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1.
Matsuo E Kanno S Matsumoto S Tsuneizumi K 《Bioscience, biotechnology, and biochemistry》2010,74(10):2120-2123
Human nucleostemin (NS) is a nucleolar protein involved in cell-cycle progression and ribosomal biogenesis. While four NS orthologs have been reported in Drosophila melanogaster, their roles in development have yet to be determined. Here we describe evidence that Drosophila nucleostemin 2 (ns2) plays a significant role in early eye development and is essential for cell survival in vivo. 相似文献
2.
3.
Several nucleolar proteins, such as ARF, ribosomal protein (RP) L5, L11, L23 and S7, have been shown to induce p53 activation by inhibiting MDM2 E3 ligase activity and consequently to trigger cell cycle arrest and/or apoptosis. Our recent study revealed another nucleolar protein called nucleostemin (NS), a nucleolar GTP binding protein, as a novel regulator of the p53-MDM2 feedback loop. However, unlike other known nucleolar regulators of this loop, NS surprisingly plays a dual role, as both up and downregulations of its levels could turn on p53 activity. Here, we try to offer some prospective views for this unusual phenomenon by reconciling previously and recently published studies in the field in hoping to better depict the role of NS in linking the p53 pathway with ribosomal biogenesis during cell growth and proliferation as well as to propose NS as another potential molecular target for anti-cancer drug development.Key words: ribosomal biogenesis, nucleolar stress, nucleostemin, p53, MDM2, cell cycle, cell growth 相似文献
4.
Four nucleostemin-like proteins (nucleostemin (NS) 1–4) were identified previously in Drosophila melanogaster. NS1 and NS2 are nucleolar proteins, while NS3 and NS4 are cytoplasmic proteins. We showed earlier that NS1 (homologous to human GNL3) enriches within the granular components (GCs) of Drosophila nucleoli and is required for efficient maturation or nucleolar release of the 60S subunit. Here, we show that NS2 is homologous to the human nucleostemin-like protein, Ngp1 (GNL2), and that endogenous NS2 is expressed in both progenitor and terminally differentiated cell types. Exogenous GFP-NS2 enriched within nucleolar GCs versus endogenous fibrillarin that marked the dense fibrillar components (DFCs). Like NS1, depletion of NS2 in midgut cells blocked the release of the 60S subunit as detected by the accumulation of GFP-RpL11 within nucleoli, and this likely led to the general loss of 60S subunits as shown by immunoblot analyses of RpL23a and RpL34. At the ultrastructural level, nucleoli in midgut cells depleted of NS2 displayed enlarged GCs not only on the nucleolar periphery but interspersed within the DFCs. Depletion of NS2 caused ribosome stress: larval midgut cells displayed prominent autophagy marked by the appearance of autolysosomes containing mCherry-ATG8a and the appearance of rough endoplasmic reticulum (rER)-derived isolation membranes. Larval imaginal wing disc cells depleted of NS2 induced apoptosis as marked by anti-caspase 3 labeling; loss of these progenitor cells resulted in defective adult wings. We conclude that nucleolar proteins NS1 and NS2 have similar but non-overlapping roles in the final maturation or nucleolar release of 60S ribosomal subunits. 相似文献
5.
Liudmila Romanova Anthony Grand Liying Zhang Samuel Rayner Nobuko Katoku-Kikyo Steven Kellner Nobuaki Kikyo 《The Journal of biological chemistry》2009,284(8):4968-4977
Nucleostemin is a nucleolar protein widely expressed in proliferating
cells. Nucleostemin is involved in the regulation of cell proliferation, and
both depletion and overexpression of nucleostemin induce cell cycle arrest
through the p53 signaling pathway. Although the presence of p53-independent
functions of nucleostemin has been previously suggested, the identities of
these additional functions remained to be investigated. Here, we show that
nucleostemin has a novel role as an integrated component of ribosome
biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large
protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal
subunit in a sucrose gradient. This complex contains proteins related to
pre-rRNA processing, such as Pes1, DDX21, and EBP2, in addition to several
ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is
dependent on the presence of nucleostemin in the nucleolus. Furthermore, the
knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S
rRNA. This is accompanied by a substantial decrease of protein synthesis as
well as the levels of rRNAs and some mRNAs. In addition, overexpressed
nucleostemin significantly promotes the processing of 32 S pre-rRNA.
