Nucleophosmin Protein Research Articles

The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo.

Nucleophosmin (NPM), a nucleolar multifunctional phosphoprotein, acts as a stress sensor in different cell types. NPM can be actively secreted by inflammatory cells, however its biology on endothelium remains unexplored. In this study, we show for the first time that NPM is secreted by human vein endothelial cells (HUVEC) in the early response to serum deprivation and that NPM acts as a pro-inflammatory and angiogenic molecule both in vitro and in vivo. Accordingly, 24 h of serum starvation condition induced NPM relocalization from the nucleus to cytoplasm. Interestingly, NPM was increasingly excreted in HUVEC-derived conditioned media in a time dependent fashion upon stress conditions up to 24 h. The secretion of NPM was unrelated to cell necrosis within 24 h. The treatment with exogenous and recombinant NPM (rNPM) enhanced migration as well as the Intercellular Adhesion Molecule 1 (ICAM-1) but not Vascular cell adhesion protein 1 (VCAM-1) expression and it did not affect cell proliferation. Notably, in vitro tube formation by Matrigel assay was significantly increased in HUVEC treated with rNPM compared to controls. This result was confirmed by the in vivo injection of Matrigel plug assay upon stimulation with rNPM, displaying significant enhanced number of functional capillaries in the plugs. The stimulation with rNPM in HUVEC was also associated to the increased expression of master genes regulating angiogenesis and migration, including Vascular Endothelial Growth Factor-A (VEGF-A), Hepatocyte Growth Factor (HGF), Stromal derived factor-1 (SDF-1), Fibroblast growth factor-2 (FGF-2), Platelet Derived Growth Factor-B (PDGF-B), and Matrix metallopeptidase 9 (MMP9). Our study demonstrates for the first time that NPM is physiologically secreted by somatic cells under stress condition and in the absence of cell necrosis. The analysis of the biological effects induced by NPM mainly related to a pro-angiogenic and inflammatory activity might suggest an important autocrine/paracrine role for NPM in the regulation of both phenomena.

Nucleophosmin Protein Dephosphorylation by DUSP3 Is a Fine-Tuning Regulator of p53 Signaling to Maintain Genomic Stability.

The dual-specificity phosphatase 3 (DUSP3), an atypical protein tyrosine phosphatase (PTP), regulates cell cycle checkpoints and DNA repair pathways under conditions of genotoxic stress. DUSP3 interacts with the nucleophosmin protein (NPM) in the cell nucleus after UV-radiation, implying a potential role for this interaction in mechanisms of genomic stability. Here, we show a high-affinity binding between DUSP3-NPM and NPM tyrosine phosphorylation after UV stress, which is increased in DUSP3 knockdown cells. Specific antibodies designed to the four phosphorylated NPM’s tyrosines revealed that DUSP3 dephosphorylates Y29, Y67, and Y271 after UV-radiation. DUSP3 knockdown causes early nucleolus exit of NPM and ARF proteins allowing them to disrupt the HDM2-p53 interaction in the nucleoplasm after UV-stress. The anticipated p53 release from proteasome degradation increased p53-Ser15 phosphorylation, prolonged p53 half-life, and enhanced p53 transcriptional activity. The regular dephosphorylation of NPM’s tyrosines by DUSP3 balances the p53 functioning and favors the repair of UV-promoted DNA lesions needed for the maintenance of genomic stability.

Early nucleolar responses differentiate mechanisms of cell death induced by oxaliplatin and cisplatin

