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• Research Paper Volume 13, Issue 16 pp 20277-20301

  Comprehensive analysis of ubiquitin-proteasome system genes related to prognosis and immunosuppression in head and neck squamous cell carcinoma

  Relevance score: 3.7695408

  Juncheng Wang, Jianing Li, Luan Zhang, Yuexiang Qin, Fengyu Zhang, Rulong Hu, Huihong Chen, Yongquan Tian, Zhifeng Liu, Yuxi Tian, Xin Zhang

  Keywords: head and neck squamous cell carcinoma (HNSCC), ubiquitin proteasome system (UPS), the cancer genome atlas (TCGA) database, gene expression omnibus (GEO) database, immunosuppression

  Published in Aging on August 16, 2021

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  The ubiquitin-proteasome system (UPS) with a capacity of degrading multiple intracellular proteins is an essential regulator in tumor immunosurveillance. Tumor cells that escape from recognition and destruction of immune system have been consistently characterized an important hallmark in the setting of tumor progression. Little know about the exact functions of UPS-related genes (UPSGs) and their relationships with antitumor immunity in head and neck squamous cell carcinoma (HNSCC) patients. In this study, for the first time, we comprehensively identified 114 differentially expressed UPSGs (DEUPSGs) and constructed a prognostic risk model based on the eight DEUPSGs (BRCA1, OSTM1, PCGF2, PSMD2, SOCS1, UCHL1, UHRF1, and USP54) in the TCGA-HNSCC database. This risk model was validated using multiple data sets (all P < 0.05). The high-risk score was found to be an independently prognostic factor in HNSCC patients and was significantly correlated with T cells suppression. Accordingly, our risk model can act as a prognostic signature and provide a novel concept for improving the precise immunotherapy for patients with HNSCC.

• Research Paper Volume 13, Issue 1 pp 1176-1185

  The 1316T>C missenses mutation in MTHFR contributes to MTHFR deficiency by targeting MTHFR to proteasome degradation

  Relevance score: 4.9263377

  Xi Liu, Yu Li, Menghan Wang, Xiaojun Wang, Limin Zhang, Tao Peng, Wenping Liang, Zhe Wang, Hong Lu

  Keywords: molecular mechanisms, MTHFR deficiency, proteasome degradation, pathogenic mutation, single nucleotide polymophorism

  Published in Aging on December 3, 2020

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  5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare hereditary disease characterized by defects in folate and homocysteine metabolism. Individuals with inherited MTHFR gene mutations have a higher tendency to develop neurodegeneration disease as Alzheimer’ disease and atherosclerosis. MTHFR is a rate-limiting enzyme catalyzing folate production, various SNPs/mutations in the MTHFR gene have been correlated to MTHFR deficiency. However, the molecular mechanisms underpinning the pathogenic effects of these SNPs/mutations have not been clearly understood. In the present study, we reported a severe MTHFR deficiency patient with late-onset motor dysfunction and sequenced MTHFR gene exons of the family. The patient carries an MD-associating SNP (rs748289202) in one MTHFR allele and the rs545086633 SNP with unknown disease relevance in the other. The rs545086633 SNP (p.Leu439Pro) results in an L439P substitution in MTHFR protein, and drastically decreases mutant protein expression by promoting proteasomal degradation. L⁴³⁹ in MTHFR is highly conserved in vertebrates. Our study demonstrated that p.Leu439Pro in MTHFR is the first mutation causing significant intracellular defects of MTHFR, and rs545086633 should be examined for the in-depth diagnosis and treatment of MD.

• Research Paper Volume 12, Issue 22 pp 22949-22974

  In vitro and in vivo efficacy of the novel oral proteasome inhibitor NNU546 in multiple myeloma

  Relevance score: 5.627314

  Hui Zhou, Meng Lei, Wang Wang, Mengjie Guo, Jia Wang, Haoyang Zhang, Li Qiao, Huayun Feng, Zhaogang Liu, Lijuan Chen, Jianhao Hou, Xueyuan Wang, Chenxi Gu, Bo Zhao, Evgeny Izumchenko, Ye Yang, Yongqiang Zhu

  Keywords: proteasome inhibitor, multiple myeloma, oral drug, mechanism research

  Published in Aging on November 16, 2020

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  Proteasome inhibition demonstrates highly effective impact on multiple myeloma (MM) treatment. Here, we aimed to examine anti-tumor efficiency and underlying mechanisms of a novel well tolerated orally applicable proteasome inhibitor NNU546 and its hydrolyzed pharmacologically active form NNU219. NNU219 showed more selective inhibition to proteasome catalytic subunits and less off-target effect than bortezomib ex vivo. Moreover, intravenous and oral administration of either NNU219 or NNU546 led to more sustained pharmacodynamic inhibitions of proteasome activities compared with bortezomib. Importantly, NNU219 exhibited potential anti-MM activity in both MM cell lines and primary samples in vitro. The anti-MM activity of NNU219 was associated with induction of G2/M-phase arrest and apoptosis via activation of the caspase cascade and endoplasmic reticulum stress response. Significant growth-inhibitory effects of NNU219 and NNU546 were observed in 3 different human MM xenograft mouse models. Furthermore, such observation was even found in the presence of a bone marrow microenvironment. Taken together, these findings provided the basis for clinical trial of NNU546 to determine its potential as a candidate for MM treatment.

• Research Paper Volume 11, Issue 11 pp 3624-3638

  Proteasome-dependent degradation of intracellular carbamylated proteins

  Relevance score: 5.414916

  Aurore Desmons, Anaïs Okwieka, Manon Doué, Laëtitia Gorisse, Vincent Vuiblet, Christine Pietrement, Philippe Gillery, Stéphane Jaisson

  Keywords: carbamylation, cell aging, homocitrulline, nonenzymatic post-translational modifications, proteasome, proteostasis

  Published in Aging on June 6, 2019

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  Carbamylation, which corresponds to the binding of isocyanic acid to the amino groups of proteins, is a nonenzymatic post-translational modification responsible for alterations of protein structural and functional properties. Tissue accumulation of carbamylation-derived products and their role in pathological processes such as atherosclerosis or chronic renal failure have been previously documented. However, few studies have focused on the carbamylation of intracellular proteins and their subsequent role in cellular aging. This study aimed to determine the extent of intracellular protein carbamylation, its impact on cell functions and the ability of cells to degrade these modified proteins. Fibroblasts were incubated with cyanate or urea and the carbamylation level was evaluated by immunostaining and homocitrulline quantification. The results showed that carbamylated proteins accumulated intracellularly and that all proteins were susceptible. The presence of intracellular carbamylated proteins did not modify cell proliferation or type I collagen synthesis nor did it induce cell senescence, but it significantly decreased cell motility. Fibroblasts were able to degrade carbamylated proteins through the ubiquitin-proteasome system. In conclusion, intracellular proteins are susceptible to carbamylation but their accumulation does not seem to deeply affect cell function, owing largely to their elimination by the ubiquitin-proteasome system.

  

  Evidence for the presence of carbamylated proteins inside human skin cells. An anti-HCit immunolabelling was realized in a skin section from a 77-year old man counter-stained with DAPI and hematoxylin-eosin. Epidermal area was screened by light microscopy thanks to hematoxylin-eosin counterstaining (a). Nuclei were highlighted by DAPI staining (b, blue) permitting confirmation of the presence of HCit inside skin cells by immunofluorescence analysis (c, green). An example of a cell exhibiting a positive intracellular labelling for HCit is indicated by arrows. Observations were realized at 10x magnification.

  
  
  

  

  Intracellular accumulation of carbamylated proteins after long-term incubations with urea or cyanate. Confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions, open bars) or with 20 mmol/L urea (grey bars) or 0.5 mmol/L cyanate (black bars). HCit content was determined by LC-MS/MS in total cell extracts (a) and in cytoplasmic and membrane fractions (b). The data are presented as means ± SEM (n=6) compared using the Mann-Whitney U test (ns: non significant, **: p<0.01).

  
  
  

  

  Localization of intracellular carbamylated proteins. Fibroblasts were seeded in chambered coverglass system and incubated for 5 days with DMEM containing 0.5% (v/v) FBS and 5 mmol/L cyanate. At the end of incubation, cells were fixed with 4% (v/v) paraformaldehyde and permeabilized with 0.25% (v/v) Triton X-100 before immunolabelling of carbamylated proteins using an anti-HCit polyclonal antibody (a). Cells were also labelled using ActinRed 555 ReadyProbes® in order to identify actin fibers (b). Colocalization points between HCit and actin labelling were identified using ImageJ software (c). In a second set of experiments, fibroblasts were incubated in the same conditions without (control) or with 5 mmol/L cyanate before preparing total cell extracts which were then used for β-actin immunoprecipitation. The immunoprecipitates were submitted to acid hydrolysis before HCit quantification by LC-MS/MS (d). The data are presented as means ± SEM (n=4) compared using the Mann-Whitney U test (**: p<0.01).

  
  
  

  

  Effect of intracellular protein carbamylation on cell function and senescence. Confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions) or with 20 mmol/L urea or 0.5 mmol/L cyanate. (a) Proliferation: cells were then seeded in 96-well plates at a density of 1,500 cells per well and incubated for 1, 2, 4 and 7 days with DMEM with 10% (v/v) FBS and carbamylating agents. Cell number was evaluated using a WST-1 assay by measuring absorbance at 450 nm. The data presented are means ± SEM (n=6) compared using the Mann-Whitney U test. No significant difference was found between the three conditions (control: dotted line,•; urea: grey line ■; cyanate: black line, ■). (b) Cell migration: cells were seeded in 24-well plates at a density of 15,000 cells per well and incubated for 24h at 37°C with DMEM containing 0.5% (v/v) FBS. Pictures of cells were taken every 30 min over the incubation period and each cell (n=58) was followed separately in order to calculate the migration speed. The data are presented as means ± SEM compared using the Mann-Whitney U test (*:p<0.05, **:p<0.01). (c) Expression of type I collagen mRNAs: at the end of the 4 weeks-incubation, RNA was isolated from confluent cells and then submitted to RT-qPCR analysis for evaluating the expression of COL1A1 and COL1A2 genes. Data represent the relative mRNA expression normalized to EEF1A1 gene and are expressed as means ± SEM (n=4). The Mann-Whitney U test was used to compare the three conditions: control (open bars), urea (grey bars) and cyanate (black bars). ns: not significant. (d) Senescence: cell senescence was determined by measuring the SA-β-galactosidase activity using a C₁₂FDG fluorogenic substrate and by detection of senescent cells by flow cytometry. Each plot represents the results of 20,000 events acquired per condition. Incubation of cells with rotenone was used as a positive control of cell senescence whereas a negative control without addition of the fluorogenic substrate was performed.

