DOCSLIB.ORG

Overexpression of SIRT3 Disrupts Mitochondrial Proteostasis and Cell Cycle Progression

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Overexpression of SIRT3 Disrupts Mitochondrial Proteostasis and Cell Cycle Progression Protein Cell 2016, 7(4):295–299 DOI 10.1007/s13238-016-0251-z Protein & Cell LETTER Overexpression of SIRT3 disrupts mitochondrial proteostasis and cell cycle progression Dear Editor, down-regulated in ccRCC tissues (Fig. 1B), suggesting that the low expression of SIRT3 is important for ccRCC As a mitochondrial deacetylase, SIRT3 deacetylates many progression. enzymes involved in central metabolism and maintains To understand the role of SIRT3 in tumorigenesis and mitochondrial proteostasis (Verdin et al., 2010; Papa and progression of ccRCC, stable cells overexpressing SIRT3 Cell Germain, 2014). Substrates of SIRT3 include components of were established in 293T cells. The overexpression of SIRT3 & the respiratory complexes, proteins involved in fatty acid in 293T cells (SIRT3-OE) was examined by Western blotting oxidation and TCA cycle (Yu et al., 2012). SIRT3 activates (Fig. S1), confirming that the expression level of SIRT3 in MnSOD to maintain reactive oxygen species (ROS) home- SIRT3-OE cells was four fold higher than that in control cells. ostasis and a loss of SIRT3 contributes to the age-associ- The SIRT3 overexpression in 293T led to a decrease in ated diseases (McDonnell et al., 2015; Qiu et al., 2010). proliferation rates (Fig. 1C). The ROS level in SIRT3-OE Protein SIRT3 plays dual roles functioning as a tumor suppressor or cells is two and half fold higher than that in the control cells a promoter in tumorigenesis and progression (Alhazzazi as detected using the CellROX® Deep Red kit (Fig. 1D). To et al., 2011). On one hand, SIRT3 regulates the cellular ROS determine the susceptibility of SIRT3-OE cells to oxidative level and maintains genomic stability, and mediates meta- stress, cells were treated with various concentrations of bolic reprogramming to prevent tumorigenesis (Finley and hydrogen peroxide for 12 h. The cell viability was measured Haigis, 2012). As a result, the low expression of SIRT3 has using CCK-8 assay. The effects of hydrogen peroxide were been found in breast cancer, glioblastoma, colon cancer, represented as the percentage of viable cells after 12 h osteosarcoma, prostate, and ovarian cancers (Kim et al., treatment (Fig. 1E). When cells were treated with 400 µmol/L 2010; Finley and Haigis, 2012). On the other hand, SIRT3 is H2O2 for 12 h, the percentages of viable cells were 20% and a prosurvival factor that modulates p53 activities and is 90% for the control and SIRT3-OE cells, respectively upregulated in oral cancer, the node-positive breast cancer, (Fig. 1E). This declares that SIRT3-OE cells are more and bladder cancer (Ashraf et al., 2006; Alhazzazi et al., resistant to H2O2 treatment. 2011). These results suggest that SIRT3 possesses the Next, proteomic analysis was carried out on SIRT3-OE tumor-type dependent function and its precise role needs to and control cells in biological replicates. Equal amounts of be elucidated in the context of a specific cancer. Clear cell proteins from SIRT3-OE and the control cells were in-solu- renal cell carcinoma (ccRCC) is the most common histo- tion digested and labeled with TMT reagents. The generated logical subtype of renal cancer (Cohen and McGovern, tryptic peptides were fractionated using off-line HPLC and 2005). The aims of the present study were to examine the each fraction was further analyzed by nano-LC-MS/MS. expression of SIRT3 in ccRCC and to characterize effects of Differentially expressed proteins were identified and quanti- SIRT3 on tumorigenesis and progression using 293T human fied using the TMT-based quantification. We identified 7536 embryonic kidney cells as the model system that has cancer proteins