Collectively, these biochemical and functional studies demonstrate a novel
role of nucleostemin in ribosome biogenesis. This is a key aspect of the role
of nucleostemin in regulating cell proliferation.Nucleostemin (NS)2
is a nucleolar protein preferentially expressed in actively proliferating
cells. The structure of NS is characterized by two GTP-binding domains, which
are involved in the regulation of its dynamic shuttling between the nucleolus
and nucleoplasm (1). NS was
originally identified as a nucleolar protein prominently expressed in rat
neural stem cells and down-regulated during differentiation of these cells
in vitro (2). The same
authors also found that NS is widely expressed in neural precursor cells in
early mouse embryos as well as in a variety of cancer cells and stem cells,
including embryonic stem cells and a hematopoietic stem cell-enriched
fraction. NS is generally down-regulated in the early stage of differentiation
before exit from the cell cycle. In addition, knockdown of NS significantly
inhibits proliferation of cortical stem cells and cancer cells. These initial
observations led to suggestions that NS is involved in multipotency in stem
cells as well as in the regulation of cancer and stem cell proliferation
(2).Recent work, however, has demonstrated that NS is in fact widely expressed
in many types of normal proliferating cells at levels similar to those in
malignant cells. For instance, NS is expressed in normal kidney cells and
renal carcinoma cells at comparable levels as detected in histological
sections (3). The expression of
NS is significantly up-regulated when normal T lymphocytes are activated by
concanavalin A (3) and when
bone marrow stem cells are stimulated by fibroblast growth factor 2
(4). Cells in NS-null mouse
embryos fail to enter the S phase, resulting in embryonic death at the
blastocyst stage (5,
6). In early Xenopus
embryos NS is also expressed in the sites of active cell proliferation and
local depletion of NS results in a decrease in proliferating neural progenitor
cells (6). Based on these
observations, it was proposed that expression of NS is more closely linked
with cell proliferation than with the malignant state or differentiation
status of a cell.Several studies have provided evidence that the p53 signaling pathway is
involved in the G1 arrest of the cell cycle induced by the
down-regulation of NS. Physical interaction between NS and p53 was initially
reported by Tsai and McKay (2).
Later, it was shown that the G1 arrest requires the presence of p53
(7). In the most recent study
Dai et al. (8) showed
that knockdown of NS enhances the interaction between the p53-binding protein
MDM2 and the ribosomal protein L5 or L11, preventing MDM2 from inducing
ubiquitylation-based p53 degradation. However, other studies have also
suggested that NS may have a p53-independent role in the regulation of cell
proliferation. For instance, the depletion of p53 from NS-null blastocysts did
not rescue them from the embryonic lethality
(6). In addition, NS partial
loss-of-function in mouse fibroblasts did not result in any change in the p53
level (5). Furthermore,
knockdown of L5 and L11 only partially rescued the G1 arrest in NS
knockdown cells (8). Finally,
the fact that NS is primarily localized in the nucleolus, whereas the
p53-mediated mechanism occurs in the nucleoplasm, suggests that NS might have
an additional role more directly relevant to nucleolar functions.To identify novel functions of NS, we purified an endogenous NS complex
from HeLa cell extract and investigated whether NS interacts with other
proteins not described previously. Identification of the components of this
complex and the alterations of the expression level of NS in HeLa cells led us
to uncover a novel role of NS in the processing of rRNA. Our findings not only
provide supporting evidence for the hypothesis that NS has a p53-independent
function but also demonstrate that NS is critical for ribosome biogenesis, one
of the most fundamental processes common for all cell types. 相似文献
6.
Farinaz Nazmi Mohammad Amin Moosavi Marveh Rahmati Mohammad Ali Hoessinpour-Feizi 《Bioinformation》2015,11(7):353-358
Human GNL3 (nucleostemin) is a recently discovered nucleolar protein with pivotal functions in maintaining genomic integrity
and determining cell fates of various normal and cancerous stem cells. Recent reports suggest that targeting this GTP-binding
protein may have therapeutic value in cancer. Although, sequence analyzing revealed that nucleostemin (NS) comprises 5
permuted GTP-binding motifs, a crystal structure for this protein is missing at Protein Data Bank (PDB). Obviously, any attempt
for predicting of NS structure can further our knowledge on its functional sites and subsequently designing molecular inhibitors.