  
  
  

  

  Intracellular degradation of carbamylated proteins. Confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS and 0.5 mmol/L cyanate in order to induce intracellular protein carbamylation. Cells were then incubated in the same conditions (with 0.5 mM cyanate, solid line) or without cyanate (dotted line) for two additional weeks. HCit content was determined at each time point. The data are presented as means ± SEM (n=6) and the two conditions (with or without cyanate) were compared using the Mann-Whitney U test (**: p<0.01).

  
  
  

  

  Impact of carbamylation on proteasome proteolytic activities and on the ubiquitination process. (a) Evaluation of proteasome proteolytic activity after incubation of cells with urea or cyanate: confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions, open bars) or with 20 mmol/L urea (grey bars) or 0.5 mmol/L cyanate (black bars). Chymotrypsin-like, caspase-like and trypsin-like activities have been measured in cell extracts using the corresponding Proteasome-Glo™ assays. The data are presented as means ± SEM (n=6) and compared using the Mann-Whitney U test (ns: non significant, **: p<0.01). (b) Ubiquitination level of intracellular proteins after incubation of cells with urea or cyanate: confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions) or with 20 mmol/L urea or 0.5 mmol/L cyanate, and cell extracts were prepared and submitted to western-blot analysis using an anti-ubiquitin antibody. (c) Anti-HCit and anti-ubiquitin immunolabellings were performed using fibroblasts previously seeded (10,000 cells/well) in chambered coverglass system and incubated for 2 days with DMEM containing 0.5% (v/v) FBS and 5 mmol/L cyanate. At the end of incubation, cells were fixed with 4% (v/v) paraformaldehyde and permeabilized with 0.25% (v/v) Triton X-100 before immunolabelling of proteins using both anti-HCit and anti-ubiquitin antibodies. Colocalization points between HCit and ubiquitin labelling were identified using ImageJ software.

  
  
  

  

  Role of proteasome in the degradation of carbamylated proteins. Confluent fibroblasts were incubated for 7 and 14 days at 37°C with DMEM containing 0.5% (v/v) FBS, 0.5 mmol/L sodium cyanate with or without proteasome inhibitors (10 nmol/L Bortezomib (Bz) or 500 nM MG-132). HCit content in total cell extracts was determined by LC-MS/MS. The data are presented as means ± SEM (n=6) and compared using the Mann-Whitney U test (ns: non significant, **: p<0.01).

  
  
  

• Research Paper Volume 10, Issue 12 pp 3821-3833

  Caloric restriction rescues yeast cells from alpha-synuclein toxicity through autophagic control of proteostasis

  Relevance score: 6.1432314

  Belém Sampaio-Marques, Hélder Pereira, Ana R. Santos, Alexandra Teixeira, Paula Ludovico

  Keywords: caloric restriction, aging, ubiquitin-proteasome system, autophagy, alpha-synuclein

  Published in Aging on December 7, 2018

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  α-Synuclein (SNCA) is a presynaptic protein that is associated with the pathophysiology of synucleinopathies, including Parkinson’s disease. SNCA is a naturally aggregation-prone protein, which may be degraded by the ubiquitin-proteasome system (UPS) and by lysosomal degradation pathways. Besides being a target of the proteolytic systems, SNCA can also alter the function of these pathways further, contributing to the progression of neurodegeneration. Deterioration of UPS and autophagy activities with aging further aggravates this toxic cycle. Caloric restriction (CR) is still the most effective non-genetic intervention promoting lifespan extension. It is known that CR-mediated lifespan extension is linked to the regulation of proteolytic systems, but the mechanisms underlying CR rescue of SNCA toxicity remain poorly understood. This study shows that CR balances UPS and autophagy activities during aging. CR enhances UPS activity, reversing the decline of the UPS activity promoted by SNCA, and keeps autophagy at homeostatic levels. Maintenance of autophagy at homeostatic levels appears to be relevant for UPS activity and for the mechanism underlying rescue of cells from SNCA-mediated toxicity by CR.

  

  Caloric restriction abrogates α-synuclein (SNCA)-induced toxicity by upregulating ubiquitin-proteasome system activity. (A) Chronological lifespan (CLS) and SNCA levels of stationary wild type cells harbouring the vector control or expressing the human SNCA grown under regular (2% glucose) or CR (0.5% glucose) conditions. (B) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. (C) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UB^G76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (D) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. (E) Ubiquitination profile determined by Western blotting using an anti-mono and polyubiquitination antibody. (F) Graphical representation of the intensity of total UB/Pgk1 obtained by densitometric analysis. (G) RPN4 and (H) RPN5 mRNA relative expression levels. Three reference genes (ACT1-actin, PDA1-alpha subunit of pyruvate dehydrogenase and TDH2-isoform 2 of glyceraldehyde-3-phosphate dehydrogenase) were used as internal standards and for the normalization of mRNA expression levels. Significance was determined by two-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) between cells grown under regular or CR conditions expressing vector control or SNCA. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).

  
  
  

  

  Autophagy inhibition leads to upregulation of the ubiquitin-proteasome system activity in α-synuclein (SNCA)-expressing cells under caloric restriction. (A) Chronological lifespan (CLS) and SNCA levels of SNCA-expressing stationary wild type cells, under caloric restriction (CR, 0.5% glucose) conditions, in the presence or absence of chloroquine (CQ), an inhibitor of autophagy. (B) Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS. Significance was determined by two-way ANOVA (*p≤0.05, ****p≤0.0001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of CQ. (C) RPN4 mRNA relative expression levels as described in the legend of Figure 1. (D) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. (E) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UB^G76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (F) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. (G) Ubiquitination profile determined by Western blotting using an anti-mono and polyubiquitination antibody. (H) Graphical representation of the intensity of total UB/Pgk1 obtained by densitometric analysis. Statistical significance represented in (C), (D), (F) and (H) was determined by Student's t-test (*p≤0.05, ***p≤0.001, ****p≤0.0001) comparing caloric restricted vector control or SNCA-expressing cells in the presence or absence of CQ. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).

  
  
  

  

  Pharmacological inhibition of the ubiquitin-proteasome system activity decreases lifespan of α-synuclein (SNCA)-expressing cells grown under caloric restriction but has no major impact on autophagy. (A) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. Significance was determined by two-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001) between cells grown under CR (0.5% glucose) conditions expressing vector control or SNCA in the presence or absence of bortezomib (Bort). (B) RPN4 mRNA relative expression levels as described in the legend of Figure 1. (C) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UB^G76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (D) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. Statistical significance represented in (B) and (D) was determined by Student's t-test (*p≤0.05, **p≤0.01, ***p≤0.001) comparing caloric restricted vector control or SNCA-expressing cells in the presence or absence of Bort. (E) Chronological lifespan (CLS) and SNCA levels of pdr5Δ cells expressing SNCA grown under CR conditions, in the presence or absence of Bort. (F) Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS. Significance was determined by two-way ANOVA (***p≤0.001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of Bort. Autophagy flux assessed by the GFP-Atg8 processing assay (immunoblotting analysis with antibody against GFP) of caloric restricted cells expressing vector control (G) or SNCA (H) in the absence or presence of Bort. Blots represented in (G) are from the same gel, as in (H). (I) Densitometric analysis of the ratio between the free GFP versus the total GFP. Bands were quantified by Quantity One software. Significance of the data was determined by two-way ANOVA (**p≤0.01, ***p≤0.001, ****p≤0.0001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of Bort. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).

  
  
  

  

  Genetic inhibition of the ubiquitin-proteasome system activity decreases lifespan of α-synuclein (SNCA)-expressing cells grown under caloric restriction conditions but has no major impact on autophagy. Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS of caloric restricted vector control and SNCA expressing wild type cells compared with RPN4 (A) and UMP1 (B) deleted cells. (C) Autophagy flux assessed by the GFP-Atg8 processing assay (immunoblotting analysis with antibody against GFP) of wild type, rpn4Δ and ump1Δ caloric restricted cells expressing vector control or SNCA. Blots from same strain were run in the same gel. Densitometric analysis of the ratio between the free GFP versus the total GFP of rpn4Δ (D) and ump1Δ (E) cells. Data from wild type cells is repeated in (D) and (E) for easier interpretation. Bands were quantified by Quantity One software. (F) Mean and maximum lifespans determined from curve fitting of the survival data from CLS of wild type and cue5Δ cells expressing the vector control or SNCA in normal (2% glucose) or CR (0.5% glucose) growth conditions. (G) Mean and maximum lifespans determined from curve fitting of the survival data from CLS of wild type cells expressing the vector control or SNCA in the absence or presence of chloroquine (CQ). Significance was determined by Student's t-test (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) comparing wild type with each mutant strain in the same conditions. For (G) the comparison was done between vector control or SNCA-expressing cells in the presence or absence of CQ. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).

  
  
  

• Editorial Volume 10, Issue 10 pp 2544-2546

  Mutant ubiquitin reduces Aβ plaques

  Relevance score: 4.6169524

  Bert M. Verheijen, Fred W. van Leeuwen

  Keywords: mutant ubiquitin, ubiquitin-proteasome system, amyloid β, Aβ plaques, γ-secretase, proteostasis, Alzheimer’s disease

  Published in Aging on October 15, 2018

  

  Mutant ubiquitin (UBB⁺¹) reduces Aβ plaques in APPPS1 mice. (A) Schematic diagram of amyloid precursor protein (APP) processing leading to Aβ. (B) γ-secretase is a multimeric enzyme complex that cleaves APP C-terminal fragment (CTF) to produce Aβ and an APP intracellular domain (AICD). (C) Transgenic APPPS1 mice carry two mutations that represent early-onset AD and develop age-related amyloid plaque pathology. (D) In APPPS1xUBB+1 mice, APP CTF-β levels are reduced and γ-secretase function is partially restored. (E) Besides secretases, alternate protein degradation pathways have been reported to degrade APP and Aβ.

  
  
  

• Review Volume 10, Issue 5 pp 868-901

  Origin and pathophysiology of protein carbonylation, nitration and chlorination in age-related brain diseases and aging

  Relevance score: 6.5463414

  Efstathios S. Gonos, Marianna Kapetanou, Jolanta Sereikaite, Grzegorz Bartosz, Katarzyna Naparło, Michalina Grzesik, Izabela Sadowska-Bartosz

  Keywords: oxidative stress, carbonylation, nitration, chlorination, proteasome

  Published in Aging on May 17, 2018

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  Non-enzymatic protein modifications occur inevitably in all living systems. Products of such modifications accumulate during aging of cells and organisms and may contribute to their age-related functional deterioration. This review presents the formation of irreversible protein modifications such as carbonylation, nitration and chlorination, modifications by 4-hydroxynonenal, removal of modified proteins and accumulation of these protein modifications during aging of humans and model organisms, and their enhanced accumulation in age-related brain diseases.