in two biological replicates and the false-positive stem cell-like features (Debeb et al., 2010). rate was estimated to be less than 1%. Based on the aver- Equal amounts of proteins extracted from 18 paired age reporter ion ratios (>1.5 or <0.67), 188 proteins were ccRCC lesions and associated pericarcinous tissue samples found to be differentially expressed between SIRT3-OE and were analyzed by Western blotting and the representative control cells, in which 93 proteins were down-regulated and Western blot images of eight paired samples were shown in 95 were up-regulated (Tables S1 and S2). To understand the Fig. 1A, indicating that the expression levels of SIRT3 were biological relevance of the differentially expressed proteins, lower in ccRCC than those in normal tissues. The gray scale the Gene Ontology (GO) was used to cluster differentially analysis of the Western blot data for all eighteen paired expressed proteins according to their associated biological samples showed that the SIRT3 expression was statistically processes. The annotations of gene lists are summarized via © The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn LETTER Xiaofei Wang et al. A B ** 1 2 3 4 1.5 N C N C N C N C SIRT3 1.0 β-Actin 5 6 7 8 0.5 N C N C N C N C (SIRT3/β-Actin) SIRT3 Relative SIRT3 protein 0.0 Pericarcinous tissue ccRCC tissue β-Actin C 0.7 D E Control ** Control 3 *** SIRT3-OE 0.6 *** 100 SIRT3-OE *** 0.5 80 2 0.4 60 0.3 40 Cell 0.2 1 & Absorbance in 450 nm 0.1 20 0.0 0 Percentage of viable cells (%) 0 0 12 24 36 48 60 72 84 96 production ROS of change Fold Control SIRT3-OE 200 μmol/L 400 μmol/L Culture time (h) Concentration of H2O2 Protein Figure 1. Downregulation of SIRT3 in ccRCC compared to associated pericarcinous tissues and characterization of SIRT3 overexpression cells. (A) Representative Western blot images of the expression levels of SIRT3 of eight paired samples, N (pericarcinous tissue), C (ccRCC tissue). (B) The gray scale analysis of SIRT3 presented in (A). (C) Growth curve of SIRT3-OE and the control cells. (D) Graphical representation of ROS levels of SIRT3-OE cells compared to the control cells. and (E) Survival rate of SIRT3-OE cells and the control cells treated with different concentration of H2O2 for 12 h. Data were analyzed using student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. *P < 0.05 is considered statistically significant. Error bars represent ±SEM. a pie plot based on the functional classification from Panther Figure 2. Proteomic, qPCR and Western blot analysis c as shown in Fig. 2A. One hundred and eighty eight differ- of differentially expressed proteins between SIRT3-OE entially expressed proteins participated in a variety of cellular and control cells. (A) GO analysis of the differentially processes including metabolic process, cellular process, and expressed proteins in SIRT3-OE cells compared to the cellular component organization process. The primary control cells with PANTHER (http://www.pantherdb.org). metabolic process shows the dominant difference between (B) Graphical representation of TMT ratios for proteins in SIRT3-OE and the control cells. About 25% of the down- SIRT3-OE cells compared to control cells. (C) qPCR regulated proteins are classified as mitochondrial proteins, analysis of mRNA expressions of selected mitochondrial indicating that SIRT3 overexpression has a great impact on ribosomal genes and other selected genes from SIRT3-OE the mitochondrial protein expressions. Five subunits of res- cells and control cells. (D) Western blot analysis of piratory complex IV were down-regulated in SIRT3-OE cells selected proteins from SIRT3-OE and control cells. (E) Cell including COX7C, COX6A1, COX7A2, COA7, and COA5 cycle analysis of SIRT3-OE cells and the control cells. (Fig. 2B), suggesting that the SIRT3 overexpression dis- (F) Western blotting images showing expression level