Herein, we used bioinformatics tools and could model 262 amino acids of NS (132-393 aa). Initial models were built by
MODELLER, refined with Scwrl4 program, and validated with ProsA and Jcsc databases as well as PSVS software. Then, the best
quality model was chosen for motif and domain analyzing by Pfam, PROSITE and PRINTS. The final model was visualized by
vmd program. This predicted model may pave the way for next studies regarding ligand binding states and interaction sites as
well as screening of databases for potential inhibitors. 相似文献
7.
Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells and in many tumors and tumor-derived cell lines. In coimmunoprecipitation experiments, nucleostemin is recovered with the tumor suppressor p53, and more recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway. Here, we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high degree of colocalization in the granular component of the nucleolus during interphase, and both proteins associated with prenucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that NS and NPM co-reside in complexes, and yeast two-hybrid experiments confirmed that they are interactive proteins, revealing the NPM-interactive region to be the 46-amino acid N-terminal domain of NS. In bimolecular fluorescence complementation studies, bright nucleolar signals were observed, indicating that these two proteins directly interact in the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production. 相似文献
8.
Marcin Skoreński Aleksandra Milewska Krzysztof Pyrć Marcin Sieńczyk 《Journal of enzyme inhibition and medicinal chemistry》2019,34(1):8-14
West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)P(OPh)2 displaying Ki and k2/Ki values of 0.4 µM and 28 265 M?1s?1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease. 相似文献
9.
Matsuo E Nagamine T Matsumoto S Tsuneizumi K 《Bioscience, biotechnology, and biochemistry》2011,75(8):1511-1515
Nucleostemin (NS), a nucleolar guanosine triphosphate (GTP)-binding protein, plays significant roles in cell cycle progression and ribosomal biogenesis. Drosophila Nucleostemin 2 (NS2), a member of the Drosophila NS family, regulates early eye development and is essential to cell survival in vivo, but the underlying mechanisms have yet to be clarified. Biochemical analysis using the recombinant NS2 protein indicated that NS2 has GTPase activity. Immunohistochemistry revealed that NS2 changes in subcellular locus from the nucleolus to the nucleoplasm during larval development, and that a mutation in the ATP/GTP-binding site motif A (p-loop) prevents nuclear localization of NS2 and results in cytoplasmic distribution. Furthermore, downregulation of NS2 altered the rRNA proportions between the nucleus and the cytoplasm. These results suggest that NS2 at least requires GTP to import into the nucleoplasm. 相似文献
10.
Masayuki Yamashita Eriko Nitta Go Nagamatsu Yoshiko Matsumoto Ikushima Kentaro Hosokawa Fumio Arai Toshio Suda 《Biochemical and biophysical research communications》2013
Nucleostemin is a nucleolar protein known to play a variety of roles in cell-cycle progression, apoptosis inhibition, and DNA damage protection in embryonic stem cells and tissue stem cells. However, the role of nucleostemin in hematopoietic stem cells (HSCs) is yet to be determined. Here, we identified an indispensable role of nucleostemin in mouse HSCs. Depletion of nucleostemin using short hairpin RNA strikingly impaired the self-renewal activity of HSCs both in vitro and in vivo. Consistently, nucleostemin depletion triggered apoptosis rather than cell-cycle arrest in HSCs. Furthermore, DNA damage accumulated during cultivation upon depletion of nucleostemin. The impaired self-renewal activity of HSCs induced by nucleostemin depletion was partially rescued by p53 deficiency but not by p16Ink4a or p19Arf deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs. 相似文献
11.
A multistep, GTP-driven mechanism controlling the dynamic cycling of nucleostemin 总被引:16,自引:0,他引:16
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Nucleostemin (NS) was identified as a stem cell- and cancer cell-enriched nucleolar protein that controls the proliferation of these cells. Here, we report the mechanism that regulates its dynamic shuttling between the nucleolus and nucleoplasm. The nucleolar residence of nucleostemin involves a transient and a long-term binding by the basic and GTP-binding domains, and a dissociation mechanism mediated by the COOH-terminal region. This cycle is propelled by the GTP binding state of nucleostemin. We propose that a rapid nucleostemin cycle is designed to translate extra- and intra-cellular signals into the amount of nucleostemin in the nucleolus in a bidirectional and fast manner. 相似文献
12.