  

  Selected non-enzymatic protein modifications. (A) oxidation of cysteine residues in proteins. Cysteine residues may be oxidized to sulfenic, sulfinic and sulfonic derivatives or form disulfide bonds. Oxidation to sulfenic acid and formation of disulfides is reversible; (B) modifications of cysteine residues in proteins: formation of nitrosocysteine and S-glutathionylation; (C) oxidation of methionine forms methionine sulfoxide, which may be reduced back to methionine by methionine sulfoxide reductases (MSR); (D) formation of hydroperoxides of valine, lysine and leucine; (E) formation of carbonyl derivatives of lysine, arginine, His and threonine; (F) formation of 4-hydroxynonenal adducts of cysteine, His and lysine; (G) oxidative modifications of phenylalanine; (H) modifications of tyrosine; (I) modifications of tryptophan.

  
  
  

  

  Reactions of 4-hydroxy-2,3-trans-nonenal (4-HNE) with proteins.

  
  
  

  

  Overview of the ubiquitin (Ub)/proteasome system and its substrates in relation to aging. Ub conjugation is mediated by a series of enzymes. The Ub-activating enzyme E1 transfers Ub to the active site of the E2 Ub-conjugating enzyme and the E3 Ub-ligase ligate Ub to the target protein. The ubiquitinated protein is targeted to the 26S proteasome for degradation. The 26S proteasome consists of the 20S catalytic core and of one or two 19S regulatory particles. The 20S proteasome consists of 28 subunits that are divided to two outer α and two central β rings. The immunoproteasome is induced in response to the immunomodulatory cytokine interferon-gamma (IFN-gamma) or in response to the increased OS that is observed during aging. The age-related elevation of OS also causes oxidative damage to proteins, such as carbonylation. In addition, the excessive •NO production during aging can lead to aberrant S-nitrosylation/tyrosine nitration. Nitrated proteins are prone to aggregation and may contribute to the onset and progression of various neurodegenerative diseases, including AD or PD. The accumulation of aggregated or carbonylated proteins inhibit proteasomal activity contributing the observed proteasomal dysfunction during aging and to the advancement of age-related pathologies.

  
  
  

• Editorial Volume 9, Issue 8 pp 1857-1858

  Novel therapies for multiple myeloma

  Relevance score: 5.927117

  Craig T. Wallington-Beddoe, Stuart M. Pitson

  Keywords: myeloma, therapy, proteasome inhibitor, immunomodulatory agent, monoclonal antibody

  Published in Aging on August 28, 2017

• Editorial Volume 9, Issue 7 pp 1645-1646

  Enhancing ER stress in myeloma

  Relevance score: 6.2170715

  Craig T. Wallington-Beddoe, Stuart M. Pitson

  Keywords: myeloma, ER stress, proteasome inhibitor, sphingolipids

  Published in Aging on July 30, 2017

• Research Paper Volume 9, Issue 4 pp 1153-1185

  The age- and sex-specific decline of the 20s proteasome and the Nrf2/CncC signal transduction pathway in adaption and resistance to oxidative stress in Drosophila melanogaster

  Relevance score: 5.6362123

  Laura C.D. Pomatto, Sarah Wong, Caroline Carney, Brenda Shen, John Tower, Kelvin J. A. Davies

  Keywords: 20S proteasome, protein aggregation, Nrf2, adaptive homeostasis, oxidative stress, protein oxidation

  Published in Aging on April 3, 2017

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  Hallmarks of aging include loss of protein homeostasis and dysregulation of stress-adaptive pathways. Loss of adaptive homeostasis, increases accumulation of DNA, protein, and lipid damage. During acute stress, the Cnc-C (Drosophila Nrf2 orthologue) transcriptionally-regulated 20S proteasome degrades damaged proteins in an ATP-independent manner. Exposure to very low, non-toxic, signaling concentrations of the redox-signaling agent hydrogen peroxide (H₂O₂) cause adaptive increases in the de novo expression and proteolytic activity/capacity of the 20S proteasome in female D. melanogaster (fruit-flies). Female 20S proteasome induction was accompanied by increased tolerance to a subsequent normally toxic but sub-lethal amount of H₂O₂, and blocking adaptive increases in proteasome expression also prevented full adaptation. We find, however, that this adaptive response is both sex- and age-dependent. Both increased proteasome expression and activity, and increased oxidative-stress resistance, in female flies, were lost with age. In contrast, male flies exhibited no H₂O₂ adaptation, irrespective of age. Furthermore, aging caused a generalized increase in basal 20S proteasome expression, but proteolytic activity and adaptation were both compromised. Finally, continual knockdown of Keep1 (the cytosolic inhibitor of Cnc-C) in adults resulted in older flies with greater stress resistance than their age-matched controls, but who still exhibited an age-associated loss of adaptive homeostasis.

  

  Hydrogen peroxide resistance declines with age. (A-D) Males and females of the Actin-GS-255B strain crossed to the w[1118] strain were aged to 3 days or 60 days and then transferred to vials containing kimwipes soaked in 1.0M to 8.0M H₂O₂ dissolved in 5% sucrose solution, and flies were scored as dead when completely immobile, as described previously [43]. (A) 3 day old females. (B) 3 day old males. (C) 60 day old females. (D) 60 day old males.

  
  
  

  

  Adaptation to hydrogen peroxide declines with age in females. Progeny of the Actin-GS-255B strain crossed to the w[1118] strain were aged to 3 days and 60 days prior to H₂O₂ pretreatment. Following recovery, flies were fed H₂O₂ challenge dose: 4.4M in 3 day old males and females, 2M in 60 day old females, and 1M in 60 day old males. (A) 3 day old females showed increased survival following pretreatment. (B) 3 day old males showed no change in survival following pretreatment. (C) With age, 60 day old females no longer show increased survival. (D) 60 day old males show no change in adaptation following pretreatment. Statistical difference in survival (p < 0.05) was calculated using the Log-Rank test. Statistical summary is located in Supplementary Table S1.

  
  
  

  

  Adaptive de novo expression of the 20S proteasome diminishes with age in a sex-dependent manner. (A,B) Virgin females of the Actin-GS-255B strain were crossed to males of the w[1118] strain and progeny were assayed for 20Sα expression without H₂O₂ pretreatment, or after pretreatment with [10µM or 100µM] H₂O₂. (A) 3 day and 60 day aged females. (B) 3 day and 60 day aged males. All samples were compared to the 3 day old 0µM H₂O₂ controls (C) Basal expression of the 20S proteasome α subunits was measured between 3 day old females, 60 day old females, 3 day old males, and 60 day old males, with samples normalized to the 3 day old female. Western blots were performed in triplicate, normalized to Actin-HRP, and quantified using ImageJ. Error bars denote standard error of the mean (S.E.M) values. * P <0.05 and ** P < 0.01, relative to control using one-way ANOVA. Asterisks color indicates the age of the sample, young female (pink *) and aged female (black *). All statistical significance was calculated relative to the young controls.

  
  
  

  

  Adaptive proteolytic capacity of the 20S proteasome diminishes with age in a sex-dependent manner. Virgin females of the Actin-GS-255B strain were crossed to males of the w[1118] strain and progeny were assayed for the proteolytic activity of the three catalytic subunits of the 20S proteasome in 3 day-old (red or blue) and 60 day-old flies (checked pattern). Caspase-like activity in (A) Females and males. Trypsin-like activity in (B) Females and males. Chymotrypsin-like activity in (C) Females and males. (D) Proteolytic degradation of oxidized [³H] hemoglobin in flies pretreated with hydrogen peroxide at 3 days and 60 days. Statistical significance for proteolysis of oxidized substrate was compared to young control females. Error bars denote standard error of the mean (S.E.M) values. * P <0.05, ** P < 0.01, *** P < 0.001 relative to control using one-way ANOVA. Asterisk color corresponds to young females (pink *), aged females (black *), young males (green #), and aged males (black #). Statistical significance was calculated relative to the young control females (A-D).

  
  
  

  

  The adaptive expression of the 20S proteasome is age and tissue-dependent in females. Body segments collected from females of the Actin-GS-255B strain crossed to the w[1118] strain were used as controls, or were pretreated with either 10µM or 100µM hydrogen peroxide. (A,B) 20Sα expression in female abdominal tissue following pretreatment. (A) 3 day old. (B) 60 day old. (C,D) 20Sα expression in female head following pretreatment. (C) 3 day old. (D) 60 day old. (E,F) 20Sα expression in female thorax following pretreatment. (E) 3 day old. (F) 60 day old. Western blots were performed in triplicate, normalized to Actin-HRP, and quantified using ImageJ. The bar charts represent the quantification. Error bars denote standard error of the mean (S.E.M) values. * P <0.05, ** P <0.01, *** P < 0.001, relative to the female control using one-way ANOVA. Statistical significance is indicated in young females with pink asterisks (pink *).

  
  
  

  

  Tissue-specific differences of the adaptive proteolytic capacity, and age-dependent changes in basal activity, of the 20S proteasome in females. Body segments collected from female progeny of the Actin-GS-255B strain crossed to the w[1118] strain were used as controls, or were pretreated with either 10µM or 100µM hydrogen peroxide. Individual proteolytic capacity of the 20S proteasome (caspase/peptidyl glutamyl-peptide hydrolyzing-like activity, trypsin-like, and chymotrypsin-like activity) was measured in the abdomen, head, and thorax. (A-C) Abdomen isolated from 3 day old (pink) and 60 day old (black) females following hydrogen peroxide pretreatment. (A) Caspase-like activity. (B) Trypsin-like activity. (C) Chymotrypsin-like activity. (D-F) Head isolated from 3 day old (pink) and 60 day old (black) females following hydrogen peroxide pretreatment. (D) Caspase-like activity. (E) Trypsin-like activity. (F) Chymotrypsin-like activity. (G-I) Thorax isolated from 3 day old (pink) and 60 day old (black) females following hydrogen peroxide pretreatment. (G) Caspase-like activity. (H) Trypsin-like activity. (I) Chymotrypsin-like activity. Error bars indicate the standard error of the mean (S.E.M) values. * P <0.05, ** P <0.01, *** P < 0.001, relative to the young female control using one-way ANOVA. Statistical significance is shown with pink asterisks (pink *) for young females and black asterisks (black *) in aged females.