of rupted the integrity of the respiratory complexes. We also SIRT3 in HSP60-KN cells compared to the control cells, identified that three proteins in the fatty acid β-oxidation WCL (whole cell lysates), Mito (mitochondria); and pathway were downregulated in SIRT3-OE cells including (G) mRNA expression level of SIRT3 in HSP60-KN cells ’ t enoyl-CoA hydratase, very long-chain specific acyl-CoA and control cells. Data were analyzed using student s - test. *P < 0.05, **P < 0.01 and ***P < 0.001. Error bars dehydrogenase and hydroxyacyl-coenzyme A dehydroge- represent ±SEM. nase. More importantly, proteomic analysis showed that nine 296 © The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn Deciphering functions of SIRT3 in ccRCC LETTER A Nitrogen compound Developmental process 9.9% Multicellular metabolic process 1.8% organismal Locomotion 0.5% Vitamin metabolic Response to stimulus 3.8% process 5.3% process 3.8% Phosphate-containing Biological Biological compound metabolic regulation 7.7% adhesion 0.5% Coenzyme metabolic process 5.3% process 8.8% Reproduction Metabolic 2.2% process 31.3% Apoptotic process 1.1% Localization 4.4% Cellular process 22.0% Generation of Immune system Primary metabolic precursor metabolites Cellular component organization process 31.3% process 75.4% Catabolic and energy 7.0% or biogenesis 9.9% process 1.8% B C Control 0.8 1.2 SIRT3-OE *** *** *** ** ***** **** *** *** *** ** *** 0.6 0.9 Cell & 0.4 0.6 Fold changes 0.2 0.3 (SIRT3-OE/Control) Relative mRNA level Relative mRNA 0.0 0.0 Protein CDK1CDK4SDHB COA7COA5HADH SDHBCOA7CDK1CDK4 MRPL1 MRPS6MRPS9 COX7C ECHS1 MRPL1 MRPS6MRPS9 MRPL17MRPL34MRPL45MRPL49 MRPS21MRPS22 COX6A1COX7A2 ACADVL MRPL17MRPL34MRPL45MRPL49 MRPS21MRPS22 D E *** Control 60 AMOT SIRT3-OE ** VIM 40 EEF1A2 CDK1 20 * CDK4 Percentage of cells (%) β-Actin 0 G1S G2-M F G WCL Mito 0.005 Control Ctrl HSP60-KNCtrl HSP60-KN HSP60-KN 0.004 SIRT3 0.003 β-Actin 0.002 mRNA level mRNA 0.001 COX4 0.000 SIRT3 © The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn 297 LETTER Xiaofei Wang et al.
Load more

Recommended publications
  • Supplementary Materials: Evaluation of Cytotoxicity and Α-Glucosidase Inhibitory Activity of Amide and Polyamino-Derivatives of Lupane Triterpenoids
    Supplementary Materials: Evaluation of cytotoxicity and α-glucosidase inhibitory activity of amide and polyamino-derivatives of lupane triterpenoids Oxana B. Kazakova1*, Gul'nara V. Giniyatullina1, Akhat G. Mustafin1, Denis A. Babkov2, Elena V. Sokolova2, Alexander A. Spasov2* 1Ufa Institute of Chemistry of the Ufa Federal Research Centre of the Russian Academy of Sciences, 71, pr. Oktyabrya, 450054 Ufa, Russian Federation 2Scientific Center for Innovative Drugs, Volgograd State Medical University, Novorossiyskaya st. 39, Volgograd 400087, Russian Federation Correspondence Prof. Dr. Oxana B. Kazakova Ufa Institute of Chemistry of the Ufa Federal Research Centre of the Russian Academy of Sciences 71 Prospeсt Oktyabrya Ufa, 450054 Russian Federation E-mail: [email protected] Prof. Dr. Alexander A. Spasov Scientific Center for Innovative Drugs of the Volgograd State Medical University 39 Novorossiyskaya st. Volgograd, 400087 Russian Federation E-mail: [email protected] Figure S1. 1H and 13C of compound 2. H NH N H O H O H 2 2 Figure S2. 1H and 13C of compound 4. NH2 O H O H CH3 O O H H3C O H 4 3 Figure S3. Anticancer screening data of compound 2 at single dose assay 4 Figure S4. Anticancer screening data of compound 7 at single dose assay 5 Figure S5. Anticancer screening data of compound 8 at single dose assay 6 Figure S6. Anticancer screening data of compound 9 at single dose assay 7 Figure S7. Anticancer screening data of compound 12 at single dose assay 8 Figure S8. Anticancer screening data of compound 13 at single dose assay 9 Figure S9. Anticancer screening data of compound 14 at single dose assay 10 Figure S10.