Recent reports provide evidence that the platinum chemotherapeutic oxaliplatin causes cell death via ribosome biogenesis stress, while cisplatin causes cell death via the DNA damage response (DDR). Underlying differences in mechanisms that might initiate disparate routes to cell death by these two broadly used platinum compounds have not yet been carefully explored. Additionally, prior studies had demonstrated that cisplatin can also inhibit ribosome biogenesis. Therefore, we sought to directly compare the initial influences of oxaliplatin and cisplatin on nucleolar processes and on the DDR. Using pulse-chase experiments, we found that at equivalent doses, oxaliplatin but not cisplatin significantly inhibited ribosomal RNA (rRNA) synthesis by Pol I, but neither compound affected rRNA processing. Inhibition of rRNA synthesis occurred as early as 90 min after oxaliplatin treatment in A549 cells, concurrent with the initial redistribution of the nucleolar protein nucleophosmin (NPM1). We observed that the nucleolar protein fibrillarin began to redistribute by 6 h after oxaliplatin treatment and formed canonical nucleolar caps by 24 h. In cisplatin-treated cells, DNA damage, as measured by γH2AX immunofluorescence, was more extensive, whereas nucleolar organization was unaffected. Taken together, our results demonstrate that oxaliplatin causes early nucleolar disruption via inhibition of rRNA synthesis accompanied by NPM1 relocalization and subsequently causes extensive nucleolar reorganization, while cisplatin causes early DNA damage without significant nucleolar disruption. These data support a model in which, at clinically relevant doses, cisplatin kills cells via the canonical DDR, and oxaliplatin kills cells via ribosome biogenesis stress, specifically via rapid inhibition of rRNA synthesis.

Ulcerative Colitis-Derived Colonoid Culture: A Multi-Mineral-Approach to Improve Barrier Protein Expression.

BackgroundRecent studies demonstrated that Aquamin®, a calcium-, magnesium-rich, multi-mineral natural product, improves barrier structure and function in colonoids obtained from the tissue of healthy subjects. The goal of the present study was to determine if the colonic barrier could be improved in tissue from subjects with ulcerative colitis (UC).MethodsColonoid cultures were established with colon biopsies from 9 individuals with UC. The colonoids were then incubated for a 2-week period under control conditions (in culture medium with a final calcium concentration of 0.25 mM) or in the same medium supplemented with Aquamin® to provide 1.5 – 4.5 mM calcium. Effects on differentiation and barrier protein expression were determined using several approaches: phase-contrast and scanning electron microscopy, quantitative histology and immunohistology, mass spectrometry-based proteome assessment and transmission electron microscopy.ResultsAlthough there were no gross changes in colonoid appearance, there was an increase in lumen diameter and wall thickness on histology and greater expression of cytokeratin 20 (CK20) along with reduced expression of Ki67 by quantitative immunohistology observed with intervention. In parallel, upregulation of several differentiation-related proteins was seen in a proteomic screen with the intervention. Aquamin®-treated colonoids demonstrated a modest up-regulation of tight junctional proteins but stronger induction of adherens junction and desmosomal proteins. Increased desmosomes were seen at the ultrastructural level. Proteomic analysis demonstrated increased expression of several basement membrane proteins and hemidesmosomal components. Proteins expressed at the apical surface (mucins and trefoils) were also increased as were several additional proteins with anti-microbial activity or that modulate inflammation. Finally, several transporter proteins that affect electrolyte balance (and, thereby affect water resorption) were increased. At the same time, growth and cell cycle regulatory proteins (Ki67, nucleophosmin, and stathmin) were significantly down-regulated. Laminin interactions, matrix formation and extracellular matrix organization were the top three up-regulated pathways with the intervention.ConclusionA majority of individuals including patients with UC do not reach the recommended daily intake for calcium and other minerals. To the extent that such deficiencies might contribute to the weakening of the colonic barrier, the findings employing UC tissue-derived colonoids here suggest that adequate mineral intake might improve the colonic barrier.

Flow cytometry in detection of Nucleophosmin 1 mutation in acute myeloid leukemia patients: A reproducible tertiary hospital experience.

Nucleophosmin 1 (NPM1) mutation is one of the most frequent gene mutations in adult acute myeloid leukemia (AML), being detected in 35% of all cases and in up to 60% of patients with normal karyotype AML. AML with mutated NPM1 has distinct pathology, immunophenotyping, and confirmed favorable prognostic significance. Hence, AML with mutated NPM1 is a separate entity in the revised 2016 World Health Organization classification. This study aimed to evaluate the use of a reproducible flow cytometry approach in the assay of mutant NPM1 protein in AML patients and to correlate flow cytometric results with the NPM1 gene mutation. Eighty-nine newly diagnosed AML patients were evaluated for the expression of mutant NPM1 using flow cytometry and for the presence of NPM1 exon 12 mutations using high-resolution melting polymerase chain reaction (HRM PCR). The NPM1 mutation was found in 35 (39.3%) patients by HRM PCR. These patients showed a significantly higher level of percentage of positive-stained cells (% positive cells) and normalized median fluorescence intensity (MFI) for mutant NPM1 by flow cytometry than the negative mutation group. Flow cytometric detection of mutant NPM1 offers a possible tool to indicate NPM1 mutational status.

