Human Ccr4–Not complexes contain variable deadenylase subunits

The Ccr4–Not complex is evolutionarily conserved and important for regulation of mRNA synthesis and decay. The composition of the yeast complex has been well described. Orthologues of the yeast Ccr4–Not components have been identified in human cells including multiple subunits with mRNA deadenylase activity. In the present study, we examine the composition of the human Ccr4–Not complex in an in-depth proteomic approach using stable cell lines expressing tagged CNOT proteins. We find at least four different variants of the human complex, consisting of seven stable core proteins and mutually exclusive associated mRNA deadenylase subunits. Interestingly, human CNOT4 is in a separate ~200 kDa complex. Furthermore, analyses of associated proteins indicate involvement of Ccr4–Not complexes in splicing, transport and localization of RNA molecules. Taken together, human Ccr4–Not complexes are heterogeneous in composition owing to differences in their deadenylase subunits, which may reflect the multi-functionality of these complexes in cellular processes.

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Disclosing the Interactome of Leukemogenic NUP98-HOXA9 and SET-NUP214 Fusion Proteins Using a Proteomic Approach

Cells ◽

10.3390/cells9071666 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1666

Author(s):

Adélia Mendes ◽

Ramona Jühlen ◽

Sabrina Bousbata ◽

Birthe Fahrenkrog

Keyword(s):

Nuclear Export ◽

Fusion Proteins ◽

Cellular Transformation ◽

Tumor Suppressor P53 ◽

Cellular Processes ◽

Proteomic Approach ◽

Associated Proteins ◽

Nuclear Export Receptor ◽

Druggable Targets ◽

Metabolic Regulators

The interaction of oncogenes with cellular proteins is a major determinant of cellular transformation. The NUP98-HOXA9 and SET-NUP214 chimeras result from recurrent chromosomal translocations in acute leukemia. Functionally, the two fusion proteins inhibit nuclear export and interact with epigenetic regulators. The full interactome of NUP98-HOXA9 and SET-NUP214 is currently unknown. We used proximity-dependent biotin identification (BioID) to study the landscape of the NUP98-HOXA9 and SET-NUP214 environments. Our results suggest that both fusion proteins interact with major regulators of RNA processing, with translation-associated proteins, and that both chimeras perturb the transcriptional program of the tumor suppressor p53. Other cellular processes appear to be distinctively affected by the particular fusion protein. NUP98-HOXA9 likely perturbs Wnt, MAPK, and estrogen receptor (ER) signaling pathways, as well as the cytoskeleton, the latter likely due to its interaction with the nuclear export receptor CRM1. Conversely, mitochondrial proteins and metabolic regulators are significantly overrepresented in the SET-NUP214 proximal interactome. Our study provides new clues on the mechanistic actions of nucleoporin fusion proteins and might be of particular relevance in the search for new druggable targets for the treatment of nucleoporin-related leukemia.

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Role of microRNAs in the molecular diagnosis of cancer

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2010.1659 ◽

2010 ◽

Vol 1 (1) ◽

pp. 4 ◽

Cited By ~ 4

Author(s):

Simona Giglio ◽

Andrea Vecchione

Keyword(s):

Unknown Origin ◽

Rna Molecules ◽

Cellular Processes ◽

Non Coding Rna ◽

Evolutionarily Conserved ◽

Diagnosis Of Cancer ◽

Diagnosis And Classification ◽

Tumor Types ◽

Cycle Regulation

MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small non-coding RNA molecules of about 22 nucleotides in length that function as posttranscriptional gene regulators. They are involved in numerous cellular processes including development, cell differentiation, cell cycle regulation and apoptosis. There is increasing evidence to show that miRNAs are mutated or differentially expressed in many types of cancer and specific functions of the miRNAs are now becoming apparent. Here we discuss the current literature on potential usefulness of miRNAs as diagnostic markers, emphasizing the involvement of specific miRNAs in particular tumor types, highlighting their potential role in distinguishing benign from malignant tissues and/or the different subtypes of the same tumor and/or in diagnosis and classification of tumor of unknown origin.

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Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes

Cells ◽

10.3390/cells10071667 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1667

Author(s):

Laura Abaandou ◽

David Quan ◽

Joseph Shiloach

Keyword(s):

Cell Line ◽

Cell Lines ◽

Hek293 Cell ◽

Chinese Hamster ◽

Production Performance ◽

Cellular Processes ◽

Hek293 Cell Line ◽

Global Engineering ◽

Genomic Tools ◽

Serum Free Suspension

The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line’s most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.

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Tracking the Antibody Immunome in Sporadic Colorectal Cancer by Using Antigen Self-Assembled Protein Arrays

Cancers ◽

10.3390/cancers13112718 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2718

Author(s):

María González-González ◽

José María Sayagués ◽

Luis Muñoz-Bellvís ◽

Carlos Eduardo Pedreira ◽

Marcello L. R. de Campos ◽

...

Keyword(s):

Colorectal Cancer ◽

Clinical Utility ◽

Genetic Alterations ◽

Western World ◽

Sporadic Colorectal Cancer ◽

Protein Arrays ◽

Humoral Immune ◽

Cellular Processes ◽

Healthy Donors ◽

Associated Proteins

Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.

