRF2 complex. Increased free NRF2 enters the nucleus. On one hand, NRF2 promotes the activity of PPAR𝛾and CEBP𝛼, thus promoting adipogenesis. On the other hand, NRF2 itself upregulates the expression of the antioxidative enzyme GSTA4. When faced with lipid peroxidation, enhanced GSTA4 catalyzes the GSH-mediated 4-HNE clearance, preventing the 4-HNE-mediated protein carbonylation, thereby protecting adipocyte function. Overall, reduction of Chchd10 enhances adipogenesis and the anti-oxidant capacity of adipocytes by activating the TDP43/Raptor/p62/Keap1/NRF2 axis, which modulates WAT remodeling and maintains WAT homeostasis in response to excess energy intake.

Article Snippet: The membranes were blocked with TBST containing 10% bovine serum albumin (BSA) and probed with primary antibodies against Chchd10 (25671-1-AP) and GAPDH (60004-1-Ig) from Proteintech; HSP90 (#4874), PPARγ (#2443), CEBPα (#2295), p62 (#5114), Keap1 (#8047), Lamin B1 (#13 435), and p-p62 (S349, #16 177) from Cell Signaling Technology; NRF2 (ab31163) and nucleophosmin 1 (NPM1, ab15440) from Abcam, FAS (sc-48357), Raptor (sc-81537), and TDP43 (sc376311) from Santa Cruz; GSTA4 (#PA5-106329) from Invitrogen, and pp62 (S351, MBS9212103) from MyBioSource at dilutions recommended by the suppliers.

Techniques: Expressing, Phospho-proteomics, Activity Assay

Journal: Cells

Article Title: Chronic IL-1-Exposed LNCaP Cells Evolve High Basal p62-KEAP1 Complex Accumulation and NRF2/KEAP1-Dependent and -Independent Hypersensitive Nutrient Deprivation Response.

doi: 10.3390/cells14030192

Figure Lengend Snippet: Figure 1. Chronic IL-1 sublines have basally high accumulation of the p62-KEAP1 interaction. (A) Mass spectrometry using gel LCMS was performed for p62 immunoprecipitates from LNCaP-1, LNas1, and LNbs1 cells grown in normal growth medium containing 10% serum. Mass spectrometry was analyzed using Scaffold software. Values shown are Scaffold “exclusive spectrum count”. LNas1 and LNbs1 show high basal accumulation of the p62-KEAP1 interaction in 10% serum, whereas no KEAP1 interaction is detected in LNCaP-1 parentals. (B) p62 immunoprecipitation (IP) was performed for LNCaP-1, LNas1, and LNbs1 cells grown in normal growth medium containing 10% serum, followed by western blot for p62 and KEAP1. LNas1 and LNbs1 show high basal accumulation of the p62-KEAP1 interaction. IgG IP is the negative control for p62 IP. ND = not detected.

Article Snippet: Primary antibodies: p62 (Abnova, Walnut, CA, USA; L2011-2C11), KEAP1 (Cell Signaling, Danvers, MA, USA; 8074T), p-p62 (Ser349) (Cell Signaling, Danvers, MA, USA; 95697), GCLC (Cell Signaling, Danvers, MA; 48005S), HO-1 (Cell Signaling, Danvers, MA, USA; 43966S), and β-actin (Santa Cruz, Santa Cruz, CA, USA; sc-69879).

Techniques: Mass Spectrometry, Software, Immunoprecipitation, Western Blot, Negative Control

Journal: Cells

Article Title: Chronic IL-1-Exposed LNCaP Cells Evolve High Basal p62-KEAP1 Complex Accumulation and NRF2/KEAP1-Dependent and -Independent Hypersensitive Nutrient Deprivation Response.