    [Show full text]
  • Analysis of Gene Expression Data for Gene Ontology
    ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Robert Daniel Macholan May 2011 ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION Robert Daniel Macholan Thesis Approved: Accepted: _______________________________ _______________________________ Advisor Department Chair Dr. Zhong-Hui Duan Dr. Chien-Chung Chan _______________________________ _______________________________ Committee Member Dean of the College Dr. Chien-Chung Chan Dr. Chand K. Midha _______________________________ _______________________________ Committee Member Dean of the Graduate School Dr. Yingcai Xiao Dr. George R. Newkome _______________________________ Date ii ABSTRACT A tremendous increase in genomic data has encouraged biologists to turn to bioinformatics in order to assist in its interpretation and processing. One of the present challenges that need to be overcome in order to understand this data more completely is the development of a reliable method to accurately predict the function of a protein from its genomic information. This study focuses on developing an effective algorithm for protein function prediction. The algorithm is based on proteins that have similar expression patterns. The similarity of the expression data is determined using a novel measure, the slope matrix. The slope matrix introduces a normalized method for the comparison of expression levels throughout a proteome. The algorithm is tested using real microarray gene expression data. Their functions are characterized using gene ontology annotations. The results of the case study indicate the protein function prediction algorithm developed is comparable to the prediction algorithms that are based on the annotations of homologous proteins.
    [Show full text]
  • 1 AGING Supplementary Table 2
    SUPPLEMENTARY TABLES Supplementary Table 1. Details of the eight domain chains of KIAA0101. Serial IDENTITY MAX IN COMP- INTERFACE ID POSITION RESOLUTION EXPERIMENT TYPE number START STOP SCORE IDENTITY LEX WITH CAVITY A 4D2G_D 52 - 69 52 69 100 100 2.65 Å PCNA X-RAY DIFFRACTION √ B 4D2G_E 52 - 69 52 69 100 100 2.65 Å PCNA X-RAY DIFFRACTION √ C 6EHT_D 52 - 71 52 71 100 100 3.2Å PCNA X-RAY DIFFRACTION √ D 6EHT_E 52 - 71 52 71 100 100 3.2Å PCNA X-RAY DIFFRACTION √ E 6GWS_D 41-72 41 72 100 100 3.2Å PCNA X-RAY DIFFRACTION √ F 6GWS_E 41-72 41 72 100 100 2.9Å PCNA X-RAY DIFFRACTION √ G 6GWS_F 41-72 41 72 100 100 2.9Å PCNA X-RAY DIFFRACTION √ H 6IIW_B 2-11 2 11 100 100 1.699Å UHRF1 X-RAY DIFFRACTION √ www.aging-us.com 1 AGING Supplementary Table 2. Significantly enriched gene ontology (GO) annotations (cellular components) of KIAA0101 in lung adenocarcinoma (LinkedOmics). Leading Description FDR Leading Edge Gene EdgeNum RAD51, SPC25, CCNB1, BIRC5, NCAPG, ZWINT, MAD2L1, SKA3, NUF2, BUB1B, CENPA, SKA1, AURKB, NEK2, CENPW, HJURP, NDC80, CDCA5, NCAPH, BUB1, ZWILCH, CENPK, KIF2C, AURKA, CENPN, TOP2A, CENPM, PLK1, ERCC6L, CDT1, CHEK1, SPAG5, CENPH, condensed 66 0 SPC24, NUP37, BLM, CENPE, BUB3, CDK2, FANCD2, CENPO, CENPF, BRCA1, DSN1, chromosome MKI67, NCAPG2, H2AFX, HMGB2, SUV39H1, CBX3, TUBG1, KNTC1, PPP1CC, SMC2, BANF1, NCAPD2, SKA2, NUP107, BRCA2, NUP85, ITGB3BP, SYCE2, TOPBP1, DMC1, SMC4, INCENP. RAD51, OIP5, CDK1, SPC25, CCNB1, BIRC5, NCAPG, ZWINT, MAD2L1, SKA3, NUF2, BUB1B, CENPA, SKA1, AURKB, NEK2, ESCO2, CENPW, HJURP, TTK, NDC80, CDCA5, BUB1, ZWILCH, CENPK, KIF2C, AURKA, DSCC1, CENPN, CDCA8, CENPM, PLK1, MCM6, ERCC6L, CDT1, HELLS, CHEK1, SPAG5, CENPH, PCNA, SPC24, CENPI, NUP37, FEN1, chromosomal 94 0 CENPL, BLM, KIF18A, CENPE, MCM4, BUB3, SUV39H2, MCM2, CDK2, PIF1, DNA2, region CENPO, CENPF, CHEK2, DSN1, H2AFX, MCM7, SUV39H1, MTBP, CBX3, RECQL4, KNTC1, PPP1CC, CENPP, CENPQ, PTGES3, NCAPD2, DYNLL1, SKA2, HAT1, NUP107, MCM5, MCM3, MSH2, BRCA2, NUP85, SSB, ITGB3BP, DMC1, INCENP, THOC3, XPO1, APEX1, XRCC5, KIF22, DCLRE1A, SEH1L, XRCC3, NSMCE2, RAD21.
