A multistep, GTP-driven mechanism controlling the dynamic cycling of nucleostemin

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.     

Critical Role of Nucleostemin in Pre-rRNA Processing

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.     

Nucleolar trafficking of nucleostemin family proteins: common versus protein-specific mechanisms

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins.     

Nucleostemin: Another nucleolar “Twister” of the p53-MDM2 loop

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     

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics,SH3 Binding,and Phosphorylation

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein’s interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191–447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.     

Drosophila Nucleostemin 2 Proved Essential for Early Eye Development and Cell Survival

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.     

Nucleostemin is indispensable for the maintenance and genetic stability of hematopoietic stem cells

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 p16^Ink4a or p19^Arf deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs.     

A genetic approach for analyzing the co-operative function of the tRNA mimicry complex,eRF1/eRF3, in translation termination on the ribosome

During termination of translation in eukaryotes, a GTP-binding protein, eRF3, functions within a complex with the tRNA-mimicking protein, eRF1, to decode stop codons. It remains unclear how the tRNA-mimicking protein co-operates with the GTPase and with the functional sites on the ribosome. In order to elucidate the molecular characteristics of tRNA-mimicking proteins involved in stop codon decoding, we have devised a heterologous genetic system in Saccharomyces cerevisiae. We found that eRF3 from Pneumocystis carinii (Pc-eRF3) did not complement depletion of S. cerevisiae eRF3. The strength of Pc-eRF3 binding to Sc-eRF1 depends on the GTP-binding domain, suggesting that defects of the GTPase switch in the heterologous complex causes the observed lethality. We isolated mutants of Pc-eRF3 and Sc-eRF1 that restore cell growth in the presence of Pc-eRF3 as the sole source of eRF3. Mapping of these mutations onto the latest 3D-complex structure revealed that they were located in the binding-interface region between eRF1 and eRF3, as well as in the ribosomal functional sites. Intriguingly, a novel functional site was revealed adjacent to the decoding site of eRF1, on the tip domain that mimics the tRNA anticodon loop. This novel domain likely participates in codon recognition, coupled with the GTPase function.     

Drosophila nucleostemin 2 proved essential for early eye development and cell survival

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.     

5,5′- and 6,6′-Dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase

The discovery of 5,5′- and 6,6′-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC₅₀ <0.10 μM). In vitro DMPK data for selected compounds as well as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.     

Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways

Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate different environmental and intracellular signals, including metabolic status, to decide the fate of cellular morphology.     

Nucleostemin

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.     

Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity

Hepatitis C virus (HCV) frequently establishes persistent infections that can develop into severe liver disease. The HCV NS3/4A serine protease is not only essential for viral replication but also cleaves multiple cellular targets that block downstream interferon activation. Therefore, NS3/4A is an ideal target for the development of anti-HCV drugs and inhibitors. In the current study, we generated a novel NS3/4A/Lap/LC-1 triple-transgenic mouse model that can be used to evaluate and screen NS3/4A protease inhibitors. The NS3/4A protease could be conditionally inducibly expressed in the livers of the triple-transgenic mice using a dual Tet-On and Cre/loxP system. In this system, doxycycline (Dox) induction resulted in the secretion of Gaussia luciferase (Gluc) into the blood, and this secretion was dependent on NS3/4A protease-mediated cleavage at the 4B5A junction. Accordingly, NS3/4A protease activity could be quickly assessed in real time simply by monitoring Gluc activity in plasma. The results from such monitoring showed a 70-fold increase in Gluc activity levels in plasma samples collected from the triple-transgenic mice after Dox induction. Additionally, this enhanced plasma Gluc activity was well correlated with the induction of NS3/4A protease expression in the liver. Following oral administration of the commercial NS3/4A-specific inhibitors telaprevir and boceprevir, plasma Gluc activity was reduced by 50% and 65%, respectively. Overall, our novel transgenic mouse model offers a rapid real-time method to evaluate and screen potential NS3/4A protease inhibitors.     

Loss of <Emphasis Type="Italic">Drosophila</Emphasis> nucleostemin 2 (NS2) blocks nucleolar release of the 60S subunit leading to ribosome stress

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.     

Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors

The mechanism and kinetics of the interactions between ligands and immobilized full‐length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3–NS4A interaction consisted of a high‐affinity (K_D = 50 nM) and a low‐affinity (K_D = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism‐based inhibitor VX 950 exhibited a time‐dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN‐191 showed no signs of time‐dependent interactions, but ITMN‐191 had the highest affinity of the tested compounds, with both the slowest dissociation (k_off) and fastest association rate, closely followed by BILN 2061. The k_off for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti‐HCV lead discovery and optimization. Copyright © 2010 John Wiley & Sons, Ltd.     

Nucleostemin和PUMA在骨肉瘤细胞的表达及意义

目的研究Nucleostemin(NS)和p53上调凋亡调控因子(P53 up-regulated modulator of apoptosis,PUMA)在骨肉瘤中的表达。探讨其表达与骨肉瘤的病理分型,临床外科分期的关系。方法收集30例骨肉瘤患者手术标本,采用免疫组织化学SABC法检测这组标本中NS和PUMA的表达,应用图像分析系统测量其阳性表达的平均光密度值。结果 1.NS蛋白定位于细胞核,NS在骨肉瘤组表达高于骨软骨瘤组(P<0.05),在骨肉瘤组随Ennking分期的递增而增加(P<0.05),NS的表达与骨肉瘤的病理分型无关(P>0.05)。2.PUMA定位于细胞质,在骨肉瘤组表达明显低于骨软骨瘤组(P<0.05),其表达与骨肉瘤Ennking分期和病理分型无关(P>0.05)。结论 NS和PUMA在骨肉瘤发生和发展过程中起着重要的作用。