Compensatory Point Mutations in the Human Immunodeficiency Virus Type 1 Gag Region That Are Distal from Deletion Mutations in the Dimerization Initiation Site Can Restore Viral Replication

The dimerization initiation site (DIS), downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3′ end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.     

Changes in Protein Expression in Two Cholangiocarcinoma Cell Lines Undergoing Formation of Multicellular Tumor Spheroids In Vitro

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.     

Optimising non-viral gene delivery in a tumour spheroid model

BACKGROUND: Our current understanding of how the unique tumour microenvironment influences the efficacy of gene delivery is limited. The current investigation systematically examines the efficiency of several non-viral gene transfer agents to transfect multicellular tumour spheroids (MCTS), an in vitro model that displays a faithful three-dimensional (3D) representation of solid tumour tissue. METHODS: Using a luciferase reporter assay, gene transfer to MCTS was optimised for 22 kDa linear and 25 kDa branched polyethyleneimine (PEI), the cationic lipids Lipofectamine(trade mark) and DCChol : DOPE, and the physical approach of tissue electroporation. Confocal microscopy was used to take optical tissue slices to identify the tissue localisation of green fluorescent protein (GFP) reporter gene expression and the distribution of fluorescently labelled complexes. A MCTS model of quiescent tumour regions was used to establish the influence of cellular proliferation status on gene transfer efficiency. RESULTS: Of the polyplexes tested, 22 kDa linear PEI provided optimal gene delivery, with gene expression peaking at 46 h. Despite being the optimal vector tested, PEI-mediated transfection was limited to cells at the MCTS periphery. Using fluorescent PEI, it was found that complexes could only penetrate the outer 3-5 proliferating cell layers of the MCTS, sparing the deeper quiescent cells. Gene delivery in an MCTS model comprised entirely of quiescent cells demonstrated that in addition to being inaccessible to the vector, quiescent tumour regions are inherently less susceptible to PEI-mediated transfection than proliferating regions. This 'resistance' to transfection observed in quiescent cells was overcome through the use of electroporation. Despite the improved efficacy of electroporation in quiescent tissue, the gene expression was still confined to the outer regions of MCTS. The results suggest that limited access to central regions of an MCTS remain a significant barrier to gene delivery. CONCLUSIONS: This data provides new insights into tumour-specific factors affecting non-viral gene transfer and highlights the difficulties in delivering genes to avascular tumour regions. The MCTS model is a useful system for the initial screening of future gene therapy strategies for solid tumours.     

Comparative structural effects of HIV-1 Gag and nucleocapsid proteins in binding to and unwinding of the viral RNA packaging signal

The major RNA binding region of the HIV-1 Gag polyprotein is the nucleocapsid (NC) domain, which is responsible for the specific capture of the genomic RNA genome during viral assembly. The Gag polyprotein has other RNA chaperone functions, which are mirrored by the isolated NC protein after physiological cleavage from Gag. Gag, however, is suggested to have superior nucleic acid chaperone activity. Here we investigate the interaction of Gag and NC with the core RNA structure of the HIV-1 packaging signal (Ψ), using 2-aminopurine substitution to create a series of modified RNAs based on the Ψ helix loop structure. The effects of 2-aminopurine substitution on the physical and structural properties of the viral Ψ were characterized. The fluorescence properties of the 2-aminopurine substitutions showed features consistent with the native GNAR tetraloop. Dissociation constants (K(d)) of the two viral proteins, measured by fluorescence polarization (FP), were similar, and both NC and Gag affected the 2-aminopurine fluorescence of bases close to the loop binding region in a similar fashion. However, the influence of Gag on the fluorescence of the 2-aminopurine nucleotides at the base of the helix implied a much more potent helix destabilizing action on the RNA stem loop (SL) versus that seen with NC. This was further supported when the viral Ψ SL was tagged with a 5' fluorophore and 3' quencher. In the absence of any viral protein, minimal fluorescence was detected; addition of NC yielded a slight increase in fluorescence, while addition of the Gag protein yielded a large change in fluorescence, further suggesting that, compared to NC, the Gag protein has a greater propensity to affect RNA structure and that Ψ helix unwinding may be an intrinsic step in RNA encapsidation.     

