Publications by authors named "Sami Yokoo"

16 Publications

  • Page 1 of 1

Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients.

Biochim Biophys Acta Proteins Proteom 2021 Aug 9;1869(8):140659. Epub 2021 Apr 9.

Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, São Paulo, Brazil. Electronic address:

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.
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http://dx.doi.org/10.1016/j.bbapap.2021.140659DOI Listing
August 2021

ADAM17 cytoplasmic domain modulates Thioredoxin-1 conformation and activity.

Redox Biol 2020 10 24;37:101735. Epub 2020 Sep 24.

Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, São Paulo, Brazil. Electronic address:

The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cyto) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys at the C-terminal of ADAM17cyto with the Cys of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.
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http://dx.doi.org/10.1016/j.redox.2020.101735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513893PMC
October 2020

Combining discovery and targeted proteomics reveals a prognostic signature in oral cancer.

Nat Commun 2018 09 5;9(1):3598. Epub 2018 Sep 5.

Departamento de Diagnóstico Oral, Faculdade de Odontologia de Piracicaba Universidade Estadual de Campinas (UNICAMP), Piracicaba, 13414-903, SP, Brazil.

Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor-node-metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis.
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http://dx.doi.org/10.1038/s41467-018-05696-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125363PMC
September 2018

Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity.

Antioxid Redox Signal 2018 09 27;29(8):717-734. Epub 2018 Feb 27.

1 Laboratório Nacional de Biociências , LNBio, CNPEM, Campinas, Brazil .

Aims: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17.

Results: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1 and catalytic site mutant Trx-1 rescued ADAM17 activity, although the interaction with Trx-1 was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1 mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1 mutant showed similar oxidant levels to Trx-1, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity.

Innovation: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases.

Conclusion: This unexpected Trx-1 behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717-734.
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http://dx.doi.org/10.1089/ars.2017.7297DOI Listing
September 2018

GRP78 protects a disintegrin and metalloprotease 17 against protein-disulfide isomerase A6 catalyzed inactivation.

FEBS Lett 2017 11 11;591(21):3567-3587. Epub 2017 Oct 11.

Institute of Biochemistry, Christian-Albrechts University, Kiel, Germany.

The shedding of ectodomains is a crucial mechanism in many physiological and pathological events. A disintegrin and metalloprotease-17 (ADAM17) is a key sheddase involved in essential processes, such as development, regeneration, and immune defense. ADAM17 exists in two conformations which differ in their disulfide connection in the membrane-proximal domain (MPD). Protein-disulfide isomerases (PDIs) on the cell surface convert the open MPD into a rigid closed form, which corresponds to inactive ADAM17. ADAM17 is expressed in its open activatable form in the endoplasmic reticulum (ER) and consequently must be protected against ER-resident PDI activity. Here, we show that the chaperone 78-kDa glucose-regulated protein (GRP78) protects the MPD against PDI-dependent disulfide-bond isomerization by binding to this domain and, thereby, preventing ADAM17 inhibition.
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http://dx.doi.org/10.1002/1873-3468.12858DOI Listing
November 2017

Mass spectrometry-based proteomics revealed Glypican-1 as a novel ADAM17 substrate.

J Proteomics 2017 01 27;151:53-65. Epub 2016 Aug 27.

Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil. Electronic address:

ADAM17 (a disintegrin and metalloproteinase 17) is a plasma membrane metalloprotease involved in proteolytic release of the extracellular domain of many cell surface molecules, a process known as ectodomain shedding. Through this process, ADAM17 is implicated in several aspects of tumor growth and metastasis in a broad range of tumors, including head and neck squamous cell carcinomas (HNSCC). In this study, mass spectrometry-based proteomics approaches revealed glypican-1 (GPC1) as a new substrate for ADAM17, and its shedding was confirmed to be metalloprotease-dependent, induced by a pleiotropic agent (PMA) and physiologic ligand (EGF), and inhibited by marimastat. In addition, immunoblotting analysis of GPC1 in the extracellular media from control and ADAM17shRNA pointed to a direct involvement of ADAM17 in the cleavage of GPC1. Moreover, mass spectrometry-based interactome analysis of GPC1 revealed biological functions and pathways related mainly to cellular movement, adhesion and proliferation, which were events also modulated by up regulation of full length and cleavage GPC1. Altogether, we showed that GPC1 is a novel ADAM17 substrate, thus the function of GPC1 may be modulated by proteolysis signaling.

