?Np73 is capable of inducing apoptosis by co-ordinately activating several BH3-only proteins

Inactivation of p53 is one of the most relevant events in human cancer, since it allows transformed cells to escape their own proliferation control and leave them irresponsive to drugs that aim to damage their DNA. When p53 falls, other members of its family may become targets to attack tumoural cells. p73 has shown capacity to mediate these attacks. However, its N-terminal truncated isoforms have been associated with oncogenesis due to their capacity to act as dominant negatives of p53 and the transactivation (TA) isoforms of p73. We previously found a relationship between the overexpression of N-terminus-truncated p73 isoform (∆Np73) and that of the proapoptotic gene Bcl-2-interacting killer (BIK). In the present report we demonstrate that ∆Np73-α has the capacity to induce apoptosis through the co-ordinated activation of a group of genes harbouring GC-rich elements in their regulatory regions. ∆Np73-α synergizes with specificity protein (Sp1) on these elements but the overall response of these genes probably depends on the additional presence of consensus p53 elements. We explore the domains of ∆Np73-α involved in this transactivation capacity and found divergences with the previously described functions for them. Moreover, we found that the transforming mutation V12 of HRas impairs this transactivation capacity of ∆Np73-α, further supporting the anti-tumoural function of this later. Our data add complexity to the action of p73 on the induction of apoptosis and tumourogenesis, opening new interpretations to the expression profile of p73 isoforms in different human neoplasias.     

Overexpression of ΔNp63 in a human nasopharyngeal carcinoma cell line downregulates CKIs and enhances cell proliferation

p63 belongs to a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, two types of p63 proteins are produced. The ΔNp63 isoform lacks the N‐terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. ΔNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of ΔNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a ΔNp63 overexpressing human NPC cell line (NPC‐076) to explore the possible roles of ΔNp63 in cell proliferation and cell‐cycle regulation. We found that the proliferation of NPC‐076 cell is greatly suppressed when the overexpressed ΔNp63 is silenced by specific ΔNp63 siRNA. Further studies show that ΔNp63 silencing results in the upregulation of CKIs, including p27^kip1 and p57^kip2 in both mRNA and protein levels. Cell‐cycle analysis shows that ΔNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that ΔNp63 plays important roles in the regulation of NPC‐076 cell‐cycle progression, and may play a role in the maintenance of NPC‐076 tumor cell phenotype. J. Cell. Physiol. 219: 117–122, 2009. © 2008 Wiley‐Liss, Inc.     

Isolation of the major basic nuclear protein and its localization on chromosomes of the dinoflagellate,Oxyrrhis marina

Basic nuclear proteins were extracted from isolated nuclei of Oxyrrhis marina. The HPLC pattern of the extract showed a single major peak, which consisted of a major band with an apparent molecular mass of 23 kDa on an SDS-PAGE gel. We designated this protein as Np23 because of its apparent molecular mass. The amino acid composition of this protein revealed its extremely basic nature with a high lysine content. Polyclonal antibodies were raised against Np23. Immunofluorescence microscopy showed that Np23 was localized within the nucleus of dividing and non-dividing cells as well, and immuno-electron microscopy showed that the protein was localized only on chromosomes. These data established that Np23 is the major basic chromosome protein of Oxyrrhis marina.     

Long term retention of 237Np in rats

This interim report summarizes the results of observations during the first year after a single injection of 237Np nitrate (0.2 or 1.0 mg/kg body weight) into adult female rats and further preliminary data obtained with young animals. The retention of 237Np was followed by whole body counting and serial sacrifice of groups of animals. The retention data could be fitted to three-component exponential equations which show no major differences between the two 237Np dose levels. The half-times and extrapolated initial fractions calculated from the first two exponential terms indicate that one fraction, representing about 40 per cent of the injected 237Np was excreted within the first 5 days and an additional 15 per cent within the first 5 months, while the rest was excreted with a half-time of about 3.5 years. This final long term component is assumed to indicate the rate of loss of 237Np from the skeletal compartment. In young animals both whole-body and skeletal retention of 237Np during the first 5 months of observation was about 50 per cent higher than in the adults. Several soft tissue tumours, mostly mammary tumours, have appeared to approximately the same extent in both control and 237Np treated adult rats but no osteosarcomas were detected up to 15 months after injection of 237Np.     

Reduction of Np(V) and precipitation of Np(IV) by an anaerobic microbial consortium

A combination of experimental, analytical, and modeling investigations shows that an anaerobic, sulfate-reducing consortium reduced Np(V) to Np(IV), with subsequent precipitation of a Np(IV) solid. Precipitation of Np(IV) during growth on pyruvate occurred before sulfate reduction began. H₂ stimulated precipitation of Np(IV) when added alone to growing cells, but it slowed precipitation when added along with pyruvate. Increasing concentrations of pyruvate also retarded precipitation. Accumulation of an intermediate pyruvate-fermentation product – probably succinate – played a key role in retarding Np(IV) precipitation by complexing the Np(IV). Hydrogen appears to have two roles in controlling Np precipitation: donating electrons for Np(V) reduction and modulating intermediate levels. That Np(V) is microbially reduced and subsequently precipitated under anaerobic conditions has likely beneficial implications for the containment of Np on lands contaminated by radionuclides, but complexation by fermentation intermediates can prevent immobilization by precipitation.     