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Multiplexed Proteomic Approach for Identification of Serum Biomarkers in Hepatocellular Carcinoma Patients with Normal AFP

Journal of Clinical Medicine ◽

10.3390/jcm9020323 ◽

2020 ◽

Vol 9 (2) ◽

pp. 323

Author(s):

Young-Sun Lee ◽

Eunjung Ko ◽

Eileen L. Yoon ◽

Young Kul Jung ◽

Ji Hoon Kim ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cirrhosis ◽

Cell Lines ◽

Cellular Proliferation ◽

Hepatoma Cell ◽

Multiple Reaction Monitoring ◽

Serum Levels ◽

Human Hepatoma Cell ◽

Hepatoma Cell Lines ◽

Proteomic Approach

Alpha fetoprotein (AFP) has been used as a serologic indicator of hepatocellular carcinoma (HCC). We aimed to identify an HCC-specific serum biomarker for diagnosis using a multiplexed proteomic technique in HCC patients with normal AFP levels. A total of 152 patients were included from Guro Hospital, Korea University. Among 267 identified proteins, 28 and 86 proteins showed at least a two-fold elevation or reduction in expression, respectively. Multiple reaction monitoring (MRM) analysis of 41 proteins revealed 10 proteins were differentially expressed in patients with liver cirrhosis and HCC patients with normal AFP. A combination of tripartite motif22 (Trim22), seprase, and bone morphogenetic protein1 had an area under receiver operating characteristic of 0.957 for HCC diagnosis. Real-time PCR and western blot analysis of the paired tumor/non-tumor liver tissue in HCC revealed a reduced expression of Trim22 in the tumor tissue. Also, serum levels of Trim22 were significantly reduced in HCC patients with normal AFP compared to those with liver cirrhosis (p = 0.032). Inhibition of Trim22 increased cellular proliferation in human hepatoma cell lines, whereas overexpression of Trim22 decreased cellular proliferation in hepatoma cell lines. In conclusion, the combination of three serum markers improved the chance of diagnosing HCC. MRM-based quantification of the serum protein in patients with normal AFP provides the potential for early diagnosis of HCC.

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Initiation of Protein S mRNA synthesis in human liver and various cell lines

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2005.01138.x ◽

2005 ◽

Vol 3 (2) ◽

pp. 410-412 ◽

Cited By ~ 4

Author(s):

C. J. F. DE WOLF ◽

R. M. J. CUPERS ◽

R. M. BERTINA ◽

H. L. VOS

Keyword(s):

Cell Lines ◽

Human Liver ◽

Protein S ◽

Mrna Synthesis

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Effect of Adaptation to Ethanol on Cytoplasmic and Membrane Protein Profiles of Oenococcus oeni

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2748-2755.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2748-2755 ◽

Cited By ~ 71

Author(s):

M. Graça Silveira ◽

Maja Baumgärtner ◽

Frank M. Rombouts ◽

Tjakko Abee

Keyword(s):

Membrane Protein ◽

Redox Balance ◽

Oenococcus Oeni ◽

Starter Cultures ◽

Phosphotransferase System ◽

Protein Patterns ◽

Protein Levels ◽

Proteomic Approach ◽

Associated Proteins ◽

Adaptation Response

ABSTRACT The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated. Ethanol triggers alterations in protein patterns of O. oeni cells stressed with 12% ethanol for 1 h and those of cells grown in the presence of 8% ethanol. Levels of inosine-5′-monophosphate dehydrogenase and phosphogluconate dehydrogenase, which generate reduced nicotinamide nucleotides, were decreased during growth in the presence of ethanol, while glutathione reductase, which consumes NADPH, was induced, suggesting that maintenance of the redox balance plays an important role in ethanol adaptation. Phosphoenolpyruvate:mannose phosphotransferase system (PTS) components of mannose PTS, including the phosphocarrier protein HPr and EIIMan, were lacking in ethanol-adapted cells, providing strong evidence that mannose PTS is absent in ethanol-adapted cells, and this represents a metabolic advantage to O. oeni cells during malolactic fermentation. In cells grown in the presence of ethanol, a large increase in the number of membrane-associated proteins was observed. Interestingly, two of these proteins, dTDT-glucose-4,6-dehydratase and d-alanine:d-alanine ligase, are known to be involved in cell wall biosynthesis. Using a proteomic approach, we provide evidence for an active ethanol adaptation response of O. oeni at the cytoplasmic and membrane protein levels.

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Nuclear Import of TFIIB Is Mediated by Kap114p, a Karyopherin with Multiple Cargo-binding Domains

Molecular Biology of the Cell ◽

10.1091/mbc.e04-11-0990 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3200-3210 ◽

Cited By ~ 12

Author(s):

Jennifer L. Hodges ◽

Jennifer H. Leslie ◽

Nima Mosammaparast ◽

Yurong Guo ◽

Jeffrey Shabanowitz ◽

...

Keyword(s):

Nuclear Import ◽

Binding Sites ◽

Binding Domains ◽

Transcription Factor Iib ◽

General Transcription Factor ◽

Proteomic Approach ◽

Evolutionarily Conserved ◽

Import And Export ◽

Transport Factors

Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes.

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