Publications by authors named "Asier Fullaondo"

23 Publications

  • Page 1 of 1

The mitochondrial negative regulator MCJ modulates the interplay between microbiota and the host during ulcerative colitis.

Sci Rep 2020 01 17;10(1):572. Epub 2020 Jan 17.

CIC bioGUNE. Bizkaia Science and Technology Park. bld 801 A, 48160, Derio, Bizkaia, Spain.

Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.
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http://dx.doi.org/10.1038/s41598-019-57348-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969106PMC
January 2020

An E2F7-dependent transcriptional program modulates DNA damage repair and genomic stability.

Nucleic Acids Res 2019 Aug;47(14):7716-7717

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, 48080 Bilbao, Spain.

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http://dx.doi.org/10.1093/nar/gkz587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698732PMC
August 2019

Golgi Oncoprotein Gene Expression Is Regulated by Functional E2F and CREB/ATF Promoter Elements.

Genes (Basel) 2019 03 25;10(3). Epub 2019 Mar 25.

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, 48080 Bilbao, Spain.

The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of and , two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for , a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the promoter region abrogates E2F1-mediated induction of a luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.
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http://dx.doi.org/10.3390/genes10030247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471639PMC
March 2019

Does arterial hypertension influence the onset of Huntington's disease?

PLoS One 2018 23;13(5):e0197975. Epub 2018 May 23.

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain.

Huntington's disease (HD) age of onset (AO) is mainly determined by the length of the CAG repeat expansion in the huntingtin gene. The remaining AO variability has been attributed to other little-known factors. A factor that has been associated with other neurodegenerative diseases is arterial hypertension (AHT). The aim of this study is to evaluate the contribution of AHT to the AO of HD. We used data from a cohort of 630 European HD patients with adult onset collected by the REGISTRY project of the European Huntington's Disease Network. Multiple linear regression and ANOVA, controlling for the CAG repeat number of the expanded allele (CAGexp) of each patient, were performed to assess the association between the AHT condition and the AO of the motor symptoms (mAO). The results showed a significant association between AHT and mAO, especially when we only considered the patients diagnosed with AHT prior to manifesting any HD signs (pre-HD AHT). Remarkably, despite the low number of cases, those patients developed motor symptoms 5-8 years later than normotensive patients in the most frequent CAGexp range (40-44). AHT is an age-related condition and consequently, the age of the patient at the time of data collection could be a confounder variable. However, given that most pre-HD AHT patients included in our study had started treatment with antihypertensive drugs prior to the onset of HD, and that antihypertensive drugs have been suggested to confer a neuroprotective effect in other neurodegenerative diseases, raises the interest in elucidating the impact of AHT and/or AHT treatment in HD age of onset in further studies. A confirmation of our results in a larger sample set would open the possibility to significantly improve HD management.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197975PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965871PMC
November 2018

An E2F7-dependent transcriptional program modulates DNA damage repair and genomic stability.

Nucleic Acids Res 2018 05;46(9):4546-4559

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, 48080 Bilbao, Spain.

The cellular response to DNA damage is essential for maintaining the integrity of the genome. Recent evidence has identified E2F7 as a key player in DNA damage-dependent transcriptional regulation of cell-cycle genes. However, the contribution of E2F7 to cellular responses upon genotoxic damage is still poorly defined. Here we show that E2F7 represses the expression of genes involved in the maintenance of genomic stability, both throughout the cell cycle and upon induction of DNA lesions that interfere with replication fork progression. Knockdown of E2F7 leads to a reduction in 53BP1 and FANCD2 foci and to fewer chromosomal aberrations following treatment with agents that cause interstrand crosslink (ICL) lesions but not upon ionizing radiation. Accordingly, E2F7-depleted cells exhibit enhanced cell-cycle re-entry and clonogenic survival after exposure to ICL-inducing agents. We further report that expression and functional activity of E2F7 are p53-independent in this context. Using a cell-based assay, we show that E2F7 restricts homologous recombination through the transcriptional repression of RAD51. Finally, we present evidence that downregulation of E2F7 confers an increased resistance to chemotherapy in recombination-deficient cells. Taken together, our results reveal an E2F7-dependent transcriptional program that contributes to the regulation of DNA repair and genomic integrity.
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http://dx.doi.org/10.1093/nar/gky218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961008PMC
May 2018

Proteome-wide search for functional motifs altered in tumors: Prediction of nuclear export signals inactivated by cancer-related mutations.

