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

Cells ◽  
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.

scholarly journals Human Ccr4–Not complexes contain variable deadenylase subunits

2009 ◽  
Vol 422 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Nga-Chi Lau ◽  
Annemieke Kolkman ◽  
Frederik M. A. van Schaik ◽  
Klaas W. Mulder ◽  
W. W. M. Pim Pijnappel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mrna Synthesis ◽  
Rna Molecules ◽  
Cellular Processes ◽  
Proteomic Approach ◽  
Core Proteins ◽  
Evolutionarily Conserved ◽  
Associated Proteins ◽  
Stable Core ◽  
Human Complex

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.

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

Cancers ◽  
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.

scholarly journals PSPC1 is a new contextual determinant of aberrant subcellular translocation of oncogenes in tumor progression

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Yaw-Dong Lang ◽  
Yuh-Shan Jou
Keyword(s):  
Nuclear Export ◽  
Synergistic Effects ◽  
Therapeutic Option ◽  
Nucleocytoplasmic Shuttling ◽  
Innovative Strategy ◽  
Fda Approval ◽  
Cargo Proteins ◽  
The Common ◽  
Contextual Determinants ◽  
Nuclear Export Receptor

AbstractDysregulation of nucleocytoplasmic shuttling is commonly observed in cancers and emerging as a cancer hallmark for the development of anticancer therapeutic strategies. Despite its severe adverse effects, selinexor, a selective first-in-class inhibitor of the common nuclear export receptor XPO1, was developed to target nucleocytoplasmic protein shuttling and received accelerated FDA approval in 2019 in combination with dexamethasone as a fifth-line therapeutic option for adults with relapsed refractory multiple myeloma (RRMM). To explore innovative targets in nucleocytoplasmic shuttling, we propose that the aberrant contextual determinants of nucleocytoplasmic shuttling, such as PSPC1 (Paraspeckle component 1), TGIF1 (TGF-β Induced Factor Homeobox 1), NPM1 (Nucleophosmin), Mortalin and EBP50, that modulate shuttling (or cargo) proteins with opposite tumorigenic functions in different subcellular locations could be theranostic targets for developing anticancer strategies. For instance, PSPC1 was recently shown to be the contextual determinant of the TGF-β prometastatic switch and PTK6/β-catenin reciprocal oncogenic nucleocytoplasmic shuttling during hepatocellular carcinoma (HCC) progression. The innovative nucleocytoplasmic shuttling inhibitor PSPC1 C-terminal 131 polypeptide (PSPC1-CT131), which was developed to target both the shuttling determinant PSPC1 and the shuttling protein PTK6, maintained their tumor-suppressive characteristics and exhibited synergistic effects on tumor suppression in HCC cells and mouse models. In summary, targeting the contextual determinants of nucleocytoplasmic shuttling with cargo proteins having opposite tumorigenic functions in different subcellular locations could be an innovative strategy for developing new therapeutic biomarkers and agents to improve cancer therapy.

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

2004 ◽  
Vol 70 (5) ◽  
pp. 2748-2755 ◽  
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.

scholarly journals Nucleocytoplasmic Shuttling of Endocytic Proteins

2001 ◽  
Vol 153 (7) ◽  
pp. 1511-1518 ◽  
Author(s):  
Manuela Vecchi ◽  
Simona Polo ◽  
Viviane Poupon ◽  
Jan-Willem van de Loo ◽  
Alexandre Benmerah ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Export ◽  
Myeloid Leukemia ◽  
Nuclear Translocation ◽  
Initial Rate ◽  
Nucleocytoplasmic Shuttling ◽  
Cellular Processes ◽  
Transactivation Assay ◽  
Ordered Assembly ◽  
Endocytic Proteins

Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and α-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation.

scholarly journals Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

2011 ◽  
Vol 441 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Iraia García-Santisteban ◽  
Sonia Bañuelos ◽  
Jose A. Rodríguez
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
The Novel ◽  
Sequence Motifs ◽  
Leptomycin B ◽  
Specific Amino Acid ◽  
Green Fluorescent ◽  
Nuclear Export Receptor