Biological Significance: Inhibition of metalloproteases as a therapeutic approach has failed because there is limited knowledge of the degradome of individual proteases as well as the cellular function of cleaved substrates. Using different proteomic techniques, this study uncovered novel substrates that can be modulated by ADAM17 in oral squamous cell carcinoma cell line. Glypican-1 was validated as a novel substrate for ADAM17, with important function in adhesion, proliferation and migration of carcinoma cells. Therefore, this study opens new avenues regarding the proteolysis-mediated function of GPC1 by ADAM17.
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http://dx.doi.org/10.1016/j.jprot.2016.08.017DOI Listing
January 2017

Different interactomes for p70-S6K1 and p54-S6K2 revealed by proteomic analysis.

Proteomics 2016 10 12;16(20):2650-2666. Epub 2016 Sep 12.

Laboratory of Metabolic Disorders, School of Applied Sciences, University of Campinas, Limeira, São Paulo, Brazil.

S6Ks are major effectors of the mTOR (mammalian target of rapamycin) pathway, signaling for increased protein synthesis and cell growth in response to insulin, AMP/ATP levels, and amino acids. Deregulation of this pathway has been related to disorders and diseases associated with metabolism, such as obesity, diabetes, and cancer. S6K family is composed of two main members, S6K1 and S6K2, which comprise different isoforms resulted from alternative splicing or alternative start codon use. Although important molecular functions have been associated with p70-S6K1, the most extensively studied isoform, the S6K2 counterpart lacks information. In the present study, we performed immunoprecipitation assays followed by mass spectrometry (MS) analysis of FLAG-tagged p70-S6K1 and p54-S6K2 interactomes, after expression in HEK293 cells. Protein lists were submitted to CRAPome (Contaminant Repository for Affinity Purification) and SAINT (Significance Analysis of INTeractome) analysis, which allowed the identification of high-scoring interactions. By a comparative approach, p70-S6K1 interacting proteins were predominantly related to "cytoskeleton" and "stress response," whereas p54-S6K2 interactome was more associated to "transcription," "splicing," and "ribosome biogenesis." Moreover, we have found evidences for new targets or regulators of the S6K protein family, such as proteins NCL, NPM1, eIF2α, XRCC6, PARP1, and ILF2/ILF3 complex. This study provides new information about the interacting networks of S6Ks, which may contribute for future approaches to a better understanding of the mTOR/S6K pathway.
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http://dx.doi.org/10.1002/pmic.201500249DOI Listing
October 2016

EEF1D modulates proliferation and epithelial-mesenchymal transition in oral squamous cell carcinoma.

Clin Sci (Lond) 2016 05 28;130(10):785-99. Epub 2016 Jan 28.

Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, SP, Brazil

EEF1D (eukaryotic translation elongation factor 1δ) is a subunit of the elongation factor 1 complex of proteins that mediates the elongation process during protein synthesis via enzymatic delivery of aminoacyl-tRNAs to the ribosome. Although the functions of EEF1D in the translation process are recognized, EEF1D expression was found to be unbalanced in tumours. In the present study, we demonstrate the overexpression of EEF1D in OSCC (oral squamous cell carcinoma), and revealed that EEF1D and protein interaction partners promote the activation of cyclin D1 and vimentin proteins. EEF1D knockdown in OSCC reduced cell proliferation and induced EMT (epithelial-mesenchymal transition) phenotypes, including cell invasion. Taken together, these results define EEF1D as a critical inducer of OSCC proliferation and EMT.
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http://dx.doi.org/10.1042/CS20150646DOI Listing
May 2016

Integrative analysis to select cancer candidate biomarkers to targeted validation.

Oncotarget 2015 Dec;6(41):43635-52

Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil.

Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS.
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http://dx.doi.org/10.18632/oncotarget.6018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791256PMC
December 2015

Agrin and perlecan mediate tumorigenic processes in oral squamous cell carcinoma.

PLoS One 2014 15;9(12):e115004. Epub 2014 Dec 15.

Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil.

Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115004PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266612PMC
May 2016

Integrated proteomics identified up-regulated focal adhesion-mediated proteins in human squamous cell carcinoma in an orthotopic murine model.

PLoS One 2014 23;9(5):e98208. Epub 2014 May 23.

Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil.

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098208PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032327PMC
June 2015

ADAM17 mediates OSCC development in an orthotopic murine model.

Mol Cancer 2014 Feb 5;13:24. Epub 2014 Feb 5.

Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas 13083-970, Brazil.