Sci Rep 2016 05 12;6:25869. Epub 2016 May 12.

University of the Basque Country (UPV/EHU), Department of Genetics, Physical Anthropology and Animal Physiology, Leioa, 48940, Spain.

Large-scale sequencing projects are uncovering a growing number of missense mutations in human tumors. Understanding the phenotypic consequences of these alterations represents a formidable challenge. In silico prediction of functionally relevant amino acid motifs disrupted by cancer mutations could provide insight into the potential impact of a mutation, and guide functional tests. We have previously described Wregex, a tool for the identification of potential functional motifs, such as nuclear export signals (NESs), in proteins. Here, we present an improved version that allows motif prediction to be combined with data from large repositories, such as the Catalogue of Somatic Mutations in Cancer (COSMIC), and to be applied to a whole proteome scale. As an example, we have searched the human proteome for candidate NES motifs that could be altered by cancer-related mutations included in the COSMIC database. A subset of the candidate NESs identified was experimentally tested using an in vivo nuclear export assay. A significant proportion of the selected motifs exhibited nuclear export activity, which was abrogated by the COSMIC mutations. In addition, our search identified a cancer mutation that inactivates the NES of the human deubiquitinase USP21, and leads to the aberrant accumulation of this protein in the nucleus.
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http://dx.doi.org/10.1038/srep25869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4865848PMC
May 2016

LDLR and PCSK9 Are Associated with the Presence of Antiphospholipid Antibodies and the Development of Thrombosis in aPLA Carriers.

PLoS One 2016 28;11(1):e0146990. Epub 2016 Jan 28.

Department of Genetics, Physical Anthropology and Animal Physiology, School of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.

Introduction: The identification of the genetic risk factors that could discriminate non- thrombotic from thrombotic antiphospholipid antibodies (aPLA) carriers will improve prognosis of these patients. Several human studies have shown the presence of aPLAs associated with atherosclerotic plaque, which is a known risk factor for thrombosis. Hence, in order to determine the implication of atherosclerosis in the risk of developing thrombosis in aPLA positive patients, we performed a genetic association study with 3 candidate genes, APOH, LDLR and PCSK9.

Material & Methods: For genetic association study we analyzed 190 aPLA carriers -100 with non-thrombotic events and 90 with thrombotic events- and 557 healthy controls. Analyses were performed by χ2 test and were corrected by false discovery rate. To evaluate the functional implication of the newly established susceptibility loci, we performed expression analyses in 86 aPLA carrier individuals (43 with thrombotic manifestations and 43 without it) and in 45 healthy controls.

Results: Our results revealed significant associations after correction in SNPs located in LDLR gene with aPLA carriers and thrombotic aPLA carriers, when compared with healthy controls. The most significant association in LDLR gene was found between SNP rs129083082 and aPLA carriers in recessive model (adjusted P-value = 2.55 x 10-3; OR = 2.18; 95%CI = 1.49-3.21). Furthermore, our work detected significant allelic association after correction between thrombotic aPLA carriers and healthy controls in SNP rs562556 located in PCSK9 gene (adjusted P-value = 1.03 x 10-2; OR = 1.60; 95%CI = 1.24-2.06). Expression level study showed significantly decreased expression level of LDLR gene in aPLA carriers (P-value <0.0001; 95%CI 0.16-2.10; SE 0.38-1.27) in comparison to the control group.

Discussion: Our work has identified LDLR gene as a new susceptibility gene associated with the development of thrombosis in aPLA carriers, describing for the first time the deregulation of LDLR expression in individuals with aPLAs. Besides, thrombotic aPLA carriers also showed significant association with PCSK9 gene, a regulator of LDLR plasma levels. These results highlight the importance of atherosclerotic processes in the development of thrombosis in patients with aPLA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146990PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731066PMC
July 2016

Exploring Genetic Factors Involved in Huntington Disease Age of Onset: E2F2 as a New Potential Modifier Gene.