Abstract 2372: DUSP3-NPM-P53 axis: a new regulator of genomic stability of cells under genotoxic stress

Abstract The dual-specificity phosphatase 3 (DUSP3) is an atypical tyrosine phosphatase that dephosphorylates residues of p-Tyrosine and p-Threonine in protein substrates. DUSP3 is overexpressed in some human tumors, where it regulates cell cycle progression and has preferably nuclear localization. DUSP3 contributes to cellular genomic stability through mechanisms that begin to be clarified here. Our group used biochemical, bioinformatics, proteomics and interactome approaches to identify and validate nuclear proteins interacting with DUSP3 under cellular conditions of genotoxic stress promoted by UV-radiation. One of these targets is the nucleophosmin (NPM) protein, which plays key roles in nucleotide excision repair (NER) pathway. Surface Plasmon Resonance analyses showed a very strong physical bimolecular interaction between NPM-DUSP3. In order to verify the involvement of this phosphatase in DNA repair, DUSP3 knockdown was carried out in MRC-5 (normal fibroblasts) or XPA (NER-deficient cell line lacking the XPA protein) cell lines. By alkaline comet assays we found DUSP3 knockdown caused delay in DNA repair even in XPA cells, which is not to repair UV-radiation induced damage. This effect was supported by immuno-slot-blot assays specifically measuring the distortions of CPD and 6,4-photoproducts (6-4-PP) caused by UV-radiation on cells genome. Once again, MRC-5 cells DUSP3 silenced exhibited delay in the repair of these lesions, whereas XPA cells were not able to repair them even after 72h of the UVC exposure, but the accumulation of CPD and 6-4-PP lesions was even higher under DUSP3 silencing. Next we verify that NPM is a substrate of DUSP3 by immunoprecipitating tyrosine-phosphorylated NPM, and which levels are increased in both cells under DUSP3 knockdown. Using phospho-specific antibodies designed against the four tyrosine residues of NPM, immunoblotings revealed that DUSP3 specifically dephosphorylates tyrosine 29, 67 and 271 residues of NPM after UV radiation altering its subnuclear localization. Furthermore immunoblotting and imunofluorescence assays revealed an unexpected increase in phospho-p53-Ser15 and p53 transcriptional activity in DUSP3 deficient cells after exposure to UV radiation, which can be correlated to nuclear NPM functions in the repair of UV-promoted DNA lesions. Even so, we observed that DUSP3 knockdown also causes an earlier nucleolus-nucleoplasm translocation of NPM after genotoxic stress that is followed by an earlier ARF translocation and co-localization of both proteins with HDM2 and p53 at nucleoplasm. This leads to disruption of HDM2-p53 interaction, to non-degradation and earlier phosphorylation of p53-Ser15. Interestingly, using cycloheximide to inhibit protein synthesis, we confirmed that the half-life of p53 and NPM is longer in the DUSP3-deficient cells. Altogether our data highlight DUSP3 unknown roles in different signaling modules regulating biological processes involved in genomic stability through an unusual target, the NPM-p53 axis. Citation Format: Fábio L. Forti, Lilian C. Russo. DUSP3-NPM-P53 axis: a new regulator of genomic stability of cells under genotoxic stress [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2372.

Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD

BackgroundThe C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated.Methods and resultsUsing DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls.ConclusionsCollectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

Nucleophosmin1 (NPM1) abnormality in hematologic malignancies, and therapeutic targeting of mutant NPM1 in acute myeloid leukemia.

