The CacyBP/SIP Protein is Sumoylated in Neuroblastoma NB2a Cells

The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.     

Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

The membrane orientation of the NB protein of influenza B virus, a small (Mr, approximately 18,000) glycoprotein with a single internal hydrophobic domain, was investigated by biochemical and genetic means. Cell fractionation and protein solubility studies indicate NB is an integral membrane protein, and NB has been shown to be a dimer under nonreducing conditions. Treatment of infected-cell surfaces with proteinase K and endoglycosidase F and immunoprecipitation with a site-specific antibody suggests that the 18-amino-acid NH2-terminal region of NB is exposed at the cell surface. Oligonucleotide-directed mutagenesis to eliminate each of the four potential sites of N-linked glycosylation and expression of the mutant NB proteins in eucaryotic cells suggest that the two sites adjacent to the NH2 terminus are glycosylated. This provides further evidence that NB, which lacks a cleavable NH2-terminal signal sequence, has an exposed NH2 terminus at the cell surface.     

Retinoic acid confers resistance to p53-dependent apoptosis in SH-SY5Y neuroblastoma cells by modulating nuclear import of p53.

Many cell lines derived from neuroblastoma (NB) carry the wild-type p53 gene with a p53-dependent apoptotic pathway that is responsive to DNA damaging agents. A recent study has demonstrated that retinoic acid (RA) pretreatment of NB cells promotes chemoresistance to apoptosis induced by chemotherapeutic agents. We examine here the possible contribution of the p53 pathway to the chemoresistance response associated with the RA treatment in NB cells. Upon treatment with RA (1-10 microM) for 4 days, the human NB cells, SH-SY5Y, developed resistance selectively to p53-dependent apoptotic stimuli including gamma-irradiation, etoposide, and 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine (H-7). Interestingly, RA affected the ability of H-7 to induce nuclear accumulation of the p53 protein without altering its effect on elevating the steady-state level of p53, suggesting that drug-induced up-regulation and nuclear accumulation of the wild-type p53 protein are separable processes. The modulation of nuclear import of p53 protein by RA may thus represent a potential mechanism by which certain tumor cells with the wild-type p53 gene develop resistance to chemotherapeutic agents.     

The coiled-coil and nucleotide binding domains of the Potato Rx disease resistance protein function in pathogen recognition and signaling

Plant genomes encode large numbers of nucleotide binding and leucine-rich repeat (NB-LRR) proteins, some of which mediate the recognition of pathogen-encoded proteins. Following recognition, the initiation of a resistance response is thought to be mediated by the domains present at the N termini of NB-LRR proteins, either a Toll and Interleukin-1 Receptor or a coiled-coil (CC) domain. In order to understand the role of the CC domain in NB-LRR function, we have undertaken a systematic structure-function analysis of the CC domain of the potato (Solanum tuberosum) CC-NB-LRR protein Rx, which confers resistance to Potato virus X. We show that the highly conserved EDVID motif of the CC domain mediates an intramolecular interaction that is dependent on several domains within the rest of the Rx protein, including the NB and LRR domains. Other conserved and nonconserved regions of the CC domain mediate the interaction with the Ran GTPase-activating protein, RanGAP2, a protein required for Rx function. Furthermore, we show that the Rx NB domain is sufficient for inducing cell death typical of hypersensitive plant resistance responses. We describe a model of CC-NB-LRR function wherein the LRR and CC domains coregulate the signaling activity of the NB domain in a recognition-specific manner.     

Signals transduced via insulin-like growth factor I receptor (IGF(R)) mediate resistance to retinoic acid-induced cell growth arrest in a human neuroblastoma cell line