PLoS One 2015 6;10(7):e0131573. Epub 2015 Jul 6.

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain.

Age of onset (AO) of Huntington disease (HD) is mainly determined by the length of the CAG repeat expansion (CAGexp) in exon 1 of the HTT gene. Additional genetic variation has been suggested to contribute to AO, although the mechanism by which it could affect AO is presently unknown. The aim of this study is to explore the contribution of candidate genetic factors to HD AO in order to gain insight into the pathogenic mechanisms underlying this disorder. For that purpose, two AO definitions were used: the earliest age with unequivocal signs of HD (earliest AO or eAO), and the first motor symptoms age (motor AO or mAO). Multiple linear regression analyses were performed between genetic variation within 20 candidate genes and eAO or mAO, using DNA and clinical information of 253 HD patients from REGISTRY project. Gene expression analyses were carried out by RT-qPCR with an independent sample of 35 HD patients from Basque Country Hospitals. We found suggestive association signals between HD eAO and/or mAO and genetic variation within the E2F2, ATF7IP, GRIN2A, GRIN2B, LINC01559, HIP1 and GRIK2 genes. Among them, the most significant was the association between eAO and rs2742976, mapping to the promoter region of E2F2 transcription factor. Furthermore, rs2742976 T allele patient carriers exhibited significantly lower lymphocyte E2F2 gene expression, suggesting a possible implication of E2F2-dependent transcriptional activity in HD pathogenesis. Thus, E2F2 emerges as a new potential HD AO modifier factor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131573PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493078PMC
March 2016

Prediction of nuclear export signals using weighted regular expressions (Wregex).

Bioinformatics 2014 May 9;30(9):1220-7. Epub 2014 Jan 9.

Department of Communications Engineering, University of the Basque Country (UPV/EHU), Alda. Urquijo s/n Bilbao, 48013 and Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n Leioa, 48940, Spain.

Motivation: Leucine-rich nuclear export signals (NESs) are short amino acid motifs that mediate binding of cargo proteins to the nuclear export receptor CRM1, and thus contribute to regulate the localization and function of many cellular proteins. Computational prediction of NES motifs is of great interest, but remains a significant challenge.

Results: We have developed a novel approach for amino acid motif searching that can be used for NES prediction. This approach, termed Wregex (weighted regular expression), combines regular expressions with a position-specific scoring matrix (PSSM), and has been implemented in a web-based, freely available, software tool. By making use of a PSSM, Wregex provides a score to prioritize candidates for experimental testing. Key features of Wregex include its flexibility, which makes it useful for searching other types of protein motifs, and its fast execution time, which makes it suitable for large-scale analysis. In comparative tests with previously available prediction tools, Wregex is shown to offer a good rate of true-positive motifs, while keeping a smaller number of potential candidates.
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http://dx.doi.org/10.1093/bioinformatics/btu016DOI Listing
May 2014

E2F2 and CREB cooperatively regulate transcriptional activity of cell cycle genes.

Nucleic Acids Res 2013 Dec 12;41(22):10185-98. Epub 2013 Sep 12.

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, UPV/EHU, Bilbao 48940, Spain and Department of Biochemistry and Molecular Biology, University of the Basque Country, UPV/EHU, Bilbao 48940, Spain.

E2F2 is essential for the maintenance of T lymphocyte quiescence. To identify the full set of E2F2 target genes, and to gain further understanding of the role of E2F2 in transcriptional regulation, we have performed ChIP-chip analyses across the genome of lymph node-derived T lymphocytes. Here we show that during quiescence, E2F2 binds the promoters of a large number of genes involved in DNA metabolism and cell cycle regulation, concomitant with their transcriptional silencing. A comparison of ChIP-chip data with expression profiling data on resting E2f2(-)(/)(-) T lymphocytes identified a subset of 51 E2F2-specific target genes, most of which are upregulated on E2F2 loss. Luciferase reporter assays showed a retinoblastoma-independent role for E2F2 in the negative regulation of these target genes. Importantly, we show that the DNA binding activity of the transcription factor CREB contributes to E2F2-mediated repression of Mcm5 and Chk1 promoters. siRNA-mediated CREB knockdown, expression of a dominant negative KCREB mutant or disruption of CREB binding by mutating a CRE motif on Mcm5 promoter, relieved E2F2-mediated transcriptional repression. Taken together, our data uncover a new regulatory mechanism for E2F-mediated transcriptional control, whereby E2F2 and CREB cooperate in the transcriptional repression of a subset of E2F2 target genes.
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http://dx.doi.org/10.1093/nar/gkt821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905855PMC
December 2013

The nuclear protein ALY binds to and modulates the activity of transcription factor E2F2.