  
  
  

  

  Males show no tissue-specific or age-related adaptive changes in 20S proteasome protein levels. Body segments were collected from males of the Actin-GS-255B strain crossed to the w[1118] strain that were used as controls, or that were pretreated with either 10µM or 100µM hydrogen peroxide. (A,B) 20Sα expression in male abdominal tissue following pretreatment. (A) 3 day old. (B) 60 day old. (C,D) 20Sα expression in male head following pretreatment. (C) 3 day old. (D) 60 day old. (E,F) 20Sα expression in male thorax following pretreatment. (E) 3 day old. (F) 60 day old. Western blots were performed in triplicate, normalized to Actin-HRP, and quantified using ImageJ. The bar charts represent the quantification. Error bars denote standard error of the mean (S.E.M) values, relative to the male control using one-way ANOVA.

  
  
  

  

  Males show no tissue-specific differences in the adaptive proteolytic capacity of the 20S proteasome, but do exhibit age-dependent changes in proteasomal basal activity. Body segments were collected from male progeny of the Actin-GS-255B strain crossed to the w[1118] strain that were used as controls, or that were pretreated with either 10µM or 100µM hydrogen peroxide. Individual proteolytic capacity of the 20S proteasome (caspase/peptidyl glutamyl-peptide hydrolyzing-like activity, trypsin-like, and chymotrypsin-like activity) was measured in the abdomen, head, and thorax. (A-C) Abdomen isolated from 3 day old (green) and 60 day old (black) males following hydrogen peroxide pretreatment. (A) Caspase-like activity. (B) Trypsin-like activity. (C) Chymotrypsin-like activity. (D-F) Head isolated from 3 day old (green) and 60 day old (black) males following hydrogen peroxide pretreatment. (D) Caspase-like activity. (E) Trypsin-like activity. (F) Chymotrypsin-like activity. (G-I) Thorax isolated from 3 day old (green) and 60 day old (black) males following hydrogen peroxide pretreatment. (G) Caspase-like activity. (H) Trypsin-like activity. (I) Chymotrypsin-like activity.

  
  
  

  

  Adaptation is dependent upon the 20S proteasome. Progeny of the Actin-GS-255B strain crossed to the β₁ or β₂ RNAi strains were aged for 5 days in the absence or presence of RU486 prior to H₂O₂ pretreatment. (A-D). The purpose of the experiment was not to completely knockdown the entire pool of 20S proteasome, but only to block the transcription/translation-dependent adaptive increase in proteasome expression following hydrogen peroxide pretreatment. Thus, we used RNAi conditions that blocked increased proteasome expression, without depressing basal proteasome protein levels. Using this approach, we found at least a 50% decrease in mRNA in RNAi strains, and within proteasome western blots and activity, we found blockage of the adaptive increase. After pretreatment, proteolytic capacity of the individual subunits of the 20S proteasome (trypsin-like, caspase/peptidyl glutamyl-peptide hydrolyzing-like activity, and chymotrypsin-like activity) were measured in whole fly lysate. (A-B) Proteolytic capacity in β₁ RNAi flies in the absence (black) “control” or presence (pink in females or blue in males, denoted with “+RU486”) of RU486. (A) Females. (B) Males. (C-D) Proteolytic capacity in β₂ RNAi flies in the absence (black) “control” or presence (pink in females or blue in males, denoted with “+RU486”) of RU486. (C) Females. (D) Males. (E,G) Females of the β₁ and β₂ RNAi strains raised in the absence of RU486 were either not pretreated “control” (black circle) or were pretreated with either 10µM H₂O₂ (grey squares) or 100µM H₂O₂ (grey circles) for 8 hours, followed by a 16-hour recovery prior to H₂O₂ [4.4M] challenge. Females of the β₁ and β₂ RNAi strains raised in the presence of RU486 were either not pretreated “+RU486” (pink triangle) or were pretreated with either 10µM H₂O₂ (pink diamonds) or 100µM H₂O₂ (pink squares) for 8 hours, followed by a 16-hour recovery prior to H₂O₂ [4.4M] challenge. (F,H) Males of the β₁ and β₂ RNAi strains raised in the absence of RU486 were either not pretreated “control” (black circle) or were pretreated with either 10µM H₂O₂ (grey circles) or 100µM H₂O₂ (grey triangles) for 8 hours, followed by a 16-hour recovery prior to H₂O₂ [4.4M] challenge. Males of the β₁ and β₂ RNAi strains raised in the presence of RU486 were either not pretreated “+RU486” (green circle) or were pretreated with either 10µM H₂O₂ (blue square) or 100µM H₂O₂ (blue circle) for 8 hours, followed by a 16-hour recovery prior to H₂O₂ [4.4M] challenge. Statistical difference in survival (p < 0.05) was calculated using the Log-Rank test. Statistical summary is located in Supplementary Table S2.

  
  
  

  

  Decline in 20S proteasome induction is accompanied by an accumulation of oxidized proteins. Carbonyl content was detected with a DNP antibody in progeny of the Actin-GS-255B strain crossed to w[1118] strain following [0, 10, or 100µM] H₂O₂ pre-treatment for 8 hours, followed by a 16-hour recovery to allow for adaption before challenged with H₂O₂ [4.4M] for an additional 24 hours. (A) Carbonyl content showed significant decrease following H₂O₂ pretreatment in 3 day old females. (B) Carbonyl content showed no significant change in 60 day old females, irrespective H₂O₂ pretreatment. (C) Carbonyl content was measured in 3 day old males that were pre-treated with H₂O₂. (D) 60 day old males showed no change in carbonyl content upon H₂O₂ pre-treatment and subsequent recovery. Western blots were performed in triplicate and carbonyl content was normalized to Actin-HRP. Error bars denote standard error of the mean (S.E.M) values. * P <0.05, ** P <0.01, *** P < 0.001, relative to the young control using one-way ANOVA. Statistical significance is shown with asterisks (pink *) in young females.

  
  
  

  

  Loss of proteasomal subunits or regulators impacts lifespan. (A,B) To control for the effect of RU486 on males and females, lifespan of progeny from the Actin-GS-255B strain crossed to w[1118] strain raised in the absence/control (black line) or presence of RU486, pink line for females and blue line for males. (A) Females. (B) Males. (C-F) Effect of removal of proteasome subunits on life span. The Actin-GS-255B strain was crossed to the β₁ RNAi or β₂ RNAi strains and the progeny were assayed for life span in the absence/control (black line) or presence (pink line in females and blue line in males) of RU486, as indicated. (C) β₁ RNAi females. (D) β₁ RNAi males. (E) β₂ RNAi females. (F) β₂ RNAi males. (G,H) Effect of removal of the Cap-n-collar (CncC)/Nrf2 orthologue upon lifespan. The Actin-GS-255B strain was crossed to the CncC RNAi strain. Male and female lifespan was measured in the absence/control (black line) or presence (pink line for females and blue line for males) of RU486. (G) CncC RNAi female. (H) CncC RNAi male. (I,J) Effect of removal of Keap1 upon lifespan. The Actin-GS-255B strain was crossed to the Keap1 RNAi strain. Males and female lifespan was assessed in the absence/control (black line) and presence (pink line for females and blue line for males) of RU486. (I) Keap1 RNAi female. (J) Keap1 RNAi male. Statistical difference in survival (p < 0.05) was calculated using the Log-Rank test. Statistical summary is located in Supplementary Table S3

  
  
  

  

  Hydrogen peroxide stress resistance improves with continual knockdown of Keap1 in aged (60 days) flies. In panels A and B, male and female progeny of the Actin-GS-255B strain crossed to Keap1 RNAi strain were collected and aged to 60 days. (A) Females raised in the absence of RU486 were either pretreated with 10µM H₂O₂ (gray line) “10µM H₂O₂” or not pre-treated with H₂O₂ (black line) “No Additions”. Females raised in the presence of RU486 were either pretreated with 10µM H₂O₂ (red line) “10µM H₂O₂ (+RU486)” or not pretreated with H₂O₂ (pink line) “+RU486”. (B) Males raised in the absence of RU486 were either pretreated with 10µM H₂O₂ (gray line) “10µM H₂O₂” or not pre-treated with H₂O₂ (black line) “No Additions”. Males raised in the presence of RU486 were either pretreated with 10µM H₂O₂ (blue line) “10µM H₂O₂ (+RU486)” or not pretreated with H₂O₂ (green line) “+RU486”. Statistical difference in survival (p < 0.05) was calculated using the Log-Rank test. Statistical summary is located in Supplementary Table S4.

  
  
  

• Research Paper Volume 9, Issue 2 pp 508-523

  Resveratrol fuels HER2 and ERα-positive breast cancer behaving as proteasome inhibitor

  Relevance score: 5.627314

  Cristina Andreani, Caterina Bartolacci, Kathleen Wijnant, Rita Crinelli, Marzia Bianchi, Mauro Magnani, Albana Hysi, Manuela Iezzi, Augusto Amici, Cristina Marchini

  Keywords: breast cancer, Δ16HER2 mice, resveratrol, estrogen receptor, proteasome

  Published in Aging on February 26, 2017

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  The phytoestrogen resveratrol has been reported to possess cancer chemo-preventive activity on the basis of its effects on tumor cell lines and xenograft or carcinogen-inducible in vivo models. Here we investigated the effects of resveratrol on spontaneous mammary carcinogenesis using Δ16HER2 mice as HER2+/ERα+ breast cancer model. Instead of inhibiting tumor growth, resveratrol treatment (0.0001% in drinking water; daily intake of 4μg/mouse) shortened tumor latency and enhanced tumor multiplicity in Δ16HER2 mice. This in vivo tumor-promoting effect of resveratrol was associated with up-regulation of Δ16HER2 and down-regulation of ERα protein levels and was recapitulated in vitro by murine (CAM6) and human (BT474) tumor cell lines. Our results demonstrate that resveratrol, acting as a proteasome inhibitor, leads to Δ16HER2 accumulation which favors the formation of Δ16HER2/HER3 heterodimers. The consequential activation of downstream mTORC1/p70S6K/4EBP1 pathway triggers cancer growth and proliferation. This study provides evidence that resveratrol mechanism of action (and hence its effects) depends on the intrinsic molecular properties of the cancer model under investigation, exerting a tumor-promoting effect in luminal B breast cancer subtype models.

  

  Resveratrol fuels mammary tumor formation in a luminal B breast cancer in vivo model. (a) Schematic representation of the experimental workflow used for the treatment of Δ16HER2 mice with vehicle or resveratrol. (b) Kaplan-Meier disease-free survival plot for vehicle- (n= 9) and resveratrol-treated (n= 9) Δ16HER2 mice. **p ≤ 0.01, Log Rank test. (c) Tumor multiplicity in resveratrol-treated vs control mice; the number of palpable mammary tumors per mouse is represented as mean ± SD. Statistical significance was assessed by two-way ANOVA test. (d) Left panel: Representative Hematoxyilin-Eosin (H&E) and PCNA stained sections of tumors from resveratrol and vehicle treated mice. Magnification 400 X. Right panel: Quantification of PCNA staining in tumors from resveratrol and vehicle treated mice. Data are expressed as mean ± SEM. *p < 0.05; unpaired two-tailed student t test.