    [Show full text]
  • Stem Cells® Original Article
    ® Stem Cells Original Article Properties of Pluripotent Human Embryonic Stem Cells BG01 and BG02 XIANMIN ZENG,a TAKUMI MIURA,b YONGQUAN LUO,b BHASKAR BHATTACHARYA,c BRIAN CONDIE,d JIA CHEN,a IRENE GINIS,b IAN LYONS,d JOSEF MEJIDO,c RAJ K. PURI,c MAHENDRA S. RAO,b WILLIAM J. FREEDa aCellular Neurobiology Research Branch, National Institute on Drug Abuse, Department of Health and Human Services (DHHS), Baltimore, Maryland, USA; bLaboratory of Neuroscience, National Institute of Aging, DHHS, Baltimore, Maryland, USA; cLaboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA; dBresaGen Inc., Athens, Georgia, USA Key Words. Embryonic stem cells · Differentiation · Microarray ABSTRACT Human ES (hES) cell lines have only recently been compared with pooled human RNA. Ninety-two of these generated, and differences between human and mouse genes were also highly expressed in four other hES lines ES cells have been identified. In this manuscript we (TE05, GE01, GE09, and pooled samples derived from describe the properties of two human ES cell lines, GE01, GE09, and GE07). Included in the list are genes BG01 and BG02. By immunocytochemistry and reverse involved in cell signaling and development, metabolism, transcription polymerase chain reaction, undifferenti- transcription regulation, and many hypothetical pro- ated cells expressed markers that are characteristic of teins. Two focused arrays designed to examine tran- ES cells, including SSEA-3, SSEA-4, TRA-1-60, TRA-1- scripts associated with stem cells and with the 81, and OCT-3/4. Both cell lines were readily main- transforming growth factor-β superfamily were tained in an undifferentiated state and could employed to examine differentially expressed genes.
    [Show full text]
  • MRPL1 Rabbit Pab
    Leader in Biomolecular Solutions for Life Science MRPL1 Rabbit pAb Catalog No.: A4947 Basic Information Background Catalog No. Mammalian mitochondrial ribosomal proteins are encoded by nuclear genes and help in A4947 protein synthesis within the mitochondrion. Mitochondrial ribosomes (mitoribosomes) consist of a small 28S subunit and a large 39S subunit. They have an estimated 75% Observed MW protein to rRNA composition compared to prokaryotic ribosomes, where this ratio is 37kDa reversed. Another difference between mammalian mitoribosomes and prokaryotic ribosomes is that the latter contain a 5S rRNA. Among different species, the proteins Calculated MW comprising the mitoribosome differ greatly in sequence, and sometimes in biochemical 36kDa properties, which prevents easy recognition by sequence homology. This gene encodes a 39S subunit protein that belongs to the L1 ribosomal protein family. Category Primary antibody Applications WB,IHC Cross-Reactivity Human, Rat Recommended Dilutions Immunogen Information WB 1:500 - 1:2000 Gene ID Swiss Prot 65008 Q9BYD6 IHC 1:20 - 1:200 Immunogen Recombinant fusion protein containing a sequence corresponding to amino acids 51-325 of human MRPL1 (NP_064621.3). Synonyms MRPL1;BM022;L1MT;MRP-L1 Contact Product Information www.abclonal.com Source Isotype Purification Rabbit IgG Affinity purification Storage Store at -20℃. Avoid freeze / thaw cycles. Buffer: PBS with 0.02% sodium azide,50% glycerol,pH7.3. Validation Data Western blot analysis of extracts of various cell lines, using MRPL1 antibody (A4947) at 1:1000 dilution. Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution. Lysates/proteins: 25ug per lane. Blocking buffer: 3% nonfat dry milk in TBST.