《Cell cycle (Georgetown, Tex.)》2013,12(16):3247-3252
Several nucleolar proteins, such as ARF, ribosomal protein (RP) L5, L11, L23 and S7, have been shown to induce p53 activation by inhibiting MDM2 E3 ligase activity and consequently to trigger cell cycle arrest and/or apoptosis. Our recent study revealed another nucleolar protein called nucleostemin (NS), a nucleolar GTP binding protein, as a novel regulator of the p53-MDM2 feedback loop. However, unlike other known nucleolar regulators of this loop, NS surprisingly plays a dual role, as both up and down regulations of its levels could turn on p53 activity. Here, we try to offer some prospective views for this unusual phenomenon by reconciling previously and recently published studies in the field in hoping to better depict the role of NS in linking the p53 pathway with ribosomal biogenesis during cell growth and proliferation as well as to propose NS as another potential molecular target for anti-cancer drug development. 相似文献
13.
14.
Nancy S. Petersen Arthur D. Riggs Robert L. Seecof 《In vitro cellular & developmental biology. Plant》1977,13(1):36-40
Summary A simple method is presented for establishing continuous cell lines fromDrosophila melanogaster embryos. Subculturing is performed after the first 8 weeks and at 2-week intervals therafter. Initial plating densities of
5×104 to 5×105 cells per cm2 are required for maintaining the subcultures. Cell lines were established from wild-type embryos, from embryos bearing chromosomal
rearrangements and from embryos bearing recessive mutations. Permanent lines have doubling times of 24 to 48 hr and have been
maintained for as long as 13 months and 25 subcultures.
Supported in part by NSF grant BMS75-02138 and NIH grant NS09330 to. R. Seecof. 相似文献
15.
It has been shown that mammalian neurogenesis is partly controlled by multiple basic helix–loop–helix (bHLH) genes, as inDrosophila.Recently, mouse homologs ofDrosophila atonal,a proneural gene encoding a bHLH protein required for chordotonal organ and photoreceptor development, have been characterized to obtain further insights into the molecular nature of mammalian neurogenesis. Here, to assess their potential involvement in genetic neural disorders, we have determined genetic map positions for four mouseatonal-related genes,Atoh1, Atoh2, Atoh3,andNdrf,which encode MATH-1, MATH-2, MATH-3, and NDRF, respectively. Interspecific backcross analysis indicated thatAtoh1andAtoh2were located in separate positions of Chr 6 and thatAtoh3andNdrfwere mapped to Chr 10 and Chr 11, respectively. Thus, these structurally related genes are located separately on multiple chromosomes. 相似文献
16.
Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration. 相似文献
17.
T. Kozlova Lucia Perezgasga Enrique Reynaud Mario Zurita 《Development genes and evolution》1997,207(4):253-263
The hsp60 (heat-shock protein 60) gene family of molecular chaperones has been a subject of study in numerous systems due to its important role in the correct
folding of non-native proteins in development as well as after heat-shock treatment. Here we present the characterization
of the first Drosophila hsp60 homologue. Drosophila HSP60 is most closely related (72% identity across the entire protein sequence) to the mouse mitochondrial HSP60. Western
blot experiments indicate that Drosophila HSP60 is enriched in the mitochondrial fraction. The distribution of HSP60 protein is dynamic during fly embryogenesis, suggesting
that various cell types might have different HSP60 requirements. The molecular analysis of a P-element-induced mutation that
affects the l(1)10Ac locus shows that the transposon is inserted in a 3-kb intron present in the hsp60 gene. By genetic rescue experiments we prove that Drosophila HSP60 is encoded by the essential locus l(1)10Ac opening the possibility for detailed genetic analysis of HSP60 functions in the fly.
Received: 24 March 1997 / Accepted: 16 June 1997 相似文献
18.
Huang M Whang P Chodaparambil JV Pollyea DA Kusler B Xu L Felsher DW Mitchell BS 《The Journal of biological chemistry》2011,286(13):11035-11046
Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation. 相似文献
19.
Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement
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Luping Zheng Zhenguo Du Chen Lin Qianzhuo Mao Kangcheng Wu Jianguo Wu Taiyun Wei Zujian Wu Lianhui Xie 《Molecular Plant Pathology》2015,16(9):921-930
Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2‐YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2‐YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2‐YFP fluorescence were affected in fibrillarin‐silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed. 相似文献
20.
Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2 总被引:1,自引:0,他引:1
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The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here, we show that NS regulates p53 activity through the inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G(1) cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G(1) arrest. These effects require the ribosomal proteins L5 and L11, since the depletion of NS enhanced their interactions with MDM2 and the knockdown of L5 or L11 abrogated the NS depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function. 相似文献