Nucleophosmin (NPM1) is an abundant nucleolar protein that is implicated in a variety of biological processes and in the pathogenesis of several human malignancies. For hematologic malignancies, approximately one-third of anaplastic large-cell non-Hodgkin’s lymphomas were found to express a fusion between NPM1 and the catalytic domain of anaplastic lymphoma receptor tyrosine kinase. About 50–60% of acute myeloid leukemia patients with normal karyotype carry NPM1 mutations, which are characterized by cytoplasmic dislocation of the NPM1 protein. Nevertheless, NPM1 is overexpressed in various hematologic and solid tumor malignancies. NPM1 overexpression is considered a prognostic marker of recurrence and progression of cancer. Thus, NPM1 abnormalities play a critical role in several types of hematologic malignancies. This has led to intense interest in the development of an NPM1 targeting strategy for cancer therapy. The aim of this review is to summarize present knowledge on NPM1 origin, pathogenesis, and therapeutic interventions in hematologic malignancies.

Nucleophosmin contributes to vascular inflammation and endothelial dysfunction in atherosclerosis progression

ObjectiveIt is unclear whether nucleophosmin (NPM) participates in cardiovascular disease. The present study aimed to investigate the role and underlying mechanisms of NPM in atherosclerosis. MethodsLevels and location of NPM in human carotid atherosclerotic plaques and healthy controls were detected by real-time polymerase chain reaction, immunoblots, and immunofluorescence. Atherosclerotic prone ApoE–/– mice were fed with a Western diet for 16 weeks as an in vivo model. Human primary umbilical vein endothelial cells (HUVECs) were cultured as an in vitro model. ResultsCompared with controls, we found that NPM levels in human carotid atherosclerotic plaques were more than twice as high as in normal arteries, which mainly localized in endothelial cells. In vivo, adenovirus-containing NPM small hairpin RNA attenuated atherosclerotic lesion and promoted plaque stabilization in ApoE–/– mice fed a Western diet by reducing vascular inflammation, maintaining endothelial function, and decreasing macrophage infiltration. Furthermore, NPM knockdown decreased nuclear factor-κB (NF-κB) p65 phosphorylation. In cultured HUVECs, palmitic acid increased the protein levels of NPM and induced the expression of inflammatory cytokines and monocyte adhesion, whereas NPM knockdown attenuated this effect. In HUVECs, NPM protein physically interacted with NF-κB p65 subunit and promoted its nuclear transposition. NPM also increased the transcriptional activity of NF-κB p65 promoter and enhance its binding to target genes, including interleukin-1β, interleukin-6, intercellular adhesion molecule-1, and E-selectin. ConclusionsThese data provide novel evidence that NPM promotes atherosclerosis by inducing vascular inflammation and endothelial dysfunction through the NF-κB signaling pathway and suggest that NPM may be a promising target for atherosclerosis prevention and treatment.

Abstract 3136: Discovery of low abundance colorectal cancer related biomarkers by the ADAPT Biotargeting System

Abstract Disease biomarkers play an essential role in disease-diagnostics and in monitoring their responses to therapies. Identification of low to medium abundance biomarkers is one of the biggest challenges in the field. We have developed Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT), a method in which libraries of single-stranded oligodeoxynucleotides (ssODNs) are enriched for sequences that bind to targets existing in samples in various amounts. Pull-downs with enriched libraries followed by LC-MS/MS allows for the enrichment and identification of low abundance biomarkers that cannot be identified by conventional proteomics. A highly diverse library of 1011 ssODNs was subjected to multiple rounds of positive selection against formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissue with negative selection against adjacent non-cancer tissue of the same specimen. An enriched library of ~3x106 ssODNs that bound preferentially to cancer tissue was obtained and used in combination with LC-MS/MS to identify biomarkers related to colorectal cancer. Using this approach, a total of 14 proteins were identified in cancer but not in non-cancer tissue lysates. Their identification was only possible due to their enrichment by binding of the immobilized ssODN library to the cancer sample since the same proteins were undetectable by conventional proteomics. Immunohistochemical staining (IHC) of multiple colorectal cancer cases verified that several of the identified target proteins, including nucleophosmin (NPM1) and synaptotagmin-like protein 2 (SYTL2), showed higher expression in cancer tissue compared to adjacent non-cancer tissue. Our data indicate the potential of the ADAPT platform to profile small differences between cancer affected and unaffected tissue and provide a novel approach for cancer biomarker discovery. Citation Format: Tassilo Hornung, Jelena Zarkovic, Michelle Kassner, Matthew Rosenow, Mark R. Miglarese, Günter Mayer, Michael Famulok, David B. Spetzler. Discovery of low abundance colorectal cancer related biomarkers by the ADAPT Biotargeting System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3136.