  
  
  

  

  Resveratrol treatment induces HER2 over-expression and ERα down-regulation in HER2+/ERα+ mammary carcinomas. (a) Representative western blot analysis of ERα and β-actin (loading control) in spontaneous mammary tumors from Δ16HER2 mice supplemented or not with resveratrol (left panel), and relative densitometry quantification from three independent experiments (right panel). The significance was determined by unpaired two-tailed student t test, *p < 0.05. (b) Representative western blot analysis of HER2 and β-actin (loading control) in spontaneous mammary tumors from Δ16HER2 mice, supplemented or not with resveratrol (left panel), and relative densitometry quantification from three independent experiments (right panel). The significance was determined by unpaired two-tailed student t test, *p < 0.05. (c) Left panel: representative immunofluorescence images of tumor sections from control and resveratrol supplemented mice stained with an antibody anti-HER2 (green) and DRAQ5 dye (red) for nuclei staining. Magnification 400 X. Right panel: quantification of HER2 staining in tumors from resveratrol and vehicle treated mice. Data are expressed as mean ± SEM. *p < 0.05; unpaired two-tailed student t test. (d) Resveratrol treatment induces HER2 over-expression in lung metastases. Left panel: representative immunofluorescence images of lung metastasis sections from control and resveratrol supplemented mice stained with an antibody anti-HER2 (green) and DRAQ5 (red). Magnification 400 X. Right panel: quantification of HER2 staining in tumors from resveratrol and vehicle treated mice. Data are expressed as mean ± SEM. *p < 0.05; unpaired two-tailed student t test.

  
  
  

  

  Resveratrol triggers HER2 over-expression and ERα down-regulation in luminal B breast cancer cell lines. (a) CAM6 and (b) BT474 cells were incubated for 24 hours in the presence of vehicle or increasing concentrations of resveratrol and cell viability was determined by MTT assay. Results (including vehicle group treated with 0.02% DMSO) are expressed as percentage (%) of cell viability relative to untreated controls. Columns, mean of three separate experiments wherein each treatment was repeated in 16 wells; bars, SE. **p ≤ 0.01, ***p ≤ 0.001, one-way ANOVA followed by Bonferroni's multiple comparison test. Representative western blot analysis of HER2, ERα and β-actin (loading control) in murine CAM6 cells (c) or human BT474 cells (d), treated with resveratrol or 17β-estradiol or vehicle for 24 hours (upper panel), and relative densitometry quantification (lower panel). The significance was determined by one-way ANOVA (*p < 0.05, **p ≤ 0.01).

  
  
  

  

  Resveratrol inhibits the chymotrypsin-like activity of 20S proteasome and resulted in an increased accumulation of protein-ubiquitin conjugates. Δ16HER2 and β-actin (internal control) mRNA levels were measured by semi-quantitative RT-PCR (a) and qRT-PCR (b) analyses in spontaneous mammary tumors from Δ16HER2 mice supplemented or not with resveratrol. (c) The chymotrypsin-like activity of the 20S proteasome was measured in tumor samples, from Δ16HER2 mice treated or not with resveratrol, as described in Materials and Methods, and expressed as fluorimetric units (FU) min^-1 mg^-1. The significance was determined by unpaired two-tailed student t test, *p < 0.05. (d) Western blot analysis of 20S proteasome subunit content (upper panel) and ubiquitin-protein conjugates and free ubiquitin levels (lower panel) in tumor samples from Δ16HER2 mice treated or not with resveratrol. β-actin was used as loading control. (e) Representative western blot analysis of p53 and β-actin (loading control) in spontaneous mammary tumors from Δ16HER2 mice, supplemented or not with resveratrol, and (f) relative densitometric quantification from three independent experiments. The significance was determined by unpaired two-tailed student t test, *p < 0.05.

  
  
  

  

  Increased HER2 level induced by resveratrol results in a preferential activation of mTORC1/p70S6K pathway. Representative western blot analysis of HER2 and HER3 downstream signaling pathways in spontaneous mammary tumors from Δ16HER2 mice, treated or not with resveratrol (left panels), and densitometry quantification from three independent experiments (right panels). (a) PI3K/AKT and (b) mTOR signaling pathways were analyzed. β-actin was used as loading control. The significance was determined by t-test (*p < 0.05; **p ≤ 0.01; ***p ≤ 0.001). (c) Resveratrol promotes the direct coupling of Δ16HER2 to HER3 kinase in HER2+/ERα+ breast cancer. Δ16HER2 kinase co-immunoprecipitates with pHER3. Proteins were immunoprecipitated with anti-HER2 antibody and then probed by western blot for pHER3. Input represents 10% of the co-immunoprecipitation protein amount (30 μg).

  
  
  

  

  Proposed resveratrol’s mechanism of action in a luminal B breast cancer model. Our data show that resveratrol down-regulates ERα and lowers the chymotrypsin-like activity of the 20S proteasome in HER2+/ERα+ breast cancer, leading to an increased accumulation of Δ16HER2, which efficiently couples to HER3 and activates the PI3K-AKT-mTOR pathway. In particular, Δ16HER2/HER3 heterodimers trigger the mTORC1/p70S6K/4EBP1 signaling axis inducing an up-regulation of protein synthesis and cell growth. On the other hand, resveratrol inhibits mTORC2 and promotes phosphorylation of PTEN, reducing its catalytic activity, thereby enhancing PI3K-mediated AKT activation, while feedback loops compensate it.

  
  
  

• Research Paper Volume 8, Issue 12 pp 3321-3340

  RelA NF-κB subunit activation as a therapeutic target in diffuse large B-cell lymphoma

  Relevance score: 4.4529037

  Mingzhi Zhang, Zijun Y. Xu-Monette, Ling Li, Ganiraju C. Manyam, Carlo Visco, Alexandar Tzankov, Jing Wang, Santiago Montes-Moreno, Karen Dybkaer, April Chiu, Attilio Orazi, Youli Zu, Govind Bhagat, Kristy L. Richards, Eric D. Hsi, William W.L. Choi, J. Han van Krieken, Jooryung Huh, Maurilio Ponzoni, Andrés J.M. Ferreri, Michael B. Møller, Ben M. Parsons, Jane N. Winter, Miguel A. Piris, L. Jeffrey Medeiros, Lan V. Pham, Ken H. Young

  Keywords: NF-κB, p65, diffuse large B-cell lymphoma, TP53, GCB, gene expression profiling, proteasome inhibitor

  Published in Aging on December 8, 2016

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  It has been well established that nuclear factor kappa-B (NF-κB) activation is important for tumor cell growth and survival. RelA/p65 and p50 are the most common NF-kB subunits and involved in the classical NF-kB pathway. However, the prognostic and biological significance of RelA/p65 is equivocal in the field. In this study, we assessed RelA/p65 nuclear expression by immunohistochemistry in 487 patients with de novo diffuse large B-cell lymphoma (DLBCL), and studied the effects of molecular and pharmacological inhibition of NF-kB on cell viability. We found RelA/p65 nuclear expression, without associations with other apparent genetic or phenotypic abnormalities, had unfavorable prognostic impact in patients with stage I/II DLBCL. Gene expression profiling analysis suggested immune dysregulation and antiapoptosis may be relevant for the poorer prognosis associated with p65 hyperactivation in germinal center B-cell–like (GCB) DLBCL and in activated B-cell–like (ABC) DLBCL, respectively. We knocked down individual NF-κB subunits in representative DLBCL cells in vitro, and found targeting p65 was more effective than targeting other NF-κB subunits in inhibiting cell growth and survival. In summary, RelA/p65 nuclear overexpression correlates with significant poor survival in early-stage DLBCL patients, and therapeutic targeting RelA/p65 is effective in inhibiting proliferation and survival of DLBCL with NF-κB hyperactivation.

  

  Nuclear expression of p65 and its effect on progression-free survival (PFS) in diffuse large B-cell lymphoma (DLBCL) (A) Representative immunohistochemical analysis (IHC) and histograms for p65 nuclear expression in DLBCL. The mean expression of nuclear p65 was significantly higher in the germinal center B-cell–like (GCB) subtype than in the activated B-cell–like (ABC) subtype. (B) In overall DLBCL, high p65 nuclear expression (p65^high, ≥50% nuclear expression) was associated with a trend towards worse PFS. In patients with stage I/II DLBCL, p65^high correlated with significantly shorter PFS. In patients with stage III/IV DLBCL, p65^high did not show signficant prognostic impact. (C) p65^high correlate with significantly shorter PFS in patients with stage I/II DLBCL independent of GCB/ABC subtypes. (D) TP53 mutation status was significantly associated with higher RELA mRNA expression. (E) In patients with stage I/II DLBCL, p65^high correlate with significantly shorter PFS independent of TP53 mutation status although more significant in patients with wild-type TP53 (WT-TP53). In patients with mutated TP53 (MUT-TP53) and stage III/IV DLBCL, p65^high was associated with a trend of better PFS.

  
  
  

  

  Prognosis for p65 hyperactivation in diffuse large B-cell lymphoma (DLBCL). (A) In overall DLBCL, high p65 nuclear expression (p65^high, ≥50% nuclear expression) was associated with unfavorable progression-free survival (PFS). The adverse prognostic impact was significant in patients with an international prognostic index score (IPI) ≤2. (B) In patients with stage I/II DLBCL, p65^high correlated with significantly poorer PFS. Among p65^high DLBCL patients, disease stages did not show further prognostic impact. (C) p65^high correlated with significantly poorer PFS in patients with GCB-DLBCL and patients with wild-type TP53 (WT-TP53).

  
  
  

  

  Gene expression profiling analysis. (A) Heatmaps for comparisons between DLBCL patients with p65^high expression (IHC ≥50%) and those without p65 nuclear expression (IHC <10%) in the overall and GCB-DLBCL cohorts (FDR <0.15 and FDR <0.05, respectively). (B) BIRC5/survivin and BCL2L2 were significantly upregulated in p65^high ABC-DLBCL. (C) NOXA/PMAIP1 was significantly downregulated in p65^high ABC-DLBCL. (D-E) BTK and TNFRSF13B were significantly upregulated in the p65^high group in ABC-DLBCL but not in GCB-DLBCL. (F-H) MAP3K14/NIK, MYD88, and TNFRSF13C were significantly upregulated in the p65^high group in GCB-DLBCL but not in ABC-DLBCL.