Mol Cell Proteomics 2013 May 7;12(5):1087-98. Epub 2013 Jan 7.

Department of Biochemistry and Molecular Biology, University of the Basque Country, UPV/EHU, 48940 Leioa, Spain.

E2F transcription factors control the expression of genes involved in a variety of essential cellular processes and consequently their activity needs to be tightly regulated. Protein-protein interactions are thought to be key modulators of E2F activity. To gain insight into the mechanisms that regulate the activity of E2F2, we searched for novel proteins that associate with this transcription factor. We show that the nuclear protein ALY (THO complex 4), originally described as a transcriptional co-activator, associates with DNA-bound E2F2 and represses its transcriptional activity. The capacity of ALY to modulate gene expression was analyzed with expression microarrays by characterizing the transcriptome of E2F2 expressing HEK293T cells in which ALY was either overexpressed or silenced. We show that ALY influences the expression of more than 400 genes, including 98 genes bearing consensus E2F motifs. Thus, ALY emerges as a novel E2F2-interacting protein and a relevant modulator of E2F-responsive gene expression.
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http://dx.doi.org/10.1074/mcp.M112.024158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650323PMC
May 2013

PAnalyzer: a software tool for protein inference in shotgun proteomics.

BMC Bioinformatics 2012 Nov 5;13:288. Epub 2012 Nov 5.

Department of Communications Engineering, University of the Basque Country (UPV/EHU), Alda, Urquijo s/n, Bilbao, 48013, Spain.

Background: Protein inference from peptide identifications in shotgun proteomics must deal with ambiguities that arise due to the presence of peptides shared between different proteins, which is common in higher eukaryotes. Recently data independent acquisition (DIA) approaches have emerged as an alternative to the traditional data dependent acquisition (DDA) in shotgun proteomics experiments. MSE is the term used to name one of the DIA approaches used in QTOF instruments. MSE data require specialized software to process acquired spectra and to perform peptide and protein identifications. However the software available at the moment does not group the identified proteins in a transparent way by taking into account peptide evidence categories. Furthermore the inspection, comparison and report of the obtained results require tedious manual intervention. Here we report a software tool to address these limitations for MSE data.

Results: In this paper we present PAnalyzer, a software tool focused on the protein inference process of shotgun proteomics. Our approach considers all the identified proteins and groups them when necessary indicating their confidence using different evidence categories. PAnalyzer can read protein identification files in the XML output format of the ProteinLynx Global Server (PLGS) software provided by Waters Corporation for their MSE data, and also in the mzIdentML format recently standardized by HUPO-PSI. Multiple files can also be read simultaneously and are considered as technical replicates. Results are saved to CSV, HTML and mzIdentML (in the case of a single mzIdentML input file) files. An MSE analysis of a real sample is presented to compare the results of PAnalyzer and ProteinLynx Global Server.

Conclusions: We present a software tool to deal with the ambiguities that arise in the protein inference process. Key contributions are support for MSE data analysis by ProteinLynx Global Server and technical replicates integration. PAnalyzer is an easy to use multiplatform and free software tool.
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http://dx.doi.org/10.1186/1471-2105-13-288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548767PMC
November 2012

SIR: Deterministic protein inference from peptides assigned to MS data.

J Proteomics 2012 Jul 14;75(13):4176-83. Epub 2012 May 14.

Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal.