C9orf72 Poly(PR) Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function.

Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 invitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution invitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus.

Molecular Insights into the Role of RNA Structure in the Phase Separated Nucleolus

Ribosome biogenesis is a critical cellular process and its dysregulation is associated with altered cellular behavior, as well as morbidity and death in humans. Biogenesis and assembly of these complex molecular machines occurs in the nucleolus, a large compartmentalized membrane-less organelle formed through liquid-liquid phase separation (LLPS). The protein Nucleophosmin (NPM1) is a major constituent of the outer, granular component (GC) region of the nucleolus wherein it phase separates with ribosomal proteins (r-proteins) and ribosomal RNA (rRNA). Despite the biological importance of NPM1/rRNA interactions in ribosome assembly, the physiochemical basis of these interactions is poorly understood. Preliminary data demonstrates that rRNA structure influences rRNA/NPM1 LLPS, suggesting a structure-based regulatory mechanism. Mg2+ addition significantly enhances and alters the observed fluid properties of NPM1/rRNA droplets. Chemical (SHAPE-MaP) and biophysical studies correlate observed enhanced LLPS with an increase in nascent RNA structure. Seminal studies in the 1970s by Nomura and others (Nomura, 1973) showed that during assembly, association of r-proteins act to stabilize and/or reshape rRNA into conformations(s) conducive to later r-protein association. Data presented herein further demonstrate binding of r-proteins to rRNA abrogates rRNA/NPM1 phase separation,suggesting a mechanism wherein sequential association of r-proteins leads to the occlusion of NPM1 binding sites and allows for release of assembled pre-ribosomal particles into the nucleoplasm. Broadly, our studies provide novel insight into how RNA structure can actively regulate phase separation in the nucleolus and suggest how leukemia associated NPM1 mutations may alter nucleolar LLPS, deregulate ribosome biogenesis, and promote oncogenesis.

Inhibition of nucleolar stress response by Sirt1: A potential mechanism of acetylation-independent regulation of p53 accumulation.

The mammalian Sirt1 deacetylase is generally thought to be a nuclear protein, but some pilot studies have suggested that Sirt1 may also be involved in orchestrating nucleolar functions. Here, we show that nucleolar stress response is a ubiquitous cellular reaction that can be induced by different types of stress conditions, and Sirt1 is an endogenous suppressor of nucleolar stress response. Using stable isotope labeling by amino acids in cell culture approach, we have identified a physical interaction of between Sirt1 and the nucleolar protein nucleophosmin, and this protein–protein interaction appears to be necessary for Sirt1 inhibition on nucleolar stress, whereas the deacetylase activity of Sirt1 is not strictly required. Based on the reported prerequisite role of nucleolar stress response in stress‐induced p53 protein accumulation, we have also provided evidence suggesting that Sirt1‐mediated inhibition on nucleolar stress response may represent a novel mechanism by which Sirt1 can modulate intracellular p53 accumulation independent of lysine deacetylation. This process may represent an alternative mechanism by which Sirt1 regulates functions of the p53 pathway.

Nucleophosmin Phosphorylation as a Diagnostic and Therapeutic Target for Ischemic AKI.

Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target.Methods Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury. The biologic behavior and toxicity of NPM was assessed using phospho-NPM mutant proteins that either mimic stress-induced or normal NPM phosphorylation. Peptides designed to interfere with NPM function were used to explore NPM as a therapeutic target.Results Within hours of stress, virtually identical phosphorylation changes were detected at distinct serine/threonine sites in NPM harvested from primary renal cells, tissue, and urine. A phosphomimic NPM protein that replicated phosphorylation under stress localized to the cytosol, formed monomers that interacted with Bax, a cell death protein, coaccumulated with Bax in isolated mitochondria, and significantly increased cell death after stress; wild-type NPM or a phosphomimic NPM with a normal phosphorylation configuration did not. Three renal targeted peptides designed to interfere with NPM at distinct functional sites significantly protected against cell death, and a single dose of one peptide administered several hours after ischemia that would be lethal in untreated mice significantly reduced AKI severity and improved survival.Conclusions These findings establish phosphorylated NPM as a potential early marker of ischemic AKI that links early diagnosis with effective therapeutic interventions.