Quantitative proteomic Isotope-Coded Protein Label (ICPL) analysis reveals alteration of several functional processes in the glioblastoma

Glioblastoma (GB), the most frequent primary tumor of the central nervous system, remains one of the most lethal human cancers despite intensive researches. Current paradigm in the study of GB has been focused on inter-patient variability and on trying to isolate new classification elements or prognostic factors. Here, using ICPL, a technique for protein relative quantification by mass spectrometry, we investigated protein expression between the four regions of GB on clinically relevant biopsies from 5 patients. We identified 584 non-redundant proteins and 31 proteins were found to be up-regulated in the tumor region compared to the peri-tumoral brain tissue, among which, 24 proteins belong to an interaction network linked to 4 biological processes. The core of this network is mainly constituted of interactions between beta-actin (ACTB) with heat shock proteins (HSP90AA1, HSPA8) and 14-3-3 proteins (YWHAZ, YWHAG, YWHAB). A cluster of three isoforms of the sodium pump α-subunit (ATP1A1, ATP1A2, ATP1A3) was also identified outside this network. The differential expression observed for ACTB and 14-3-3γ was further validated by western blot and/or immunohistochemistry. Our study confirms the identity of previously proposed molecular targets, highlights several functional processes altered in GB such as energy metabolism and synaptic transmission and could thus provide added value to new therapeutic trails.     

Identification of proteins regulated by ABA in response to combined drought and heat stress in maize roots

We adopted a proteomics approach to identify and analyze the differential expression of maize root proteins associated with abscisic acid (ABA) regulation under combined drought and heat stress. Using mass spectrometry, we identified 22 major proteins that were significantly up-regulated under combined drought and heat stress. These 22 proteins were classified into 6 functional categories: disease/defense (8), metabolism (3), cell growth/division (3), signal transduction (2), transporters (2) and unclassified (4). Our previous reports showed that ABA regulates the expression of several small heat-shock proteins (sHSPs) in maize leaves subjected to the combination of drought and heat stress; however, no sHSPs were identified among the root proteins up-regulated in this study. RT-PCR and western blot analyses were used to identify six known sHSPs. The maize roots were pretreated with 100 μM of ABA, and subsequently, the expression of the 22 up-regulated proteins and 6 sHSPs was examined. 11 proteins were up-regulated in an ABA-dependent manner, 13 proteins were up-regulated in an ABA-independent manner, and 4 proteins were up-regulated but inhibited by ABA. The up-regulated proteins are interesting candidates for further physiological and molecular investigations of combination stress tolerance in maize.     

Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.     

Functional Study of miR-27a in Human Hepatic Stellate Cells by Proteomic Analysis: Comprehensive View and a Role in Myogenic Tans-Differentiation

We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA.     

万座嗜酸两面菌硫代谢相关膜蛋白基因的筛选

活性巯基在浸矿微生物硫代谢的过程中起着重要的作用,半胱氨酸残基作为蛋白质中活性巯基的提供者,为筛选硫代谢相关蛋白质基因提供了依据。本研究以极端嗜酸热古菌万座嗜酸两面菌Acidianus manzaensis为研究对象,基于其全基因组注释信息,筛选出编码富半胱氨酸残基的潜在硫代谢相关膜蛋白基因,并通过RT-qPCR实验对筛选出来的基因进行表达水平验证,同时利用生物信息学方法对其进一步分析。研究表明,与在亚铁中生长的细胞相比,单质硫培养下的细菌中与能量代谢相关的β-葡糖苷酶,与电子传递相关的ATP合成酶、NADH-辅酶Q氧化还原酶基因均表达上调,说明硫代谢途径可能与能量代谢和电子传递有着重要的联系。此外,还有三个假定蛋白基因表达上调,这三个假定膜蛋白中,ARM75161.1、ARM75436.1中的半胱氨酸都位于保守区域,且均有一个半胱氨酸残基暴露于膜外,而ARM75580.1中的半光氨酸不位于保守区域。其中ARM75436.1具有CXXXC结构域,且该结构域中半胱氨酸残基处于同一个β-折叠中。这些假定蛋白可能参与A. manzaensis中硫代谢途径。     

Proteomic analysis of CA1 and CA3 regions of rat hippocampus and differential susceptibility to intermittent hypoxia