Currently the bottom up approach is the most popular for characterizing protein samples by mass spectrometry. This is mainly attributed to the fact that the bottom up approach has been successfully optimized for high throughput studies. However, the bottom up approach is associated with a number of challenges such as loss of linkage information between peptides. Previous publications have addressed some of these problems which are commonly referred to as protein inference. Nevertheless, all previous publications on the subject are oversimplified and do not represent the full complexity of the proteins identified. To this end we present here SIR (spectra based isoform resolver) that uses a novel transparent and systematic approach for organizing and presenting identified proteins based on peptide spectra assignments. The algorithm groups peptides and proteins into five evidence groups and calculates sixteen parameters for each identified protein that are useful for cases where deterministic protein inference is the goal. The novel approach has been incorporated into SIR which is a user-friendly tool only concerned with protein inference based on imports of Mascot search results. SIR has in addition two visualization tools that facilitate further exploration of the protein inference problem.
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http://dx.doi.org/10.1016/j.jprot.2012.05.010DOI Listing
July 2012

Interleukin-2 signaling pathway analysis by quantitative phosphoproteomics.

J Proteomics 2011 Dec 23;75(1):177-91. Epub 2011 Jun 23.

Department of Biochemistry and Molecular Biology, University of the Basque Country, UPV/EHU, 48940 Leioa, Spain.

Interleukin-2 (IL-2) is major cytokine involved in T cell proliferation, differentiation and apoptosis. Association between IL-2 and its receptor (IL-2R), triggers activation of complex signaling cascade governed by tyrosine phosphorylation that culminates in transcription of genes involved in modulation of the immune response. The complete characterization of the IL-2 pathway is essential to understand how aberrant IL-2 signaling results in several diseases such as cancer or autoimmunity and also how IL-2 treatments affect cancer patients. To gain insights into the downstream machinery activated by IL-2, we aimed to define the global tyrosine-phosphoproteome of IL-2 pathway in human T cell line Kit225 using high resolution mass spectrometry combined with phosphotyrosine immunoprecipitation and SILAC. The molecular snapshot at 5min of IL-2 stimulation resulted in identification of 172 proteins among which 79 were found with increased abundance in the tyrosine-phosphorylated complexes, including several previously not reported IL-2 downstream effectors. Combinatorial site-specific phosphoproteomic analysis resulted in identification of 99 phosphorylated sites mapping to the identified proteins with increased abundance in the tyrosine-phosphorylated complexes, of which 34 were not previously described. In addition, chemical inhibition of the identified IL-2-mediated JAK, PI3K and MAPK signaling pathways, resulted in distinct alteration on the IL-2 dependent proliferation.
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http://dx.doi.org/10.1016/j.jprot.2011.06.007DOI Listing
December 2011

Differential proteomics analysis reveals a role for E2F2 in the regulation of the Ahr pathway in T lymphocytes.

Mol Cell Proteomics 2010 Oct 23;9(10):2184-94. Epub 2010 Jun 23.

Department of Biochemistry and Molecular Biology, University of the Basque Country, UPV/EHU, 48940 Leioa, Spain.

E2F transcription factors (E2F1-8) are best known for their role in cell proliferation, although it is clear that they regulate many other biological processes through the transcriptional modulation of distinct target genes. However, the specific set of genes regulated by each E2F remains to be characterized. To gain insight into the molecular pathways regulated by E2F2, we have analyzed the proteome of antigen receptor-activated T cells lacking E2F2. We report that loss of E2F2 results in a deregulated Aryl-hydrocarbon-receptor pathway. Proliferating E2F2(-/-) T lymphocytes expressed significantly higher levels of Aip, Ahr, and Arnt relative to wild-type (WT)(1) controls. The mechanism for increased levels of Aip appears straightforward, involving direct regulation of the Aip gene promoter by E2F2. Although the Ahr and Arnt promoters also bind E2F2, their regulation appears to be more complex. Nevertheless, exposure to the environmental xenobiotic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known exogenous ligand of the Ahr pathway, led to overexpression of the Ahr target gene Cyp1a1, and to increased sensitivity to TCDD-triggered apoptosis in E2F2(-/-) T cells compared with WT controls. These results suggest that E2F2 modulates cellular sensitivity to xenobiotic signals through the negative regulation of the Ahr pathway.
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http://dx.doi.org/10.1074/mcp.M110.001263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953915PMC
October 2010

Regulated increase in folding capacity prevents unfolded protein stress in the ER.