  
  
  

  

  Gene expression analysis for p65 hyperactivation in diffuse large B-cell lymphoma (DLBCL). (A) Heatmaps for gene differentially expressed between p65^high (IHC ≥50%) and p65^low (IHC <50%) patients in DLBCL overall and in ABC-DLBCL (false discovery rate <0.30 and <0.20, respectively). (B) Heatmaps for genes differentially expressed between p65^high (IHC ≥50%) and p65^low (IHC <50%) patients and between p65^high (IHC ≥50%) and p65^intermediate (IHC 10-40%) patients with germinal center B-cell–like DLBCL (false discovery rate <0.05 and <0.20, respectively).

  
  
  

  

  Molecular targeting of NF-κB in diffuse large B-cell lymphoma (DLBCL) cell lines. (A) DLBCL-MS cells were transfected with empty control vector or a pCMV-ΙκBαM vector for 24 hrs. Nuclear extracts (10 μg) were analyzed for NF-κB expression by EMSA. Cytoplasmic extracts were assessed for ΙκBα and actin protein expression by Western blotting. (B) Transfected cells from part A were also assessed for apoptosis after 24 hours of incubation using annexin V assays. (C) MS cells were transfected with plasmids expressing the p52, RelB, p65, c-Rel, or a non-specific (NS) shRNA. Forty-eight hours post-transfection, proteins were extracted and analyzed for NF-κB component inhibition by Western blot. (D) Indicated DLBCL cell lines were transfected with the validated green fluorescent protein (GFP)-plasmid–based shRNA for each of the NF-κB subunits. After 16 hours, GFP–positive cells were sorted and assessed using proliferation assays. Data represent two independent experiments with triplicate samples. Abbreviations: GCB, germinal center B-cell–like; ABC, activated B-cell–like, DN, dominant negative.

  
  
  

  

  Pharmacological inhibition of constitutive NF-κB activation in DLBCL cells. (A-B) DLBCL cells (MS) were cultured in the presence of bortezomib (BZ, 25 nM) or BAY-11 (1 µM) for the indicated time points (hours). Nuclear extracts were purified and subjected to EMSA analyzed for NF-κB DNA binding activity; cytoplasmic extracts were subjected to immunobloting for pIκBa and actin. (C) DLBCL-MS cells cultured in the presence of bortezomib (BZ, 25 nM) or BAY-11 (1 µM) for 24 hours and then analyzed for p50 (red) and p65 (green) protein expression by confocal microscopy analysis. Topro-3 (blue) serves as a nuclear staining marker.

  
  
  

  

  Inhibition of NF-κB in DLBCL cells leads to cell growth inhibition, G0/G1 cell cycle arrest, and apoptosis. (A) Representative ABC- and GCB-DLBCL cell lines were treated with bortezomib (BZ) or BAY-11 for 48 hours and cell proliferation was measured using 3H-thymidine incorporation assays. The percentages of growth inhibition of treated cells relative to untreated (control cells) were plotted. The data shown are the means and ranges of triplicate cultures from three independent experiments. (B) DLBCL-MS cells were cultured in the absence or presence of bortezomib (BZ) or BAY-11 and subjected to a 20S proteasome assay. Purified 20S proteasome was used as a positive control. Abbreviations: RLU, relative light unit; 20S pro, 20S proteasome, Neg Cont., negative control. (C) DLBCL-MS cells were cultured in the absence or presence of BZ (50 nM) or BAY-11 (1 µM) and analyzed for cell cycle profile. The percentages of cells in G0/G1, S, and G2M phases are shown. (D) DLBCL-MS cells were cultured in the absence or presence of BZ (50 nM) or BAY-11 (1 µM) for the indicated time points and then analyzed for apoptosis using annexin V assays. (E) DLBCL-MS cells were cultured in the presence of BZ (50 nM) or BAY-11 (1 µM) and in some cases with the caspase 3 inhibitor DEVD or the caspse 1 inhibitor VAD. Caspase 3 activity was measured after 24 hours of treatments. Caspase 3 activity was observed after 12 hours of treatment. Abbreviations: RLU, relative light units. (F) DLBCL-MS cells were cultured in the presence of BZ (50 nM) or BAY-11 (1 µM) for the indicated time points and cell extracts were subjected to Western blotting for a known caspase substrate, poly-(ADP-ribose) polymerase (PARP) cleavage.

  
  
  

• Research Paper Volume 8, Issue 1 pp 127-146

  Age-related deficits in skeletal muscle recovery following disuse are associated with neuromuscular junction instability and ER stress, not impaired protein synthesis

  Relevance score: 5.3724933

  Leslie M. Baehr, Daniel W.D. West, George Marcotte, Andrea G. Marshall, Luis Gustavo De Sousa, Keith Baar, Sue C. Bodine

  Keywords: aging, hindlimb unloading, anabolic resistance, ubiquitin proteasome system, autophagy

  Published in Aging on January 29, 2016

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  Age-related loss of muscle mass and strength can be accelerated by impaired recovery of muscle mass following a transient atrophic stimulus. The aim of this study was to identify the mechanisms underlying the attenuated recovery of muscle mass and strength in old rats following disuse-induced atrophy. Adult (9 month) and old (29 month) male F344BN rats underwent hindlimb unloading (HU) followed by reloading. HU induced significant atrophy of the hindlimb muscles in both adult (17-38%) and old (8-29%) rats, but only the adult rats exhibited full recovery of muscle mass and strength upon reloading. Upon reloading, total RNA and protein synthesis increased to a similar extent in adult and old muscles. At baseline and upon reloading, however, proteasome-mediated degradation was suppressed leading to an accumulation of ubiquitin-tagged proteins and p62. Further, ER stress, as measured by CHOP expression, was elevated at baseline and upon reloading in old rats. Analysis of mRNA expression revealed increases in HDAC4, Runx1, myogenin, Gadd45a, and the AChRs in old rats, suggesting neuromuscular junction instability/denervation. Collectively, our data suggests that with aging, impaired neuromuscular transmission and deficits in the proteostasis network contribute to defects in muscle fiber remodeling and functional recovery of muscle mass and strength.

  

  In vivo isometric force production at frequencies ranging from 20-125 Hz was measured in (A) adult (9 mo) and (B) old (29 mo) rats prior to hindlimb unloading (HU) (baseline, open squares), after 14 days of HU (open circles), and then after 7 (open triangles) and 13 days of reloading (open diamonds). n=6/group. Values are mean ± SEM, *p<0.05 vs old baseline value, ^#p<0.05 for old 14d HU value vs old baseline value at 40 Hz. (C) Normal cage activity of adult (open squares, n=4) and old (filled squares, n=5) rats was recorded during the dark cycle for six days prior to HU and for the first five days of reloading following 14 days of HU. Values are mean ± SEM, ^#p<0.05 vs old control.

  
  
  

  

  Adult (9 mo) and old (29 mo) male rats underwent HU for 14 days or underwent HU for 14 days and then were allowed to resume normal weight bearing activity for either 1, 3, 7, or 14 days. Average masses of the soleus (A), plantaris (C), medial gastrocnemius (D), extensor digitorum longus (E), and tibialis anterior (F) muscles of adult (open squares) and old (filled squares) rats at the various HU and reloading time points (n=6-7/group). (B) Changes in fiber cross-sectional area (CSA) were measured in the soleus of adult (open squares) and old (filled squares) rats after 14 days of HU and after 3, 7, and 14 days of reloading. Fiber CSA was determined from laminin-stained cross sections (n=5-6/group). Values are mean ± SEM, *p<0.05 vs adult control, ^#p<0.05 vs old control, ^φp<0.05 vs adult at same time point.

  
  
  

  

  (A) Using the SUnSET method, protein synthesis was measured in the soleus and tibialis anterior (TA) muscles of adult (9 mo, open squares) and old (29 mo, filled squares) rats after 14 days of HU and after 3, 7, and 14 days of reloading. Total protein, determined by stain-free imaging of the PVDF membrane, was used to normalize protein expression. Puromycin values are expressed as a percentage of each age-matched control group (n=6-7/group). Total RNA (μg/mg muscle) was measured in the soleus (B) and TA (C) muscles of adult (open squares) and old (filled squares) rats after 14 days of HU and after 1, 3, 7, and 14 days of reloading (n=6/group). Values are mean ± SEM, *p<0.05 vs adult control, ^#p<0.05 vs old control, ^φp<0.05 vs adult at same time point.

  
  
  

  

  mRNA expression (fold change relative to adult control) of HDAC4, myogenin, Gadd45a, Runx1, and acetylcholine receptor subunits alpha (AChRα) and gamma (AChRγ) were measured in the soleus (A) and tibialis anterior (TA) (B) muscles of adult (9 mo, open squares) and old (29 mo, filled squares) rats after 14 days of HU and after 1, 3, 7, and 14 days of reloading (n=5-6/group). Values are mean ± SEM, *p<0.05 vs adult control, ^#p<0.05 vs old control, ^φp<0.05 vs adult at same time point.

  
  
  

  

  Representative Western blot and quantification of total ubiquitin levels in the soleus (A) and tibialis anterior (TA) (B) muscles of adult (9 mo, open squares) and old (29 mo, filled squares) rats after 14 days of HU and after 3, 7, and 14 days of reloading. Total protein, determined by stain-free imaging of the PVDF membrane, was used to normalize protein expression. Data are expressed as a percentage relative to the adult control group (n=5-6/group). Values are mean ± SEM, *p<0.05 vs adult control, ^#p<0.05 vs old control, ^φp<0.05 vs adult at same time point.

  
  
  

  

  mRNA expression of MuRF1 and MAFbx was assessed by quantitative PCR in the soleus (A,B) and tibialis anterior (TA) (C,D) muscles of adult (9 mo, open squares) and old (29 mo, filled squares) rats after 14 days of HU and following 1, 3, 7, and 14 days of reloading. Gene expression was normalized to tissue weight. Data are expressed as a fold change relative to the adult control group. Proteolytic activity of the β1, β2, and β5 subunits of the 20S and 26S proteasome was measured in the soleus (E) and TA (F) muscles of adult (open squares) and old (filled squares) rats after 14 days of HU and after 3, 7, and 14 days of reloading. Data are expressed as a percentage relative to the activity of the adult control group for each subunit (n=4-6/group). Values are mean ± SEM, *p<0.05 vs adult control, ^#p<0.05 vs old control, ^φp<0.05 vs adult at same time point.