    [Show full text]
  • C6orf203 Controls OXPHOS Function Through Modulation of Mitochondrial Protein Biosynthesis
    bioRxiv preprint doi: https://doi.org/10.1101/704403; this version posted July 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. C6orf203 controls OXPHOS function through modulation of mitochondrial protein biosynthesis number of characters excluding Materials and Methods: 40,651 Sara Palacios-Zambrano1,2, Luis Vázquez-Fonseca1,2, Cristina González-Páramos1,2, Laura Mamblona1,2, Laura Sánchez-Caballero3, Leo Nijtmans3, Rafael Garesse1,2 and Miguel Angel Fernández-Moreno1,2,* 1 Departamento de Bioquímica, Instituto de Investigaciones Biomédicas “Alberto Sols” UAM CSIC and Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER). Facultad de Medicina, Universidad Autónoma de Madrid. Madrid 28029, Spain. 2 Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), Madrid 28041, Spain. 3 Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. * To whom correspondence should be addressed. Tel:+34 91 497 31 29; Email: [email protected] Running title “C6orf203 controls mt-proteins synthesis” bioRxiv preprint doi: https://doi.org/10.1101/704403; this version posted July 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT Mitochondria are essential organelles present in the vast majority of eukaryotic cells. Their central function is to produce cellular energy through the OXPHOS system, and functional alterations provoke so-called mitochondrial OXPHOS diseases. It is estimated that several hundred mitochondrial proteins have unknown functions. Very recently, C6orf203 was described to participate in mitochondrial transcription under induced mitochondrial DNA depletion stress conditions.
    [Show full text]
  • The Neuroprotective Role of the GM1 Oligosaccharide, Ii3neu5ac-Gg4, In
    Molecular Neurobiology (2019) 56:6673–6702 https://doi.org/10.1007/s12035-019-1556-8 The Neuroprotective Role of the GM1 Oligosaccharide, 3 II Neu5Ac-Gg4, in Neuroblastoma Cells Elena Chiricozzi1 & Margherita Maggioni1 & Erika di Biase1 & Giulia Lunghi1 & Maria Fazzari1 & Nicoletta Loberto 1 & Maffioli Elisa2 & Francesca Grassi Scalvini2 & Gabriella Tedeschi 2,3 & Sandro Sonnino1 Received: 10 January 2019 /Accepted: 13 March 2019 /Published online: 26 March 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract 3 Recently, we demonstrated that the GM1 oligosaccharide, II Neu5Ac-Gg4 (OligoGM1), administered to cultured murine Neuro2a neuroblastoma cells interacts with the NGF receptor TrkA, leading to the activation of the ERK1/2 downstream pathway and to cell differentiation. To understand how the activation of the TrkA pathway is able to trigger key biochemical signaling, we performed a proteomic analysis on Neuro2a cells treated with 50 μM OligoGM1 for 24 h. Over 3000 proteins were identified. Among these, 324 proteins were exclusively expressed in OligoGM1-treated cells. Interestingly, several proteins expressed only in OligoGM1-treated cells are involved in biochemical mechanisms with a neuroprotective potential, reflecting the GM1 neuroprotective effect. In addition, we found that the exogenous administration of OligoGM1 reduced the cellular oxidative stress in Neuro2a cells and conferred protection against MPTP neurotoxicity. These results confirm and reinforce the idea that the molecular mechanisms underlying the GM1 neurotrophic and neuroprotective effects depend on its oligosaccharide chain, suggesting the activation of a positive signaling starting at plasma membrane level. Keywords GM1 ganglioside . GM1 oligosaccharide chain . TrkA neurotrophin receptor . Plasma membrane signaling . Neuroprotection .