J Cell Sci 2010 Mar 9;123(Pt 5):787-94. Epub 2010 Feb 9.

Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.

Stimulation of thyrocytes with thyroid stimulating hormone (TSH) leads to a morphological change and a massive increase in thyroglobulin (Tg) production. Although Tg is a demanding client of the endoplasmic reticulum (ER), its increase did not result in significant accumulation of unfolded protein in the ER. Instead, ER chaperones and folding enzymes reached maximum synthesis rates immediately after TSH stimulation, before significant upregulation of Tg synthesis. The resulting increase in folding capacity before client protein production prevented cellular unfolded-protein stress, confirmed by the silence of the most conserved branch of the unfolded protein response. Thyrocytes set an example of physiological adaptation of cells to a future potentially stress-causing situation, which suggests a general strategy for both non-secretory and specialized secretory cells.
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http://dx.doi.org/10.1242/jcs.041111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823579PMC
March 2010

Microarray analysis of autoimmune diseases by machine learning procedures.

IEEE Trans Inf Technol Biomed 2009 May;13(3):341-50

Department of Computer Science and Artificial Intelligence, University of the Basque Country, 20080 San Sebastian, Spain.

Microarray-based global gene expression profiling, with the use of sophisticated statistical algorithms is providing new insights into the pathogenesis of autoimmune diseases. We have applied a novel statistical technique for gene selection based on machine learning approaches to analyze microarray expression data gathered from patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS), two autoimmune diseases of unknown genetic origin that share many common features. The methodology included a combination of three data discretization policies, a consensus gene selection method, and a multivariate correlation measurement. A set of 150 genes was found to discriminate SLE and PAPS patients from healthy individuals. Statistical validations demonstrate the relevance of this gene set from an univariate and multivariate perspective. Moreover, functional characterization of these genes identified an interferon-regulated gene signature, consistent with previous reports. It also revealed the existence of other regulatory pathways, including those regulated by PTEN, TNF, and BCL-2, which are altered in SLE and PAPS. Remarkably, a significant number of these genes carry E2F binding motifs in their promoters, projecting a role for E2F in the regulation of autoimmunity.
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http://dx.doi.org/10.1109/TITB.2008.2011984DOI Listing
May 2009

E2F2 represses cell cycle regulators to maintain quiescence.

Cell Cycle 2008 Dec 10;7(24):3915-27. Epub 2008 Dec 10.

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Bilbao, Spain.

E2F transcription factors control diverse biological processes through regulation of target gene expression. However, the mechanism by which this regulation is established, and the relative contribution of each E2F member are still poorly defined. We have investigated the role of E2F2 in regulating cellular proliferation. We show that E2F2 is required for the normal G(0)/G(1) phase because targeted disruption of the E2F2 gene causes T cells to enter S phase early and to undergo accelerated cell division. A large set of E2F target genes involved in DNA replication and cell cycle progression (such as Mcm's, cyclins and Cdc2a) that are silent in G(0) and typically transcribed late in G(1) phase are already actively expressed in quiescent T cells and MEFs lacking E2F2. The classic E2F activators, E2F1 and E2F3, are largely dispensable for this process because compound loss of E2F1(-/-) and E2F2(-/-) produces a comparably shortened G(0)/G(1) phase, with early S phase entry. Likewise, shRNA knockdown of E2F3 does not alter significantly the E2F2(-/-) phenotype. Chromatin immunoprecipitation analysis indicates that in wild-type cells the promoters of the aberrantly early-transcribed genes are occupied by E2F2 in G(0), suggesting a direct role for E2F2 in transcriptional repression. We conclude that E2F2 functions to transcriptionally repress cell cycle genes to establish the G(0) state.
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http://dx.doi.org/10.4161/cc.7.24.7379DOI Listing
December 2008

SDS-CGE of proteins in microchannels made of SU-8 films.

Electrophoresis 2006 Sep;27(18):3627-34

MEMS/MST Department, IKERLAN-IK4 S. Coop., Mondragón, Spain.