  
  
  

  

  Expression of the autophagy-related proteins phospho- and total Ulk1, p62, Atg7, Beclin, and LC3B-II was measured by Western blot and quantified after 14 days of HU and following 3, 7, and 14 days of reloading in the soleus (A) and tibialis anterior (TA) (B) muscles of adult (9 mo, open squares) and old (29 mo, filled squares) rats. Total protein, determined by stain-free imaging of the PVDF membrane, was used to normalize protein expression. Data are expressed as a percentage relative to the adult control group for each protein (n=4-6/group). Cathepsin L activity was measured by fluorometric assay after 14 days of HU and following 3, 7, and 14 days of reloading in the soleus (C) and TA (D) muscles of adult (open squares) and old (filled squares) rats. Data are expressed as a percentage relative to the activity of the adult control group (n=5-6/group). Values are mean ± SEM, *p<0.05 vs adult control, ^#p<0.05 vs old control, ^φp<0.05 vs adult at same time point.

  
  
  

  

  Representative Western blots (A) and quantification of LC3B-II protein expression in the soleus (B) and tibialis anterior (TA) (C) muscles of control and 7 day reloaded adult (9 mo) and old (29 mo) rats treated with or without colchicine. Total protein, determined by stain-free imaging of the PVDF membrane, was used to normalize protein expression. Data are expressed as a percentage relative to the adult control group for each protein (n=3-6/group). Values are mean ± SEM, *p<0.05.

  
  
  

  

  Representative Western blots and quantification of ER stress markers BiP (A), PDI (B), and CHOP (C) in the soleus and tibialis anterior (TA) muscles of adult (9 mo, open squares) and old (29 mo, filled squares) rats following 14 days of HU and after 3, 7, and 14 days of reloading. Total protein, determined by stain-free imaging of the PVDF membrane, was used to normalize protein expression. Data are expressed as a percentage relative to the adult control group for each protein (n=4-6/group). Values are mean ± SEM, *p<0.05 vs adult control, ^#p<0.05 vs old control, ^φp<0.05 vs adult at same time point.

  
  
  

• Research Paper Volume 7, Issue 10 pp 776-787

  Proteasome function is not impaired in healthy aging of the lung

  Relevance score: 7.878927

  Anne Caniard, Korbinian Ballweg, Christina Lukas, Ali Ö. Yildirim, Oliver Eickelberg, Silke Meiners

  Keywords: proteostasis, proteasome, emphysema, LMP2, immunoproteasome

  Published in Aging on October 21, 2015

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  Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age‐related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase‐like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice.

  Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging.

  

  (A) Overview over the proteasome active site nomenclature. (B) Representative H&E staining of lung from young and aged wildtype C57Bl/6 mice. Images from two individual animals are depicted per group. (C) RTqPCR analysis of proteasome subunit mRNA expression in young and aged wildtype mice. n = 8+SEM. (D) Western blot analysis of proteasome subunit expression in young and aged wildtype mice and quantification of protein expression relative to the β − actin loading control. Bar graphs show mean+SEM.

  
  
  

  

  Cleavage of luminogenic model substrates specific for the caspase-like (C-L), chymotrypsin-like (CT-L) or trypsin-like (T-L) active site of the proteasome in lung tissue lysate of (A) C57Bl/6 mice n = 8+SEM and in (B) lung tissue lysate of FVB mice. n = 6-7+SEM.

  
  
  

  

  (A) Native gel analysis of proteasome complexes with activity overlay for the chymotrypsin-like (CT-L) proteasome activity in lung tissue lysates from young and aged C57Bl/6 mice and (B) quantification thereof. (C) Native gel analysis with activity overlay for the caspase-like (C-L) proteasome activity and (D) quantification thereof. The appearance of 20S double bands is possibly due to the binding of alternative proteasome regulators such as PA28 family members [57]. Bar graphs show mean+SEM. (E) Western blot analysis for lysin-48 (K48) linked polyubiquitinated proteins in lung tissue lysates from young and aged C57Bl/6 mice. Detection of β-actin was used as a loading control. (F) Luciferase activity in lung tissue of young and aged FVB-luc reporter mice. n = 6-7+SEM.

  
  
  

  

  (A) Representative H&E staining of lungs from young and aged LMP2^−/− mice. Images from two individual animals are depicted per group (B) Western blot analysis of proteasome subunit expression in LMP2^−/− mice and (C) quantification of protein expression relative to the β − actin loading control. Bar graphs show mean+SEM.

  
  
  

  

  (A) Cleavage of luminogenic model substrates specific for the caspase-like (C-L), chymotrypsin-like (CT-L) or trypsin-like (T-L) active site of the proteasome in lung tissue lysate of LMP2^−/− mice. n = 5-8+SEM. (B) Native gel analysis of proteasome complexes with activity overlay for CT-L activity in lung tissue lysate of LMP2^−/− mice and (C) quantification thereof. Bar graphs show mean+SEM. (D) Western blot analysis for lysin-48 linked polyubiquitinated proteins in lung tissue lysates from young and aged LMP2^−/− mice (E) and quantification thereof. β-Actin was used as a loading control.

  
  
  

• Research Paper Volume 5, Issue 11 pp 802-812

  Enhancing protein disaggregation restores proteasome activity in aged cells

  Relevance score: 6.2170715

  Veronica Andersson, Sarah Hanzén, Beidong Liu, Mikael Molin, Thomas Nyström

  Keywords: Replicative aging, yeast, UPS, proteasome, disaggregation

  Published in Aging on November 12, 2013

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  The activity of the ubiquitin-proteasome system, UPS, declines during aging in several multicellular organisms. The reason behind this decline remains elusive. Here, using yeast as a model system, we show that while the level and potential capacity of the 26S proteasome is maintained in replicatively aged cells, the UPS is not functioning properly in vivo. As a consequence cytosolic UPS substrates, such as ΔssCPY* are stabilized, accumulate, and form inclusions. By integrating a pGPD-HSP104 recombinant gene into the genome, we were able to constitutively elevate protein disaggregase activity, which diminished the accumulation of protein inclusions during aging. Remarkably, this elevated disaggregation restored degradation of a 26S proteasome substrate in aged cells without elevating proteasome levels, demonstrating that age-associated aggregation obstructs UPS function. The data supports the existence of a negative feedback loop that accelerates aging by exacerbating proteostatic decline once misfolded and aggregation-prone proteins reach a critical level.

  

  (A) Representative image of bud scars in young and aged fractions. (B) Age distribution in the young and aged mother cell fractions collected. (C) The average age (arrow) of the isolated mother cells in relation to the life-span survival curve. (D) Localization of ΔssCPY*-GFP in young and aged cells. (E) Percentage of cells with ΔssCPY*-GFP foci in young and aged fractions (n=3). (F) Representative image of Hsp104-GFP distribution in young and aged cells (G) Percentage of cells with Hsp104-GFP foci in young and aged fractions (n>3). Error bars represent standard deviation. For statistical analyses, the paired two-tailed t-test was used ***P<0.001. (n= sets of analysis; Scale-bars represent 10μm).

  
  
  

  

  (A) Representative images of Hsp104-GFP distribution with and without the addition of the proteasome inhibitor MG132 (100 μM) to a growing culture. (B) Percentage of cells with Hsp104-GFP foci after partial proteasomal inhibition by MG132. (C) Relative levels of protein ubiquitination upon growing the conditional proteasomal mutant rpt4-145 (ts) at the permissive (22°C) and near non-permissive (35°C) temperature. Levels were normalized to the levels in wt cells grown at 22°C. (D) Representative images of Hsp104-GFP localization upon growth of wt and rpt4-145 cell at the permissive (22°C) and near non-permissive (35°C) temperature. (E) Percentage of wt and rpt4-145 cells with Hsp104-GFP foci after growth at the permissive (22°C) and near non-permissive (35°C) temperature. (F) The clearance of Hsp104-GFP foci was followed over time after an initial burst in aggregate formation after the temperature shift. Time point “0” depicts cells growing at 22°C and subsequent time points depict cells following the indicated time at 35°C. (G) Percentage of wt and rpt4-145 cells with Hsp104-GFP foci. Quantification of Hsp104-GFP foci formation in the experiment in “F”. Error bars represent standard deviation (n=2). For statistical analysis, the paired two-tailed t-test was used where *P<0.05, **P<0.01, ***P<0.001 and n.s = no significant difference. (n= sets of analysis; Scale-bars represent 10μm).

  
  
  

  

  (A) Representative images of Hsp104-GFP distribution in wt and ubr2Δ cells after H₂O₂ exposure (0.6 mM). Time point “0” depicts cells before stress whereas subsequent time points depict cells at the indicated time point following the addition of H₂O_2. (B) Percentage of wt and ubr2Δ cells with Hsp104-GFP foci over time after peroxide stress (n=2). (C) Representative image of Hsp104-GFP localization in young and aged (~13-15 generations) wt and ubr2Δ cells. (D) Percentage of aged wt and ubr2Δ cells containing Hsp104-GFP foci (n≥3). “Total” represents the percentage of cells containing foci independent of size, whereas “inclusions” represent the percentage of cells with large foci. Error bars represent standard deviation. For statistical analysis, the paired two-tailed t-test was used where *P<0.05, **P<0.01, ***P<0.001 and n.s = no significant difference. (n= sets of analysis; Scale-bars represent 10μm).

  
  
  

  

  (A) Degradation of the in vivo UPS substrate ub-P-βgal over time in young and aged (~13-15 generations) cells after the inhibition of protein synthesis. The starting β-gal levels were set to 1. The figure depicts representative results from one out of six independent experiments (P=7.38^E-06). (B) Relative levels of Rpt1p (19S subunit), 20S core proteins, and 26S proteasomes (based on native gels) in aged cells compared to young cells (n≥3). (C) Proteasomal capacity in total protein extracts measured as the rate of hydrolysis of the fluorogenic peptide suc-LLVY-AMC (Chymotryptic activity) depicted as the specific activity (nmol AMC/min*mg total protein). A representative figure is presented (n=3). (D) Rpt6-GFP (19S subunit) localization in young and aged (~13-15 generations) cells. Error bars represent standard deviation. For statistical analysis, the paired two-tailed t-test was used where *P<0.05, **P<0.01, ***P<0.001 and n.s = no significant difference. (n= sets of analysis; Scale-bars represent 10μm.