    [Show full text]
  • Fhl1p Protein, a Positive Transcription Factor in Pichia Pastoris, Enhances
    Zheng et al. Microb Cell Fact (2019) 18:207 https://doi.org/10.1186/s12934-019-1256-0 Microbial Cell Factories RESEARCH Open Access Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins Xueyun Zheng1,2, Yimin Zhang1,2, Xinying Zhang1,2, Cheng Li1,2, Xiaoxiao Liu1,2, Ying Lin1,2* and Shuli Liang1,2* Abstract Background: The methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scafolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins. Results: The function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verifed in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confrming Fhl1p involved in the regulation of rRNA pro- cessing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities. Conclusion: This study illustrated that the transcription factor Fhl1p could efectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.
    [Show full text]
  • Transcriptomic and Proteomic Landscape of Mitochondrial
    TOOLS AND RESOURCES Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals Inge Ku¨ hl1,2†*, Maria Miranda1†, Ilian Atanassov3, Irina Kuznetsova4,5, Yvonne Hinze3, Arnaud Mourier6, Aleksandra Filipovska4,5, Nils-Go¨ ran Larsson1,7* 1Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany; 2Department of Cell Biology, Institute of Integrative Biology of the Cell (I2BC) UMR9198, CEA, CNRS, Univ. Paris-Sud, Universite´ Paris-Saclay, Gif- sur-Yvette, France; 3Proteomics Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany; 4Harry Perkins Institute of Medical Research, The University of Western Australia, Nedlands, Australia; 5School of Molecular Sciences, The University of Western Australia, Crawley, Australia; 6The Centre National de la Recherche Scientifique, Institut de Biochimie et Ge´ne´tique Cellulaires, Universite´ de Bordeaux, Bordeaux, France; 7Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Abstract Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative *For correspondence: analyses of mitochondrial proteomes, cellular transcriptomes and targeted metabolomics of five [email protected] knockout mouse strains deficient in essential factors required for mitochondrial DNA gene (IKu¨ ); expression, leading to OXPHOS dysfunction. Moreover,
    [Show full text]
  • Supplementary Figure 1 Standardization of Gene Expression
    Supplementary Figure 1 Standardization of gene expression Notes: (A) Standardization of GSE86544, (B) standardization of GSE103479, (C) standardization of GSE102238, (D) Standardization of GSE7055. The blue bar represents the data before normalization, and the red bar represents the data after normalization. Supplementary Figure 2 Correlation between module eigengenes and clinical traits especially PNI in GSE103479 and GSE102238 datasets. Notes: (A, B) Module-trait relationships in GSE103479 and GSE102238 datasets. The correlation coefficients and corresponding P-values in the brackets are contained in each cell. The table is color- coded by correlation between eigengenes and traits according to the color legend on the right side. The modules with the most significant differences are displayed in brackets. Abbreviations: PNI, perineural invasion. Supplementary Figure 3 The expression values of CCNB2 in pancreatic cancer (GSE102238) and colon cancer (GSE103479). Notes: (A, B) CCNB2 expression values were detected in GSE102238 and GSE103479. Abbreviations: CCNB2, cyclin B2 Supplementary Table 1 Results of top 20 pathway enrichment analysis of GSE7055 Term Category Description Count Log10(P) Genes GO:0000280 GO Biological nuclear division 33 -23.4 BIRC5,BUB1B,CCNB1,CCNE1,CDC20, Processes CKS2,KIF11,MAD2L1,MYBL2,SPAST, TOP2A,TTK,PRC1,PKMYT1,PTTG1,T RIP13,DLGAP5,TACC3,SMC2,SPAG5, UBE2C,ZWINT,TPX2,FBXO5,RACGA P1,NUSAP1,SPDL1,CDCA8,CEP55,ND C1,NSFL1C,KIF18B,ASPM GO:1902850 GO Biological microtubule 15 -12.89 BIRC5,CCNB1,CDC20,KIF11,MAD2L1 Processes