Retinoic Acid (RA) has been shown to control growth and induce differentiation in a number of human neuroblastoma (NB) cell lines. However, a number of NB cell lines may be termed resistant to RA as they fail to growth arrest and differentiate. In studying the mechanism mediating RA-resistance, we noted that invariably RA-resistant NB cell lines constitutively express Insulin-like Growth Factor 2 (IGF2) (Gaetano, 1991b). The NB cell line LAN-1-15N (15N) represented an interesting model in which to study the development of RA-resistance as initially 15N cells are growth arrested by RA, however with prolonged culture (8-10 days) cells begin to proliferate. Coincidentally, RA induces IGF2 mRNA and protein secretion in 15N NB cells (Matsumoto, 1992). In this study we isolated RA-resistant 15N cell lines and analyzed their growth properties and changes in cell cycle related (cdc2, cdk2, cyclins A, B, D and E) and early response (fos and jun) gene expression to evaluate the role IGF2 may play in mediating RA resistance. We found that exogenous IGF2 stimulates growth in 15N and is capable of altering RA induced inhibition of NB cell growth. Finally we show that by blocking the Insulin-like Growth Factor Receptor (IGF1(R)) with a monoclonal antibody (alpha-IR3) in the presence of RA the growth of RAR cell lines could be completely blocked. These data are consistent with the concept that signals by IGF2 and transduced via the IGF1(R) can mediate resistance to the growth inhibiting properties of RA.     

KAP1 depletion increases PML nuclear body number in concert with ultrastructural changes in chromatin

The promyelocytic leukemia (PML) protein is the main structural component of subnuclear domains termed PML nuclear bodies (PML NBs), which are implicated in tumor suppression by regulating apoptosis, cell senescence, and DNA repair. Previously, we demonstrated that ATM kinase can regulate changes in PML NB number in response to DNA double-strand breaks (DSBs). PML NBs make extensive contacts with chromatin and ATM mediates DNA damage-dependent changes in chromatin structure in part by the phosphorylation of the KRAB-associated protein 1 (KAP1) at S824. We now demonstrate that in the absence of DNA damage, reduced KAP1 expression results in a constitutive increase in PML NB number in both human U2-OS cells and normal human diploid fibroblasts. This increase in PML NB number correlated with decreased nuclear lamina-associated heterochromatin and a 30% reduction in chromatin density as observed by electron microscopy, which is reminiscent of DNA damaged chromatin. These changes in chromatin ultrastructure also correlated with increased histone H4 acetylation, and treatment with the HDAC inhibitor TSA failed to further increase PML NB number. Although PML NB number could be restored by complementation with wild-type KAP1, both the loss of KAP1 or complementation with phospho-mutants of KAP1 inhibited the early increase in PML NB number and reduced the fold induction of PML NBs by 25-30% in response to etoposide-induced DNA DSBs. Together these data implicate KAP1-dependent changes in chromatin structure as one possible mechanism by which ATM may regulate PML NB number in response to DNA damage.     

An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to?orient the mitotic spindle during NB asymmetric division.     

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells

Methylxantine derivative, caffeine, is known to prevent the p53-dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser-15 residue. In contrast, it has been reported that caffeine induces p53-mediated apoptosis through Bax protein in non-small-cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild-type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose- and time-dependent manner. Caffeine induced G(2)/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser-15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine-induced G(2)/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser-15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.     

Germline mutations of the paired-like homeobox 2B (PHOX2B) gene in neuroblastoma

Neuroblastoma (NB) is a frequent pediatric tumor for which recurrent somatic rearrangements are known. Germline mutations of predisposing gene(s) are suspected on the basis of rare familial cases and the association of NB with other genetically determined congenital malformations of neural crest-derived cells--namely, Hirschsprung disease (HSCR) and/or congenital central hypoventilation syndrome (CCHS). We recently identified the paired-like homeobox 2B (PHOX2B) gene as the major disease-causing gene in isolated and syndromic CCHS, which prompted us to regard it as a candidate gene in NB. Here, we report on germline mutations of PHOX2B in both a familial case of NB and a patient with the HSCR-NB association. PHOX2B, therefore, stands as the first gene for which germline mutations predispose to NB.     

An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein

The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.     

PML（NLS^-）蛋白在NB4细胞及其裸鼠移植瘤中定位的验证

验证中性粒细胞弹性蛋白酶（neutrophil elastase,NE）切割PML—RARa后,PML（NLS-）蛋白的存在和定位。将质粒pCMV-HA—NE电转染NB4细胞,用Westemblot法验证质粒转染成功;提取电转染质粒成功64／NB4细胞的胞浆蛋白,用Westem blot法检测NB4细胞中PML（NLS^-）蛋白的表达;免疫荧光法和激光共聚焦检测电转染质粒成功的NB4细胞中PML（NLS^-）蛋白的表达及定位;同时,建立NB4细胞、K562细胞和电转染质粒成功的NB4细胞裸鼠皮下瘤模型,用Westem blot、免疫组化法检测PML（NLS^-）蛋白在移植瘤组织细胞中的表达与定位。结果表明,westemblot检测电转染质粒pCMV-HA-NE的NB4细胞成功表达NE蛋白：NE酶成功切割PML-RARα,Western blot检测到电转染质粒pCMV-HA-NE的NB4细胞表达PML（NLS^-）蛋白;免疫荧光和激光共聚焦均可检测到电转染质粒成功的NB4细胞中PML（NLS^-）蛋白定位于细胞胞浆：Westem blot和免疫组化法检测到电转染质粒成功的NB4细胞裸鼠移植瘤中的PML（NLS^-）蛋白的表达且定位于细胞胞浆,而NB4和K562细胞裸鼠皮下瘤中PML蛋白主要定位于胞核。综上所述,该文成功将质粒pCMV-HA—NE电转染NB4细胞并用Western blot、免疫荧光、激光共聚焦、免疫组化验证PML（NLS-）蛋白存在于NB4细胞胞浆,这一现象可以为急性早幼粒细胞白血病的临床早期诊断与治疗提供新的依据。     

Amantadine blocks channel activity of the transmembrane segment of the NB protein from influenza B

NB is short auxiliary protein with ca. 100 amino acids, encoded in the viral genome of influenza B. It is believed to be similar to M2 from influenza A and Vpu from HIV-1 in that it demonstrates ion channel activity. Channels formed by the protein can be blocked by amantadine. We have synthesized the putative transmembrane segment of NB (IRG S20 IIITICVSL I30 VILIVFGCI A40 KIFI (NB, Lee)). Reconstituted in a lipid bilayer, the peptide shows channel activity. The addition of amantadine leads to dose-dependent loss of channel activity. Channel blocking is reversible. Channel behaviour of the peptide in the presence of amantadine is in accordance with findings for the intact channel. Thus, the synthetic transmembrane peptide captures the ion channel activity of the intact NB protein.     

Application of NanoBiT for Monitoring Dimerization of the Null Hong Kong Variant of α-1-Antitrypsin,NHK, in Living Cells

In this study, we investigated expression and dimerization of an ER-associated degradation (ERAD) substrate, a null Hong Kong variant of α-1-antitrypsin (NHK) using immunoblotting assay and a novel NanoLuc complementary reporter system called the NanoBiT (NB) assay. This NB-tagged NHK made it possible to monitor the intra- and extracellular status of NHK in living cells. The values for this NB assay fluctuated in response to distinct pharmacological stimuli and co-transfection of several ERAD-related factors. We then focused on mesencephalic astrocyte-derived neurotrophic factor (MANF), an unclarified ATF6/IRE1-downstream target, and established MANF-deficient Neuro2a (N2a) cells using CRISPR/Cas9 system. MANF-deficient N2a significantly elevated OS-9 protein after tunicamycin treatment; however, no specific differences in intra- and extracellular status of NHK protein were observed between wild-type and MANF-deficient cells. Taken together, intrinsic MANF in N2a cells is not strongly associated with the accumulation and clearance of unfolded proteins within the ER under current condition, but this novel NB assay is a useful approach for characterizing the protein status including ERAD substrates.     

Designer Nodal/BMP2 Chimeras Mimic Nodal Signaling,Promote Chondrogenesis,and Reveal a BMP2-like Structure

Nodal, a member of the TGF-β superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries.     

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.     

Requirement for HDM2 activity in the rapid degradation of p53 in neuroblastoma

The wild type p53 tumor suppressor protein is rapidly degraded in normal cells by MDM2, the ubiquitin ligase that serves as the key regulator of p53 function by modulating protein stability. Cellular exposure to genotoxic stress triggers the stabilization of p53 by multiple pathways that converge upon interference with MDM2 function. In this study, we first investigated the ability of HDM2 (MDM2 human homologue) to degrade endogenous p53 in neuroblastoma (NB). Although the p53 protein in NB has been reported to be constitutively stabilized, we find that HDM2 in NB is functional and facilitates the rapid turnover of p53 in nonstressed cells via the proteasome pathway. Second, we examined the relationship between p53 and HDM2 in the adriamycin-mediated stabilization of p53 in NB. We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2. In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway.     

P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC–MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.     

Zinc depletion induces JNK/p38 phosphorylation and suppresses Akt/mTOR expression in acute promyelocytic NB4 cells

BackgroundMyeloid leukemia is associated with reduced serum zinc and increased intracellular zinc. Our previous studies found that zinc depletion by TPEN induced apoptosis with PML-RARα oncoprotein degradation in acute promyelocytic NB4 cells. The effect of zinc homeostasis on intracellular signaling pathways in myeloid leukemia cells remains unclear.ObjectiveThis study examined how zinc homeostasis affected MAPK and Akt/mTOR pathways in NB4 cells.MethodsWe used western blotting to detect the activation of p38 MAPK, JNK, ERK1/2, and Akt/mTOR pathways in NB4 cells stimulated with the zinc chelator TPEN. Whether the effects of TPEN on these pathways could be reversed by zinc or the nitric oxide donor sodium nitroprusside (SNP) was further explored by western blotting. We used Zinpyr-1 staining to assess the role of SNP on labile zinc levels in NB4 cells treated with TPEN. In additional, we evaluated expressional correlations between the zinc-binding protein Metallothionein-2A (MT2A) and genes related to MAPKs and Akt/mTOR pathways in acute myeloid leukemia (AML) based on the TCGA database.ResultsZinc depletion by TPEN activated p38 and JNK phosphorylation in NB4 cells, whereas ERK1/2 phosphorylation was increased first and then decreased. The protein expression levels of Akt and mTOR were downregulated by TPEN. The nitric oxide donor SNP promotes zinc release in NB4 cells under zinc depletion conditions. We further found that the effects of zinc depletion on MAPK and Akt/mTOR pathways in NB4 cells can be reversed by exogenous zinc supplementation or treatment with the nitric oxide donor SNP. By bioinformatics analyses based on the TCGA database, we demonstrated that MT2A expression was negatively correlated with the expression of JNK, and was positively correlated with the expression of ERK1 and Akt in AML.ConclusionOur findings indicate that zinc plays a critical role in leukemia cells and help understanding how zinc depletion induces apoptosis.     

Influence of an aggressive early enteral nutrition protocol on nitrogen balance in critically ill children

The objective was to determine stress related factors and nutritional indices affecting the nitrogen balance (NB) and the creatinine height index (CHI) in critically ill children on early enteral nutrition (EEN). Seventy-one consecutively enrolled critically ill children aged 2 to 204 months, requiring prolonged mechanical ventilation, were studied. All patients were on early intragastric nutrition (Nutrison Pediatric or Standard) from day 1 (energy intake equal to 1/2, 1, 5/4, 6/4 and 6/4 of the predicted basal metabolic rate on days 1-5, respectively). Nitrogen balance and CHI changes determined efficacy. Study patients had severe depletion of somatic protein status on stress day 1 (CHI <60%) but they reached the normal range of somatic protein status at the end of the EEN, on post-stress day 5 (CHI >80%, p <.004). On day 1, none of the patients had positive NB but after 5 days of EEN, 44 (62%) had positive NB and only 27 (38%) had negative NB (p <.0001). Multivariate stepwise regression analysis showed that only the difference of daily given-recommended dietary allowances protein and the total repleted energy were positively correlated (r(2) =.47, p <.001 and r(2) = 34, p =.003, respectively) and multiple organ system failure negatively correlated with the NB (r(2) = -.24, p <.03) on the 5th day of the EEN protocol. Our data suggest that achievement of positive protein and energy balance in relation to the basic metabolic rate using an aggressive EEN protocol improves NB during the acute phase of stress in 2/3 of critically ill children.