Background: ADAM17 is one of the main sheddases of the cells and it is responsible for the cleavage and the release of ectodomains of important signaling molecules, such as EGFR ligands. Despite the known crosstalk between ADAM17 and EGFR, which has been considered a promising targeted therapy in oral squamous cell carcinoma (OSCC), the role of ADAM17 in OSCC development is not clear.

Method: In this study the effect of overexpressing ADAM17 in cell migration, viability, adhesion and proliferation was comprehensively appraised in vitro. In addition, the tumor size, tumor proliferative activity, tumor collagenase activity and MS-based proteomics of tumor tissues have been evaluated by injecting tumorigenic squamous carcinoma cells (SCC-9) overexpressing ADAM17 in immunodeficient mice.

Results: The proteomic analysis has effectively identified a total of 2,194 proteins in control and tumor tissues. Among these, 110 proteins have been down-regulated and 90 have been up-regulated in tumor tissues. Biological network analysis has uncovered that overexpression of ADAM17 regulates Erk pathway in OSCC and further indicates proteins regulated by the overexpression of ADAM17 in the respective pathway. These results are also supported by the evidences of higher viability, migration, adhesion and proliferation in SCC-9 or A431 cells in vitro along with the increase of tumor size and proliferative activity and higher tissue collagenase activity as an outcome of ADAM17 overexpression.

Conclusion: These findings contribute to understand the role of ADAM17 in oral cancer development and as a potential therapeutic target in oral cancer. In addition, our study also provides the basis for the development of novel and refined OSCC-targeting approaches.
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http://dx.doi.org/10.1186/1476-4598-13-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928084PMC
February 2014

Identification of novel interaction between ADAM17 (a disintegrin and metalloprotease 17) and thioredoxin-1.

J Biol Chem 2012 Dec 26;287(51):43071-82. Epub 2012 Oct 26.

Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, Brasil.

ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.
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http://dx.doi.org/10.1074/jbc.M112.364513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522302PMC
December 2012

Novel processed form of syndecan-1 shed from SCC-9 cells plays a role in cell migration.

PLoS One 2012 15;7(8):e43521. Epub 2012 Aug 15.

Mass Spectrometry Laboratory, Brazilian Biosciences National Laboratory - CNPEM, Campinas, Brazil.

The extracellular milieu is comprised in part by products of cellular secretion and cell surface shedding. The presence of such molecules of the sheddome and secretome in the context of the extracellular milieu may have important clinical implications. In cancer they have been hypothesized to play a role in tumor growth and metastasis. The objective of this study was to evaluate whether the sheddome/secretome from two cell lines could be correlated with their potential for tumor development. Two epithelial cell lines, HaCaT and SCC-9, were chosen based on their differing abilities to form tumors in animal models of tumorigenesis. These cell lines when stimulated with phorbol-ester (PMA) showed different characteristics as assessed by cell migration, adhesion and higher gelatinase activity. Proteomic analysis of the media from these treated cells identified interesting, functionally relevant differences in their sheddome/secretome. Among the shed proteins, soluble syndecan-1 was found only in media from stimulated tumorigenic cells (SCC-9) and its fragments were observed in higher amount in the stimulated tumorigenic cells than stimulated non-tumorigenic cells (HaCaT). The increase in soluble syndecan-1 was associated with a decrease in membrane-bound syndecan-1 of SCC-9 cells after PMA stimuli. To support a functional role for soluble syndecan-1 fragments we demonstrated that the synthetic syndecan-1 peptide was able to induce cell migration in both cell lines. Taken together, these results suggested that PMA stimulation alters the sheddome/secretome of the tumorigenic cell line SCC-9 and one such component, the syndecan-1 peptide identified in this study, was revealed to promote migration in these epithelial cell lines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043521PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419706PMC
February 2013

Identification of FBXO25-interacting proteins using an integrated proteomics approach.

Proteomics 2010 Aug;10(15):2746-57

Department of Biochemistry and Immunology, University of São Paulo, Ribeirão Preto, Brazil.

FBXO25 is one of the 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of s-phase-kinase associated protein 1, really interesting new gene-box 1, Cullin 1, and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FAND). Combining two-step affinity purification followed by MS with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, beta-actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FAND, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions.
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http://dx.doi.org/10.1002/pmic.200900419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3034404PMC
August 2010

FBXO25-associated nuclear domains: a novel subnuclear structure.

Mol Biol Cell 2008 May 20;19(5):1848-61. Epub 2008 Feb 20.

Departments of Biochemistry and Immunology and Cellular and Molecular Biology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, São Paulo 14049-900, Brazil.

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.
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http://dx.doi.org/10.1091/mbc.e07-08-0815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2366848PMC
May 2008
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