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP–USP21 and, to a lesser extent, GFP–OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

scholarly journals AnNXF1mRNA with a retained intron is expressed in hippocampal and neocortical neurons and is translated into a protein that functions as an Nxf1 cofactor

2016 ◽  
Vol 27 (24) ◽  
pp. 3903-3912 ◽  
Author(s):  
Ying Li ◽  
Yeou-cherng Bor ◽  
Mark P. Fitzgerald ◽  
Kevin S. Lee ◽  
David Rekosh ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Neuroblastoma Cells ◽  
Nonsense Mediated Decay ◽  
Rna Granules ◽  
Neocortical Neurons ◽  
Mouse Neuroblastoma ◽  
Dendritic Mrna ◽  
Mrna Trafficking ◽  
Nuclear Export Receptor ◽  
Retained Introns

The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356–amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes.

p65 controls NF-κB activity by regulating cellular localization of IκBβ

2011 ◽  
Vol 434 (2) ◽  
pp. 253-263 ◽  
Author(s):  
Taras Valovka ◽  
Michael O. Hottiger
Keyword(s):  
Dna Binding ◽  
Nuclear Export ◽  
Cellular Localization ◽  
Nuclear Export Signal ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cellular Processes ◽  
Dna Binding Activity ◽  
Localization Signal ◽  
Export Signal

NF-κB (nuclear factor κB) controls diverse cellular processes and is frequently misregulated in chronic immune diseases or cancer. The activity of NF-κB is regulated by IκB (inhibitory κB) proteins which control nuclear–cytoplasmic shuttling and DNA binding of NF-κB. In the present paper, we describe a novel role for p65 as a critical regulator of the cellular localization and functions of NF-κB and its inhibitor IκBβ. In genetically modified p65−/− cells, the localization of ectopic p65 is not solely regulated by IκBα, but is largely dependent on the NLS (nuclear localization signal) and the NES (nuclear export signal) of p65. Furthermore, unlike IκBα, IκBβ does not contribute to the nuclear export of p65. In fact, the cellular localization and degradation of IκBβ is controlled by the p65-specific NLS and NES. The results of our present study also reveal that, in addition to stimulus-induced redistribution of NF-κB, changes in the constitutive localization of p65 and IκBβ specifically modulate activation of inflammatory genes. This is a consequence of differences in the DNA-binding activity and signal responsiveness between the nuclear and cytoplasmic NF-κB–IκBβ complexes. Taken together, the findings of the present study indicate that the p65 subunit controls transcriptional competence of NF-κB by regulating the NF-κB/IκBβ pathway.

scholarly journals Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2715
Author(s):  
Chih-Wei Chou ◽  
Xi Tan ◽  
Chia-Nung Hung ◽  
Brandon Lieberman ◽  
Meizhen Chen ◽  
...  
Keyword(s):  
Energy Homeostasis ◽  
Metabolic Stress ◽  
Primary Tumors ◽  
Coordinate Regulation ◽  
Atp Production ◽  
Metabolic Functions ◽  
Regulatory Mode ◽  
Close Proximity ◽  
Associated Proteins ◽  
Metabolic Regulators

The interplay between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) is central to maintain energy homeostasis. It remains to be determined whether there is a mechanism governing metabolic fluxes based on substrate availability in microenvironments. Here we show that menin is a key transcription factor regulating the expression of OXPHOS and glycolytic genes in cancer cells and primary tumors with poor prognosis. A group of menin-associated proteins (MAPs), including KMT2A, MED12, WAPL, and GATA3, is found to restrain menin’s full function in this transcription regulation. shRNA knockdowns of menin and MAPs result in reduced ATP production with proportional alterations of cellular energy generated through glycolysis and OXPHOS. When shRNA knockdown cells are exposed to metabolic stress, the dual functionality can clearly be distinguished among these metabolic regulators. A MAP can negatively counteract the regulatory mode of menin for OXPHOS while the same protein positively influences glycolysis. A close-proximity interaction between menin and MAPs allows transcriptional regulation for metabolic adjustment. This coordinate regulation by menin and MAPs is necessary for cells to rapidly adapt to fluctuating microenvironments and to maintain essential metabolic functions.

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