    [Show full text]
  • Supplementary Table S1. Relative Change in Proteins Associated with Heme Biosynthesis and Degradation
    Supplementary Table S1. Relative change in proteins associated with heme biosynthesis and degradation. hPXR mPxr–/– Protein Gene RIF/INH INH RIF RIF/INH p Value 5-aminolevulinate synthase Alas1 1.90 2.61 1.05 1.41 0.28 5-aminolevulinate synthase Alas2 0.86 1.38 0.73 1.18 0.018 Delta-aminolevulinic acid Alad 0.96 1.00 1.02 0.95 0.75 dehydratase Porphobilinogen deaminase Hmbs 1.04 0.99 1.10 1.05 0.67 Uroporphyrinogen-III synthase Uros 1.19 1.09 1.31 1.38 0.012 Uroporphyrinogen decarboxylase Urod 0.92 1.03 0.94 0.92 0.33 Oxygen-dependent Cpox 1.13 1.04 1.18 1.15 0.20 coproporphyrinogen-III oxidase, Protoporphyrinogen oxidase Ppox 0.69 0.81 0.85 0.83 0.013 Ferrochelatase, Fech 0.39 0.50 0.88 0.43 0.000002 Heme oxygenase 1 Hmox1 1.15 0.86 0.91 1.11 0.34 Heme oxygenase 2 Hmox2 0.96 0.98 0.89 0.88 0.22 Biliverdin reductase A Blvra 0.84 0.92 0.82 0.92 0.032 UDP-glucuronosyltransferase 1-6 Ugt1a6 1.22 0.96 1.10 1.13 0.30 NADPH--cytochrome P450 Por 1.28 0.92 1.18 1.12 0.019 reductase INH, isoniazid; RIF, rifampicin; RIF/INH, rifampicin and isoniazid. Supplementary Table S2. Relative change in protein nuclear receptors. hPXR mPxr–/– Protein Gene RIF/INH INH RIF RIF/INH p Value Aryl hydrocarbon receptor Ahr 1.09 0.91 1.00 1.26 0.092 Hepatocyte nuclear factor Hnf1a 0.87 0.97 0.82 0.79 0.027 1-alpha Hepatocyte nuclear factor Hnf4a 0.95 1.05 0.97 1.08 0.20 4-alpha Oxysterols receptor LXR- Nr1h3 0.94 1.16 1.03 1.02 0.42 alpha Bile acid receptor Nr1h4 1.05 1.17 0.98 1.19 0.12 Retinoic acid receptor Rxra 0.88 1.03 0.83 0.95 0.12 RXR-alpha Peroxisome proliferator-
    [Show full text]
  • Cardiomyopathy Is Associated with Ribosomal Protein Gene Haploinsufficiency In
    Genetics: Published Articles Ahead of Print, published on September 2, 2011 as 10.1534/genetics.111.131482 Cardiomyopathy is associated with ribosomal protein gene haploinsufficiency in Drosophila melanogaster Michelle E. Casad*, Dennis Abraham§, Il-Man Kim§, Stephan Frangakis*, Brian Dong*, Na Lin§1, Matthew J. Wolf§, and Howard A. Rockman*§** Department of *Cell Biology, §Medicine, and **Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710 1Current Address: Institute of Molecular Medicine, Peking University, Beijing, China, 100871 1 Copyright 2011. Running Title: Cardiomyopathy in Minute Drosophila Key Words: Drosophila Minute syndrome Cardiomyopathy Address for correspondence: Howard A. Rockman, M.D. Department of Medicine Duke University Medical Center, DUMC 3104 226 CARL Building, Research Drive Durham, NC, 27710 Tel: (919) 668-2520 FAX: (919) 668-2524 Email: [email protected] 2 ABSTRACT The Minute syndrome in Drosophila melanogaster is characterized by delayed development, poor fertility, and short slender bristles. Many Minute loci correspond to disruptions of genes for cytoplasmic ribosomal proteins, and therefore the phenotype has been attributed to alterations in translational processes. Although protein translation is crucial for all cells in an organism, it is unclear why Minute mutations cause effects in specific tissues. To determine whether the heart is sensitive to haploinsufficiency of genes encoding ribosomal proteins, we measured heart function of Minute mutants using optical coherence tomography. We found that cardiomyopathy is associated with the Minute syndrome caused by haploinsufficiency of genes encoding cytoplasmic ribosomal proteins. While mutations of genes encoding non-Minute cytoplasmic ribosomal proteins are homozygous lethal, heterozygous deficiencies spanning these non-Minute genes did not cause a change in cardiac function.
    [Show full text]