  
  
  

  

  (A) Relative levels of Hsp104 produced from the wt HSP104 and GPD promoters as determined by anti-Hsp104 immuno-blot analysis (n=2). (B) Percentage of cells with Ssa2-GFP foci following heat stress in the wt and Hsp104 overproducing (Hsp104↑) strains. Time point “0” represents cells after 30 min incubation at 42°C, whereas subsequent time points represent cells following the indicated time of recovery at 30°C (n=2). (C) The effect of Hsp104 overproduction on aggregate formation. Representative image of Ssa2-GFP and ΔssCPY*-GFP in young and aged, wt and Hsp104 overproducing cells. (D) Percentage of aged wt and Hsp104 overproduction cells with Ssa2-GFP or ΔssCPY*-GFP foci (n=2). (E) Relative half-life of β-gal in wt and Hsp104 overproducing young and aged cells. Values were normalized to the half-life in wt young cells (n=3). (F) Relative levels of Rpt1p (19S subunit), 20S core proteins, and proteasome specific activity (rate of hydrolysis of suc-LLVY-AMC) in Hsp104 overproducing cells compared to wt cells. (n≥2). (G) Relative levels of soluble β-gal in wt and Hsp104 overproducing cells normalized to total protein (see Experimental procedures for details) (n=2). Error bars represent standard deviation. For statistical analysis, the paired two-tailed t-test was used where *P<0.05, **P<0.01, ***P<0.001 and n.s = no significant difference. (Scale-bar represents 10μM). (H) Life-span curves of wt and Hsp104 overproducing cells. Mean replicative life-spans are: wt (28 ± 0), Hsp104 overproduction (29.5 ± 1.5) (n=2). (n= sets of analysis).

  
  
  

  

  One of many cellular functions of the UPS is to degrade native (e.g. cell cycle regulators), damaged, or aberrant proteins. If the level of damaged proteins exceeds the proteasomal capacity or if UPS degradation is somehow compromised, protein aggregates will form. The disaggregase Hsp104 can together with Hsp70/40 resolve protein aggregates. However, if the accumulation of aggregated protein is too severe, as seen in aged cells, these may interfere with the proper function of the UPS creating a negative feedback loop. This study indicates that the buildup of aggregates in aged cells can be counteracted either by increasing the amount of proteasomes present by stabilizing Rpn4, through the deletion of UBR2, or by increasing the level of disaggregation through Hsp104 overproduction. [UPS=ubiquitin proteasome system; P^N= native protein; P^D= damaged protein P^agg.= aggregated proteins].

  
  
  

• Editorial

  Boosting autophagy in anti-tumor proteasome inhibition-mediated cardiotoxicity

  Relevance score: 9.45707

  Eleni-Dimitra Papanagnou, Sentiljana Gumeni, Ioannis P. Trougakos

  Keywords: autophagy, cardiotoxicity, proteasome dysfunction, proteasome inhibitors, proteotoxicity

  Published in Aging on November 30, 1899

• Priority Research Paper pp undefined-undefined

  Long-term treatment with chloroquine increases lifespan in middle-aged male mice possibly via autophagy modulation, proteasome inhibition and glycogen metabolism

  Relevance score: 5.627314

  Thorsten R. Doeppner, Cristin Coman, Daiana Burdusel, Diana-Larisa Ancuta, Ulf Brockmeier, Daniel Nicolae Pirici, Kuang Yaoyun, Dirk M. Hermann, Aurel Popa-Wagner

  Keywords: chloroquine, longevity, middle-aged mice, toxicity, autophagy, proteasome

  Published in Aging on Invalid Date

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  Previous studies have shown that the polyamine spermidine increased the maximum life span in C. elegans and the median life span in mice. Since spermidine increases autophagy, we asked if treatment with chloroquine, an inhibitor of autophagy, would shorten the lifespan of mice. Recently, chloroquine has intensively been discussed as a treatment option for COVID-19 patients. To rule out unfavorable long-term effects on longevity, we examined the effect of chronic treatment with chloroquine given in the drinking water on the lifespan and organ pathology of male middle-aged NMRI mice. We report that, surprisingly, daily treatment with chloroquine extended the median life span by 11.4% and the maximum life span of the middle-aged male NMRI mice by 11.8%. Subsequent experiments show that the chloroquine-induced lifespan elevation is associated with dose-dependent increase in LC3B-II, a marker of autophagosomes, in the liver and heart that was confirmed by transmission electron microscopy. Quite intriguingly, chloroquine treatment was also associated with a decrease in glycogenolysis in the liver suggesting a compensatory mechanism to provide energy to the cell. Accumulation of autophagosomes was paralleled by an inhibition of proteasome-dependent proteolysis in the liver and the heart as well as with decreased serum levels of insulin growth factor binding protein-3 (IGFBP3), a protein associated with longevity. We propose that inhibition of proteasome activity in conjunction with an increased number of autophagosomes and decreased levels of IGFBP3 might play a central role in lifespan extension by chloroquine in male NMRI mice.

• Research Paper pp undefined-undefined

  Identification and validation of ubiquitin-proteasome system related genes as a prognostic signature for papillary renal cell carcinoma

  Relevance score: 5.665274

  Xin Zhang, Xinli Liu, Renhua Xiong, Han-Xiang An

  Keywords: ubiquitin-proteasome system genes, risk model, PRCC, TCGA, bioinformatics, prognosis, immunity

  Published in Aging on Invalid Date

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  Abstract: Dysregulation of the ubiquitin-proteasome system (UPS) pathway greatly affects uncontrolled proliferation, genomic instability, and carcinogenesis, particularly in those with renal papillary cell carcinoma (PRCC). However, there is little information at the molecular level about the full link between changes in the genes involved in ubiquitin-mediated proteolysis and PRCC.

  Methods: The Cancer Genome Atlas (TCGA) and GeneCards databases were utilized to find the clinical data and gene expression patterns of patients with PRCC. Univariate Cox regression analysis and absolute shrinkage and selection operator (LASSO) analyses identified a risk signature formed by ten optimal UPS genes. The predictive value of the risk signature in TCGA-PRCC cohorts was evaluated using Kaplan-Meier analysis and receiver operating characteristic (ROC) curves. By utilizing GO enrichment and the KEGG pathway, the interactions of differentially expressed genes connected to ubiquitin-mediated proteolysis were functionally examined. The protein expression of the hub genes was affirmed using the Human Protein Atlas (HPA) database. The effectiveness of particular CDC20 and UBE2C in vitro was confirmed by experimental research.

  Results: Ten of the best ubiquitin-mediated proteolysis genes (UBE2C, DDB2, CBLC, BIRC3, PRKN, UBE2O, SIAH1, SKP2, UBC, and CDC20) were detected to create a risk signature. The high-risk score group stratified was associated with advanced tumor status and poor survival of PRCC patients. 10 genes were also found to be associated with the cell cycle pathway and ubiquitin-mediated proteolysis to GO and KEGG analysis. Of these 10 genes, CDC20 and UBE2C are highly expressed in tumor tissue and correlated with cancer immunity founded on the analyses of the expression of human protein atlas and TISIDB. The downregulation of UBE2C facilitated tumor inhibition and the anti-immune effect was confirmed by in vitro experiments.

  Conclusion: Our results indicate that the risk model created from the ubiquitin-mediated proteolysis genes can be reliably and accurately predict the prognosis of PRCC patients, highlighting its targeted value for PRCC treatment. Particularly, the expression of UBE2C may be crucial for the prognosis and immunological treatment of renal cancer.

• Research Paper pp undefined-undefined

  A nomogram for predicting prognosis of multiple myeloma patients based on a ubiquitin-proteasome gene signature

  Relevance score: 6.472707

  Dexiang Ji, Yong Liu, Wenjie Sun, Qing Shi, Guoan Chen, Zhiwang Song, Yanxia Jiang

  Keywords: multiple myeloma, ubiquitin-proteasome system, prognosis, visualization, signature

  Published in Aging on Invalid Date

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  Background: Multiple myeloma (MM) is a malignant hematopoietic disease that is usually incurable. However, the ubiquitin-proteasome system (UPS) genes have not yet been established as a prognostic predictor for MM, despite their potential applications in other cancers.

  Methods: RNA sequencing data and corresponding clinical information were acquired from Multiple Myeloma Research Foundation (MMRF)-COMMPASS and served as a training set (n=787). Validation of the prediction signature were conducted by the Gene Expression Omnibus (GEO) databases (n=1040). To develop a prognostic signature for overall survival (OS), least absolute shrinkage and selection operator regressions, along with Cox regressions, were used.

  Results: A six-gene signature, including KCTD12, SIAH1, TRIM58, TRIM47, UBE2S, and UBE2T, was established. Kaplan-Meier survival analysis of the training and validation cohorts revealed that patients with high-risk conditions had a significantly worse prognosis than those with low-risk conditions. Furthermore, UPS-related signature is associated with a positive immune response. For predicting survival, a simple to use nomogram and the corresponding web-based calculator (https://jiangyanxiamm.shinyapps.io/MMprognosis/) were built based on the UPS signature and its clinical features. Analyses of calibration plots and decision curves showed clinical utility for both training and validation datasets.

  Conclusions: As a result of these results, we established a genetic signature for MM based on UPS. This genetic signature could contribute to improving individualized survival prediction, thereby facilitating clinical decisions in patients with MM.

• Research Paper pp undefined-undefined

  Cysteinyl leukotriene receptor 1 modulates retinal immune cells, vascularity and proteolytic activity in aged mice

  Relevance score: 6.1791496

  Andreas Koller, Julia Preishuber-Pflügl, Daniela Mayr, SusanneMaria Brunner, Anja-Maria Ladek, Christian Runge, Ludwig Aigner, Herbert Anton Reitsamer, Andrea Trost

  Keywords: Cysltr1, retina, proteasome activity, immune cell presence, vascular system

  Published in Aging on Invalid Date

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  Cysteinyl leukotrienes (CysLTs) modulate the immune response, the microvasculature, cell stress and the endosomal-lysosomal system, and are involved in cellular aging. Interestingly, CysLT receptor 1 (Cysltr1) is highly expressed in the retina, a tissue that is strongly affected by the aging process. Thus, we performed an introductory examination to determine a potential importance of Cysltr1 for cells in the neurovascular unit using qPCR and immunofluorescence analysis, and on proteolytic activity in the retinas of aged mice. Aged mice (~84 weeks) were treated orally with vehicle or 10 mg/kg montelukast (MTK), a specific Cysltr1 inhibitor, for 8 weeks, 5x/week. The retinas of young mice (~11 weeks) served as controls.

  Compared with young control mice, aged mice exhibited increased numbers of microglia and a reduced retinal capillary diameter, but these age-dependent changes were abrogated by MTK treatment. Retinal protein levels of the ubiquitin binding protein sequestosome-1 were amplified by aging, but were reduced by MTK treatment. Interestingly, retinal proteasome activity was decreased in aged mice, whereas Cysltr1 inhibition increased this activity.

  The reduction in immune cells caused by Cysltr1 suppression may dampen neuroinflammation, a known promoter of tissue aging. Additionally, an increase in capillary diameter after Cysltr1 inhibition could have a beneficial effect on blood flow in aged individuals. Furthermore, the increase in proteolytic activity upon Cysltr1 inhibition could prevent the accumulation of toxic deposits, which is a hallmark of aged tissue. Overall, Cysltr1 is a promising target for modulating the impact of aging on retinal tissue.