This work describes the SDS-CGE of proteins carried out in microchannels made of the negative photoresist EPON SU-8. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology allows the monolithic integration of the electrodes in the device. A high wafer fabrication yield and mass production compatibility guarantees low costs and high reliability. A poly(methyl methacrylate) (PMMA) packaging allows an easy setup and replacement of the device for electrophoresis experiments. In addition, the wire-bonding step is avoided. The electrophoretic mobilities of four proteins have been measured in microchannels filled with polyacrylamide. Different pore sizes have been tested obtaining their Ferguson plots. Finally, a separation of two proteins (20 and 36 kDa) has been carried out confirming that this novel device is suitable for protein separation. A resolution of 2.75 is obtained. This is the first time that this SU-8 microfluidic technology has been validated for SDS-CGE of proteins. This technology offers better separation performance than glass channels, at lower costs and with an easy packaging procedure.
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http://dx.doi.org/10.1002/elps.200600103DOI Listing
September 2006

Proteome analysis of yeast response to various nutrient limitations.

Mol Syst Biol 2006 16;2:2006.0026. Epub 2006 May 16.

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and 15N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose-repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids beta-oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post-transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology.
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http://dx.doi.org/10.1038/msb4100069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1681501PMC
August 2006

Differential proteome profiles in E2F2-deficient T lymphocytes.

Proteomics 2006 Apr;6 Suppl 1:S42-50

Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Spain.

E2F transcription factors are important regulators of proliferation, differentiation and apoptosis. We have previously shown that E2F2-/- mice develop late-onset autoimmune features, similar to systemic lupus erythematosus. E2F2-deficient T lymphocytes exhibit enhanced T cell receptor (TCR)-stimulated proliferation, which is presumably responsible for causing autoimmunity in E2F2-deficient mice. The comparison of E2F2-/- and wild-type T lymphocyte expression profiles by 2-DE followed by MS identification has revealed a set of deregulated proteins involved in TCR-mediated signaling, cell survival and stress responses. The deregulation of these proteins may account for the hyperproliferative phenotype that characterizes E2F2-/- T cells. Our work shows that proteomic analysis of gene-knockout strains can be a useful methodology to study the functional role of specific genes.
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http://dx.doi.org/10.1002/pmic.200500438DOI Listing
April 2006

Expression clustering reveals detailed co-expression patterns of functionally related proteins during B cell differentiation: a proteomic study using a combination of one-dimensional gel electrophoresis, LC-MS/MS, and stable isotope labeling by amino acids in cell culture (SILAC).

Mol Cell Proteomics 2005 Sep 15;4(9):1297-310. Epub 2005 Jun 15.

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands.

B cells play an essential role in the immune response. Upon activation they may differentiate into plasma cells that secrete specific antibodies against potentially pathogenic non-self antigens. To identify the cellular proteins that are important for efficient production of these antibodies we set out to study the B cell differentiation process at the proteome level. We performed an in-depth proteomic study to quantify dynamic relative protein expression patterns of several hundreds of proteins at five consecutive time points after lipopolysaccharide-induced activation of B lymphocytes. The proteome analysis was performed using a combination of stable isotope labeling using [13C6]leucine added to the murine B cell cultures, one-dimensional gel electrophoresis, and LC-MS/MS. In this study we identified 1,001 B cell proteins. We were able to quantify the expression levels of a quarter of all identified proteins (i.e. 234) at each of the five different time points. Nearly all proteins revealed changes in expression patterns. The quantitative dataset was further analyzed using an unbiased clustering method. Based on their expression profiles, we grouped the entire set of 234 quantified proteins into a limited number of 12 distinct clusters. Functionally related proteins showed a strong correlation in their temporal expression profiles. The quality of the quantitative data allowed us to even identify subclusters within functionally related classes of proteins such as in the endoplasmic reticulum proteins that are involved in antibody production.
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http://dx.doi.org/10.1074/mcp.M500123-MCP200DOI Listing
September 2005

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

J Clin Invest 2004 May;113(10):1398-407

Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Sciences, University of the Basque Country, Bilbao, Spain.

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.
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http://dx.doi.org/10.1172/JCI18879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC406522PMC
May 2004
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