Cytotoxic cationic peptides as а ligands for receptor nucleolin

Background. Chaperone proteins nucleolin (NCL, or C23) and nucleophosmin (NPM, or B23) regulate key cell functions. The most tumors are characterized by over-expression of these proteins, especially in cell nuclei and on the сell surface, as NCL. Differential expression of NCL/NPM in tumor and normal cells is the basis of selective cytotoxicity of cationic peptides – expected ligands for these proteins. Objective. Analysis of the interactions between nucleolin and some peptides with high nonspecific toxicity for tumor cells. Materials and methods. The interaction of 4 previously characterized cationic peptides with nucleolin dimer was analyzed by pair molecular docking using Maestro 11 program. Results and conclusion. It is shown that these peptides can associate with receptor nucleolin molecules, forming energy-stable complexes. In the active centre of NCL molecule were found, at least, 7 positions of amino acids, which bind to the tested peptides at a high frequency (43–100 %). This indicates the conservative structure of dimer NCL, its stable binding to peptide ligands and the possibility of design the optimal structure of cationic peptides that induce tumor cell death due to competing binding to the target proteins.

Distribution of Nucleophosmine Proteins (B23) and Hyston H4 Lysine 20 (Н4К20me3) in the Granule Cells of the Rat Brain Cerebellum

The purpose of this study was to study the relative distribution of the nucleophosmin (B23) protein and histone H4K20me3 in the granule cells of the rat cerebellar cortex. The material for the study was the samples of the cerebellum of mature male rats of the Wistar line (n = 8). Using the methods of immunocytochemistry and confocal laser microscopy for the first time, the features of the distribution of the nucleophosmin protein in the nuclei of the cerebellar granule cells were studied. In the nucleolus, a region that was characterized by the lack of the B23 protein and, supposedly, a fibrillar center was found. In addition to the nucleolus, nucleophosmin was detected (in a smaller amount) in the nucleoplasm of the cell granules. Using the method of confocal laser microscopy and three-dimensional reconstruction, we demonstrated the presence of zones of colocalization of the B23 protein and histone H4K20me3 within the nucleolus and nucleoplasm of the cell granules. It was shown that histone H4K20me3 is part of the perinucleolus heterochromatin, from which various protrusions often extend into the interior of the nucleolus. Clusters of the extranucleolus nucleophosmin were also often colocalized with heterochromatin blocks. However, nonnucleolus clusters of nucleophosmin that were not associated with heterochromatin blocks were also present. The results obtained make a significant addition to the existing ideas about the structural and functional organization of the nucleus of the cerebellar granule cells.

AML-associated mutation of nucleophosmin compromises its interaction with nucleolin.

C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.

Identification of Microprotein‐Protein Interactions via APEX Tagging

Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Genomics and proteomics technologies have led to the recent discovery of hundreds to thousands of new microproteins. Emerging evidence demonstrates that these microproteins have fundamental roles in various biological processes including metabolism, apoptosis, and development, highlighting the value of characterizing these molecules. The identification of microprotein‐protein interactions (MPIs) has proven to be a successful approach to the functional characterization of these genes; however, traditional immunoprecipitation methods result in the enrichment of non‐specific interactions for microproteins. To address this issue, we tested and applied an in situ proximity tagging method that relies on an engineered ascorbate peroxidase (APEX) to elucidate MPIs. The results demonstrated that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX‐tagging to two uncharacterized microproteins, C11orf98‐MP and MIEF‐MP revealed that they interact with nucleolar proteins nucleophosmin and nucleolin (C11orf98‐MP) and acyl carrier protein in mitochondria (MIEF‐MP) respectively, demonstrating the ability of this approach to identify novel hypothesis‐generating MPIs.Support or Funding InformationQ.C. is a postdoctoral fellow funded by the George E. Hewitt Foundation for medical research. This study was supported by the NIH with an NCI Cancer Center Support Grant P30 (CA014195 MASS core, A.S.) and R01 (GM102491, A.S.), the Leona M. and Harry B. Helmsley Charitable Trust grant (#2012‐PG‐MED002, A.S.), and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.). Imaging work in this study was supported by the Waitt Advanced Biophotonics Core Facility of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195, NINDS Neuroscience Core Grant: NS072031 and the Waitt Foundation. We also want to thank the Cancer Center Support Grant P30 CA014195 for purchasing and providing access to the MGC collection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lamin B2 Modulates Nucleolar Morphology, Dynamics, and Function.