doi: 10.3390/cells14030192

Figure Lengend Snippet: Figure 5. The p62-KEAP1 complex accumulation is upregulated by serum starvation in LNCaP parental cells and constitutive in subline cells. (A) DIA mass spectrometry was performed for LNCaP- 1, LNas1, and LNbs1 cells grown in normal growth medium containing 10% serum or 0% serum for 2 days. p62 or KEAP1 were immunoprecipitated (IP) from LNCaP-1, LNas1, or LNbs1 cells. DIA mass spectrometry was analyzed using Scaffold DIA software. Values shown are non-normalized exclusive intensity. As compared to 10% serum, p62 and KEAP1 exclusive intensity are greater in 0% serum in LNCaP-1 parental cells for both p62 IP and KEAP1 IP, suggesting that 0% serum starvation induces accumulation of the p62-KEAP1 complex in LNCaP-1 cells. An increase in exclusive intensity in 0% versus 10% serum is not observed for LNas1 or LNbs1, suggesting that serum starvation does not induce p62-KEAP1 complex formation in the sublines. (B,C) IP followed by western blot was performed for LNCaP-1, LNas1, and LNbs1 cells grown in normal growth medium containing 10% serum or 0% serum for 2 days, and blots were probed for p62, p-p62 (SER349), or KEAP1. (B) p62 or (C) KEAP1 were IP’d from LNCaP-1, LNas1, or LNbs1 cells. Input western blots show that 0% serum induces p62 accumulation in LNCaP-1 parental cells, and IP western blots show that 0% induces p62-KEAP1 interaction in LNCaP-1 parental cells. Input and IP western blots show that in comparison to parental cells, p62 levels and p62-KEAP1 complex formation are basally higher in the sublines (e.g., 10% serum). In addition, input and IP western blots show that, in comparison to 10% serum, 0% serum reduces KEAP1 accumulation in the sublines, yet the p62-KEAP1 complex formation is comparable to parental 0% and subline 10%, suggesting the complex is constitutive and stable. Finally, p-p62 (SER349) is found only in the subline p62 immunoprecipitates. Thus, KEAP1 does not bind p-p62 (SER349) in the LNCaP-1 parental or subline cells grown in 10% or 0% serum. β-actin is the western blot loading control. IgG IP is the negative control.

Article Snippet: Primary antibodies: p62 (Abnova, Walnut, CA, USA; L2011-2C11), KEAP1 (Cell Signaling, Danvers, MA, USA; 8074T), p-p62 (Ser349) (Cell Signaling, Danvers, MA, USA; 95697), GCLC (Cell Signaling, Danvers, MA; 48005S), HO-1 (Cell Signaling, Danvers, MA, USA; 43966S), and β-actin (Santa Cruz, Santa Cruz, CA, USA; sc-69879).

Techniques: Mass Spectrometry, Immunoprecipitation, Software, Western Blot, Comparison, Control, Negative Control

Journal: Cells

Article Title: Chronic IL-1-Exposed LNCaP Cells Evolve High Basal p62-KEAP1 Complex Accumulation and NRF2/KEAP1-Dependent and -Independent Hypersensitive Nutrient Deprivation Response.

doi: 10.3390/cells14030192

Figure Lengend Snippet: Figure 8. Model. Chronic IL-1 exposure selects for cells that evolve high basal accumulation of NRF2-indepedent p62-KEAP1 interaction, the function of which remains unknown. Chronic IL-1 exposure also selects for cells that have high basal HMOX1 and GCLC and are hypersensitive to serum starvation-induced NRF2-KEAP1-dependent HMOX1 repression and NRF2-KEAP1-p62-independent GCLC upregulation. We speculate that under serum starvation conditions, hypersensitive HMOX1 repression and GCLC induction may protect the chronic IL-1 sublines from ferroptosis by reducing Fe2+ and increasing glutathione production, respectively.

Article Snippet: Primary antibodies: p62 (Abnova, Walnut, CA, USA; L2011-2C11), KEAP1 (Cell Signaling, Danvers, MA, USA; 8074T), p-p62 (Ser349) (Cell Signaling, Danvers, MA, USA; 95697), GCLC (Cell Signaling, Danvers, MA; 48005S), HO-1 (Cell Signaling, Danvers, MA, USA; 43966S), and β-actin (Santa Cruz, Santa Cruz, CA, USA; sc-69879).

Techniques: