Assembly of the Trypanosoma brucei 60S ribosomal subunit nuclear export complex requires trypanosome-specific proteins P34 and P37

We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.     

Rapid up-regulation of P2Y messengers during granulocytic differentiation of HL-60 cells

HL-60 cells are human promyelocytic cells expressing two ATP receptors: the P2Y(2) and P2Y(11) subtypes. Our Northern blotting experiments have shown that P2Y(2) and P2Y(11) messengers were up-regulated in these cells, rapidly and independently of protein synthesis, following treatment with granulocytic differentiating agents such as retinoic acid, dimethylsulfoxide, granulocyte-colony stimulating factor, dibutyryl cyclic AMP and ATP. AR-C67085 and adenosine 5'-O-(3-thiotriphosphate), two potent agonists of the recombinant P2Y(11) receptor, increased intracellular cAMP concentration in HL-60 cells more potently than ATP itself. These observations support the conclusion that the effect of ATP on HL-60 cell differentiation is mediated by the P2Y(11) receptor.     

Optimization of technical conditions for the transformation of Pediococcus acidilactici P60 by electroporation

Previously reported techniques for the electrotransfer of foreign DNA into pediococci yield only a small number of transformants/mug DNA, especially when using undomesticated strains. This study reports an improved protocol for the electrotransformation of pediococci, based on trials using Pediococcus acidilactici P60 and the plasmid pRS4C1. The improved protocol yields from 2 to 3 log units more transformants than the previously reported methods, with up to (9.1+/-1.3)x10(4) transformants/mug of foreign DNA under the best conditions identified. The most important modifications proposed are an increase in electric field strength during electroporation (from 12.5 to 20kV/cm) and a reduction in lysozyme concentration during the preparation of electrocompetent cells (from 4000 to 2000U/ml): together, these two modifications greatly improve transformant yield. In addition, increasing cell culture time (from OD(600nm)=0.6 to OD(600nm)=1.0-1.2) and increasing dl-threonine concentration in the growth medium (from 20 to 40mM) also contribute to improved electrotransformation efficiency.     

ZnPcS2P2在K562和HL-60细胞中的定位研究

目的:探讨ZnPcS2P2在K562细胞,HL-60细胞亚细胞结构中的精确定位,揭示光动力学疗法(photody-namic therapy,PDT)的作用机制。方法:将K562细胞,HL-60细胞与ZnPcS2P2共同孵育5 h。应用激光扫描共聚焦显微成像系统,选择特异性细胞器荧光探针(线粒体探针若丹明Rodanmine123、溶酶体探针LysoTrackerDND-26、内质网探针Dioc6(3)采用波形比较法对光敏剂进行亚细胞定位。结果:ZnPcS2P2在K562细胞,HL-60细胞中发出的荧光与负载的Rodanmine123、Lyso-TracKer DND-26、Dioc6(3)均有部分重叠,波形均有相似之处。ZnPc-S2P2在线粒体、溶酶体、内质网均有分布。结论:线粒体是ZnPcS2P2介导的PDT(ZnPcS2P2-PDT)光损伤的主要靶点,溶酶体、内质网也是ZnPcS2P2-PDT光损伤的靶点。     

Structure-Guided Functional Characterization of DUF1460 Reveals a Highly Specific NlpC/P60 Amidase Family

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P2-purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.     

Differential inhibition of cellular and viral pp60src kinase by P1,P4-di(adenosine-5'')tetraphosphate.

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.     

P1,P4-Di(adenosine-5')tetraphosphate inhibits phosphorylation of immunoglobulin G by Rous sarcoma virus pp60src

Di(adenosine-5')oligophosphate nucleotides of general structure ApnA (n = 2-6) inhibited phosphorylation of immunoglobulin G from tumor-bearing rabbits (TBR IgG) by pp60src protein kinase purified from Rous sarcoma virus-transformed rat tumor cells. Ap4A, a nucleotide associated with eukaryotic cell proliferation, was one of the most effective inhibitors in the series, causing 50% inhibition of TBR IgG phosphorylation at 15 microM. Ap4A inhibited pp60src-dependent phosphorylation of TBR IgG in solution and immunoprecipitates, as well as the phosphorylation of tubulin, microtubule-associated proteins, and vinculin. Under similar assay conditions, Ap4A did not inhibit phosphorylation of histone H2b by cAMP- or cGMP-dependent protein kinases. Ap4A appears to interact noncovalently with the enzyme, because removal of pp60src by immunoprecipitation from solutions containing Ap4A restored activity to uninhibited levels. A 100-fold increase in ATP (4-400 nM) caused a 13-fold increase in the 50% inhibitory concentration of Ap4A (2.5-33 microM), consistent with the interpretation that Ap4A competes for an ATP-binding site on the pp60src molecule. The simplest explanation of these results is that Ap4A binds to the phosphodonor site for ATP.     

Inhibition of ligand binding and antiproliferative effects of tumor necrosis factor and lymphotoxin by soluble forms of recombinant P60 and P80 receptors.

Tumor necrosis factor (TNF) is one of the mediators of inflammatory responses. Recently, the cDNA for two distinct receptors of TNF with predicted molecular masses of 60 kDa and 80 kDa, respectively, were isolated. In this report, we compare the inhibitory effects of these two forms of recombinant soluble TNF receptors (extracellular domains) on the ligand binding and on the antiproliferative effects of TNF and lymphotoxin (LT) in a human histiocytic lymphoma cell line (U-937). Our results show that the soluble form of the p60 receptor is approximately 100-fold more potent than the soluble form of the p80 receptor in inhibiting both the antiproliferative effects of TNF as well as in blocking TNF binding to U-937 cells. In contrast, the antiproliferative effects of LT and its binding to cells is inhibited equally by both the p60 and p80 forms of the soluble receptor. Thus, overall our results indicate that the two soluble receptors differ in their ability to inhibit TNF and LT. The impotance of these soluble receptors in blocking the harmful effects of TNF and LT is discussed.     

Subunit P60 of phosphatidylinositol 3-kinase promotes cell proliferation or apoptosis depending on its phosphorylation status

The regulatory subunits (P60 in insects, P85 in mammals) determine the activation of the catalytic subunits P110 in phosphatidylinositol 3-kinases (PI3Ks) in the insulin pathway for cell proliferation and body growth. However, the regulatory subunits also promote apoptosis via an unclear regulatory mechanism. Using Helicoverpa armigera, an agricultural pest, we showed that H. armigera P60 (HaP60) was phosphorylated under insulin-like peptides (ILPs) regulation at larval growth stages and played roles in the insulin/ insulin-like growth factor (IGF) signaling (IIS) to determine HaP110 phosphorylation and cell membrane translocation; whereas, HaP60 was dephosphorylated and its expression increased under steroid hormone 20-hydroxyecdysone (20E) regulation during metamorphosis. Protein tyrosine phosphatase non-receptor type 6 (HaPTPN6, also named tyrosine-protein phosphatase corkscrew-like isoform X1 in the genome) was upregulated by 20E to dephosphorylate HaP60 and HaP110. 20E blocked HaP60 and HaP110 translocation to the cell membrane and reduced their interaction. The phosphorylated HaP60 mediated a cascade of protein phosphorylation and forkhead box protein O (HaFOXO) cytosol localization in the IIS to promote cell proliferation. However, 20E, via G protein-coupled-receptor-, ecdysone receptor-, and HaFOXO signaling axis, upregulated HaP60 expression, and the non-phosphorylated HaP60 interacted with phosphatase and tensin homolog (HaPTEN) to induce apoptosis. RNA interference-mediated knockdown of HaP60 and HaP110 in larvae repressed larval growth and apoptosis. Thus, HaP60 plays dual functions to promote cell proliferation and apoptosis by changing its phosphorylation status under ILPs and 20E regulation, respectively.     

Molecular chaperone, HSP60, and cytochrome P450 2E1 co-expression in dilated cardiomyopathy

Physiological stresses (heat, hemodynamics, genetic mutations, oxidative injury and myocardial ischemia) produce pathological states in which protein damage and misfolded protein structures are a common denominator. The specialized proteins family of antistress proteins - molecular chaperons (HSPs) - are responsible for correct protein folding, dissociating protein aggregates and transport of newly synthesized polypeptides to the target organelles for final packaging, degradation or repair. They are inducible at different cell processes such as cell division, apoptosis, signal transduction, cell differentiation and hormonal stimulation. HSPs are involved in numerous diseases including cardiovascular pathologies, revealing changes of expression and cell localization. We studied the possible changes in expression level of abundant mitochondrial chaperon Hsp60 and main human cytochrome P450 monooxygenase (2E1 isoform) at dilated cardiomyopathy (DCM) progression at the end stage of heart failure using Western blot analysis. The ischemic and normal humans' hearts were studied as control samples. We observed the decrease of Hsp60 level in cytoplasmic fraction of DCM- and ischemia-affected hearts' left ventricular and significant increase of Hsp60 in mitochondrial fractions of all hearts investigated. At the same time we detected the increase of P450 2E1 expression level in ischemic and dilated hearts' cytoplasmic fractions in comparison with normal myocardium and no detectable changes in microsomal fractions of hearts investigated which could be linked with increased level of oxidative injury for DCM heart muscle. In addition, all the changes described are accompanied by significant decrease of ATPase activity of myosin purified from DCM-affected heart in comparison with normal and ischemic myocardia as well. The data obtained allow us to propose a working hypothesis of functional link between antistress (HSPs) and antioxidative (cytochromes) systems at DCM progression.     

Evolutionary history,structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes

Background  

Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D-γ-glutamyl-meso-diaminopimelate or N-acetylmuramate-L-alanine linkages.     

Expression of human 60-kD heat shock protein (HSP60 or P1) in Escherichia coli and the development and characterization of corresponding monoclonal antibodies.

Human P1 protein, which is the homolog of the 60- to 65-kD heat shock "common" antigenic protein of numerous pathogenic organisms (synonyms: HSP60, GroEL homolog, or chaperonin), has been expressed to high level in Escherichia coli cells. A large number of well-characterized deletions of this protein spanning the entire sequence have been constructed and expressed. Methods to purify recombinant human HSP60 protein and its deletions from E. coli have been worked out. In addition, monoclonal antibodies to the human HSP60 protein have been raised and partially characterized. The availability of these materials should greatly aid in understanding the role of this highly conserved and immunologically important protein in autoimmune diseases and in cell structure and function.