ABSTRACTThe nucleolus is required for ribosome biogenesis. Human cells have 2 or 3 nucleoli associated with nucleolar organizer region (NOR)-bearing chromosomes. An increase in number and altered nucleolar morphology define cancer cells. However, the mechanisms that modulate nucleolar morphology and function are unclear. Here we show that in addition to localizing at the nuclear envelope, lamin B2 localizes proximal to nucleolin at the granular component (GC) of the nucleolus and associates with the nucleolar proteins nucleolin and nucleophosmin. Lamin B2 knockdown severely disrupted the nucleolar morphology, which was rescued to intact and discrete nucleoli upon lamin B2 overexpression. Furthermore, two mutually exclusive lamin B2 deletion mutants, ΔHead and ΔSLS, rescued nuclear and nucleolar morphology defects, respectively, induced upon lamin B2 depletion, suggesting independent roles for lamin B2 at the nucleolus and nuclear envelope. Lamin B2 depletion increased nucleolin aggregation in the nucleoplasm, implicating lamin B2 in stabilizing nucleolin within the nucleolus. Lamin B2 knockdown upregulated nucleolus-specific 45S rRNA and upstream intergenic sequence (IGS) transcripts. The IGS transcripts colocalized with aggregates of nucleolin speckles, which were sustained in the nucleoplasm upon lamin B2 depletion. Taken together, these studies uncover a novel role for lamin B2 in modulating the morphology, dynamics, and function of the nucleolus.

The Spatial Organization of the Intranuclear Structures of Human Brain Dopaminergic Neurons

We studied the intranuclear localization of protein nucleophosmin (B23) and ubiquitin in the dopaminergic neurons of human substantia nigra (n = 6, age of 25-87 years) using immunohistochemistry and confocal laser microscopy. Intranuclear ubiquitin-immunopositive bodies that morphologically correspond to Marinesco bodies were found to be present in substantia nigra dopaminergic (tyrosine hydroxylase-immunopositive) neurons but absent in non-dopaminergic neurons. The number of bodies varied from 0 to 6 per cell nucleus. Nucleophosmin (B23) was found in the neuronal nucleolus, with the nucleolus size being constant in the nigral neurons of each individual brain. All the observed neurons had only one large nucleolus with intense nucleophosmin immunoreactivity and a lightly stained region (1-2 μm in diameter) that apparently represents the giant fibrillar center (GFC). An intensely immunostained nucleophosmin-containing granule was often observed at the GFC periphery. Double labeling demonstrated that nucleophosmin-immunoreactive nucleolus and ubiquitin-immunoreactive Marinesco bodies can occur both closely to and remotely from each other. Three-dimensional reconstruction indicates that rounded Marinesco bodies are polymorphic and often have a complex shape, with some flattening and concavities, which may be associated with contact not only with the nucleolus, but also, presumably, with other intranuclear structures free of ubiquitin or nucleophosmin. Ubiquitin-immunoreactive structures with a relatively small size (up to 1 μm in length) and various clastosome-like shapes (Lafarga et al., 2002) often occur near Marinesco bodies. There were no cases of detection of ubiquitin in the nucleoli of dopaminergic neurons and nucleophosmin/B23 in typical Marinesco bodies. The obtained information may be helpful in unraveling the molecular mechanisms of the selective vulnerability of substantia nigra dopaminergic neurons to damaging factors.