The CA1 and CA3 regions of the hippocampus markedly differ in their susceptibility to hypoxia in general, and more particularly to the intermittent hypoxia that characterizes sleep apnea. Proteomic approaches were used to identify proteins differentially expressed in the CA1 and CA3 regions of the rat hippocampus and to assess changes in protein expression following a 6-h exposure to intermittent hypoxia (IH). Ninety-nine proteins were identified, and 15 were differentially expressed in the CA1 and the CA3 regions. Following IH, 32 proteins in the CA1 region and only 7 proteins in the more resistant CA3 area were up-regulated. Hypoxia-regulated proteins in the CA1 region included structural proteins, proteins related to apoptosis, primarily chaperone proteins, and proteins involved in cellular metabolic pathways. We conclude that IH-mediated CA1 injury results from complex interactions between pathways involving increased metabolism, induction of stress-induced proteins and apoptosis, and, ultimately, disruption of structural proteins and cell integrity. These findings provide initial insights into mechanisms underlying differences in susceptibility to hypoxia in neural tissue, and may allow for future delineation of interventional strategies aiming to enhance neuronal adaptation to IH.     

Virion packaging determinants and reverse transcription of SRP RNA in HIV-1 particles

Diverse retroviruses have been shown to package host SRP (7SL) RNA. However, little is known about the viral determinants of 7SL RNA packaging. Here we demonstrate that 7SL RNA is more selectively packaged into HIV-1 virions than are other abundant Pol-III-transcribed RNAs, including Y RNAs, 7SK RNA, U6 snRNA and cellular mRNAs. The majority of the virion-packaged 7SL RNAs were associated with the viral core structures and could be reverse-transcribed in HIV-1 virions and in virus-infected cells. Viral Pol proteins influenced tRNAlys,3 packaging but had little influence on virion packaging of 7SL RNA. The N-terminal basic region and the basic linker region of HIV-1 NCp7 were found to be important for efficient 7SL RNA packaging. Although Alu RNAs are derived from 7SL RNA and share the Alu RNA domain with 7SL RNA, the packaging of Alu RNAs was at least 50-fold less efficient than that of 7SL RNA. Thus, 7SL RNAs are selectively packaged into HIV-1 virions through mechanisms distinct from those for viral genomic RNA or primer tRNAlys,3. Virion packaging of both human cytidine deaminase APOBEC3G and cellular 7SL RNA are mapped to the same regions in HIV-1 NC domain.     

Proteomic investigation of metabolic shift in mammalian cell culture.

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down-regulated, and the others were up-regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down-regulated, and the precursor of the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which was up-regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, which is involved in protein turnover and degradation, was also up-regulated in the metabolically altered cells. The remaining down-regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up-regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up-regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.     

Affinities of the nucleocapsid protein for variants of SL3 RNA in HIV-1

Efficient packaging of genomic RNA into new HIV-1 virus particles requires that nucleocapsid domains of precursor proteins bind the SL3 tetraloop (G317-G-A-G320) from the 5'-untranslated region. This paper presents the affinities of 35 RNA variants of SL3 for the mature 55mer NC protein, as measured by fluorescence quenching of tryptophan-37 in the protein by nucleobases. The 1:1 complexes that form in 0.2 M NaCl have dissociation constants ranging from 8 nM (GGUG) to 20 microM (GAUA). The highly conserved (GGAG) sequence for the wild type is not the most stable (K(d) = 28 nM), suggesting that other selective pressures beyond the stability of the complex must be satisfied. The leading requirement for strong interaction is for G320, followed closely by G318. Replacing either with U, A, or C reduces affinity by a factor of 15-120. NC-domains from multiple proteins combine to recognize unpaired G(2)-loci, where two guanines are in close proximity. We have previously measured affinities of the NC protein for the important stem-loops of the major packaging domain [Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]. Comparison with the present work shows that the nature of the stem also modulates NC-RNA interactions. Placing the G(2)-loci from the apical SL2 or SL1 loops on the SL3 stem increases affinity by a factor of 2-3, while placing the SL4 loop on the SL3 stem reduces affinity 50-fold. These results are interesting in the context of RNA-protein interaction, as well as for the discovery of antiNC agents for AIDS therapy.     

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP.     

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells.

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.     

Expression of clock proteins in developing tooth

Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.