Crystalline Ferredoxin from a Blue-Green Alga, Nostoc sp.

Ferredoxin isolated from a blue-green alga, Nostoc sp., was purified and crystallized. The absorption spectrum of Nostoc ferredoxin had, in the oxidized state, peaks at 276, 331, 423, and 470 nm, a pattern characteristic of chloroplast-type ferredoxin. The 423:276 absorption ratio was 0.57. The midpoint oxidation-reduction potential of Nostoc ferredoxin was found to be –406 mV, at pH 7.5. Nostoc ferredoxin mediated the photoreduction of NADP by isolated Nostoc chromato-phores and spinach chloroplasts from which the native ferredoxin was removed. The molar ratio of Nostoc ferredoxin to chlorophyll a was about 1:50, a ratio higher than usually found in photosynthetic cells. The possible evolutionary significance of the properties of Nostoc ferredoxin compared with those of ferredoxins from other photosynthetic organisms is discussed.     

Screening of promising molecules against MurG as drug target in multi-drug-resistant-Acinetobacter baumannii - insights from comparative protein modeling,molecular docking and molecular dynamics simulation

Abstract

The UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) is located in plasma membrane which plays a crucial role for peptidoglycan biosynthesis in Gram-negative bacteria. Recently, this protein is considered as an important and unique drug target in Acinetobacter baumannii since it plays a key role during the synthesis of peptidoglycan as well as which is not found in Homo sapiens. In this study, initially we performed comparative protein modeling approach to predict the three-dimensional model of MurG based on crystal structure of UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (PDB ID: 1F0K) from E.coli K12. MurG model has two important functional domains located in N and C- terminus which are separated by a deep cleft. Active site residues are located between two domains and they are Gly20, Arg170, Gly200, Ser201, Gln227, Phe254, Leu275, Thr276, and Glu279 which play essential role for the function of MurG. In order to inhibit the function of MurG, we employed the High Throughput Virtual Screening (HTVS) and docking techniques to identify the promising molecules which will further subjected into screening for computing their drug like and pharmacokinetic properties. From the HTVS, we identified 5279 molecules, among these, 12 were passed the drug-like and pharmacokinetic screening analysis. Based on the interaction analysis in terms of binding affinity, inhibition constant and intermolecular interactions, we selected four molecules for further MD simulation to understand the structural stability of protein-ligand complexes. All the analysis of MD simulation suggested that ZINC09186673 and ZINC09956120 are identified as most promising putative inhibitors for MurG protein in A. baumannii.

Communicated by Ramaswamy H. Sarma     

HCoV-NL63N蛋白不同片段的表达纯化及血清学检测应用分析

将HCoV-NL63核衣壳蛋白N端(Np1~154aa)、C端(Cp141~306aa)基因片段克隆到原核表达载体pET30a(+)上进行原核表达,制备相应的纯化蛋白Np、Cp蛋白,利用Np、Cp蛋白建立基于Western-Blot条带印迹的HCoV-NL63抗体检测法,并与基于全长N蛋白(Nf)的HCoV-NL63抗体检测法相平行筛查了50份成年体检血清。结果显示:50份成年体检血清中,采用Nf、Np、Cp分别检出25、27、36份HCoV-NL63抗体阳性血清,检出率分别为50%、54%、72%。不同N蛋白与血清反应抗体阳性谱存在差异,其中Np与Nf检出一致率为64%,Cp与Nf检出一致率为54%,而Np与Cp检出一致率为54%。本研究表明人冠状病毒NL63在我国人群中感染常见,N蛋白C端(Cp)检出率比全长N(Nf)及N端(Np)要高,Nf、Np、Cp在抗体检测上存在不一致性。这为HCoV-NL63血清学试剂研发及免疫学研究提供了依据与实验基础。     

Characterization of two potentially universal turn motifs that shape the repeated five-residues fold--crystal structure of a lumenal pentapeptide repeat protein from Cyanothece 51142

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.     

Delta Np63 alpha expression is regulated by the phosphoinositide 3-kinase pathway

p63 is a homologue of p53 that functions to maintain progenitor cell populations in stratified epithelia. Delta Np63 alpha is overexpressed in epithelial cancers and has been shown to have oncogenic properties. We have previously reported that inhibition of epidermal growth factor receptor signaling results in a decrease in Delta Np63 alpha expression. Here, we demonstrate Delta Np63 alpha is a target of the phosphoinositide-3-kinase (PI3K) pathway downstream of the epidermal growth factor receptor. Treatment of keratinocytes with epidermal growth factor results in an increase in Delta Np63 alpha expression at the mRNA level, which is abrogated by inhibition of PI3K but not mitogen-activated protein kinase signaling. Small interfering RNA-mediated knockdown of the p110 beta catalytic subunit of PI3K results in a decrease in Delta Np63 alpha protein levels in keratinocytes. The results presented herein suggest that regulation of Delta Np63 alpha expression by the PI3K pathway plays a critical role in the survival and proliferative capacity of squamous epithelia.     

The multi-domain protein Np95 connects DNA methylation and histone modification

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ß. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.