The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway. © 2008 Federation of European Biochemical Societies.

Comparison of the coat protein, movement protein and RNA polymerase gene sequences of Australian, Chinese, and American isolates of barley yellow dwarf virus transmitted by Rhopalosiphum padi

Barley yellow dwarf luteovirus-GPV (BYDV-GPV) is a common problem in Chinese wheat crops but is unrecorded elsewhere. A defining characteristic of GPV is its capacity to be transmitted efficiently by both Schizaphis graminum and Rhopaloshiphum padi. This dual aphid species transmission contrasts with those of BYDV-RPV and BYDV-SGV, globally distributed viruses, which are efficiently transmitted only by Rhopaloshiphum padi and Schizaphis graminum respectively. The viral RNA sequences encoding the coat protein (22K) gene, the movement protein (17K) gene, the region surrounding the conserved GDD motif of the polymerase gene and the intergenic sequences between these genes were determined for GPV and an Australian isolate of BYDV-RPV (RPVa). In all three genes, the sequences of GPV and RPVa were more similar to those of an American isolate of BYDV-RPV (RPVu) than to any other luteovirus for which there is data available. RPVa and RPVu were very similar, especially their coat proteins which had 97% identity at the amino acid level. The coat protein of GPV had 76% and 78% amino acid identity with RPVa and RPVu respectively. The data suggest that RPVu and RPVa are correctly named as strains of the same serotype and that GPV is sufficiently different from either RPV strain to be considered a distinct BYDV type. The coat protein and movement protein genes of GPV are very dissimilar to SGV. The polymerase sequences of RPVu, RPVa and GPV show close affinities with those of the sobemo-like luteoviruses and little similarity with those of the carmo-like luteoviruses. The sequences of the coat proteins, movement proteins and the polymerase segments of BYDV serotypes, other than RPV and GPV, form a cluster that is separate from their counterpart sequences from dicot-infecting luteoviruses. The RPV and GPV isolates consistently fall within a dicot-infecting cluster. This suggests that RPV and GPV evolved from within this group of viruses. Since these other viruses all infect dicots it seems likely that their common ancestor infected a dicot and that RPV and GPV evolved from a virus that switched hosts from a dicot to a monocot.

The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67 348 polypeptide encoded by genome segment 8

The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSV S8 is 1 914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1 810 which is capable of encoding a protein of M(r) 67 348. The N-terminal amino acid sequence of a ~43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto- catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a ~43K major capsid protein.

Genome segment 5 of rice ragged stunt virus encodes a virion protein

The complete nucleotide sequence of the genome segment 5 (S5) of a Thai isolate of rice ragged stunt virus (RRSV) was determined. The 2682 nucleotide sequence contains a single long open reading frame capable of encoding a polypeptide with a molecular mass of ~91 kDa. Polypeptides encoded by various truncated cDNAs of S5 were expressed using the pGEX fusion protein vector and the highest level of fusion protein was obtained from a construct encoding a hydrophilic region of S5 protein. Antibodies raised against this fusion protein recognized a minor polypeptide, with a molecular mass of ~ 91 kDa, that was present in purified preparations of RRSV particles, infected insect vectors and infected rice plants. This indicates that RRSV S5 encodes a minor structural protein. Comparing the RRSV S5 sequence with sequences of other reo-viruses did not reveal any significant sequence similarities.

Sequence and identification of the barley yellow dwarf virus coat protein gene

The nucleotide sequence of the coat protein gene of barley yellow dwarf virus (BYDV, PAV serotype) was determined, and the amino acid sequence was deduced. The open reading frame, encoding a protein of relative molecular mass (Mr) 22,047, was confirmed as the coat protein gene by comparison with amino acid sequences of tryptic peptides derived from dissociated virions. In addition, a fragment of this gene expressed in Escherichia coli produced a product which was recognized by antibodies prepared against purified BYDV virions. An overlapping reading frame encoding an Mr 17,147 protein is contained completely within the coat protein gene. © 1988.

Interleukin-1β stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-β-dependent mechanism

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

The binding of nuclear factors to the as-1 element in the CaMV 35S promoter is affected by cytosine methylation in vitro

Transcriptional gene silencing (TCS) is often associated with an increased level of cytosine methylation in the affected promoters. The effect of methylation of the cauliflower mosaic virus (CaMV) 35S promoter sequence on its binding to factors present in the nuclei was analyzed by electrophoretic mobility shift assays using extracts of petunia flowers. Specific DNA-protein interactions were detected in the region of the CaMV 35S promoter that contains the as-1 element and the region between -345 and -208. The binding of protein factor(s) to the as-1 element was influenced by cytosine methylation, whereas the binding to the region between -345 and -208 was unaffected. The results suggest that cytosine methylation of the as-1 element potentially affects the activity of the CaMV 35S promoter. © Georg Thieme Verlag KG Stuttgart.

Time to face the fats : What can mass spectrometry reveal about the structure of lipids and their interactions with proteins?

Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis

As microenvironmental factors such as three-dimensionality and cell–matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

Reduced excision repair cross-complementing 1 expression associates with enhanced papilloma formation and fibroblast transformation after genetic disruption of secreted protein acidic and rich in cysteine

SPARC (secreted protein acidic and rich in cysteine)/ osteonectin/BM-40 is a matricellular protein implicated in development, cell transformation and tumorigenesis. We have examined the role of SPARC in cell transformation induced chemically with 7,12-dimethylbenz[a]anthracene (DMBA) and 12- tetradecanoylphorbol-13-acetate (TPA) in embryonic fibroblasts and in the skin of mice. Embryonic fibroblasts from SPARCnull mice showed increases in cell proliferation, enhanced sensitivity to DMBA and a higher number of DMBA/TPA-induced transformation foci. The number of DMBA-DNA adducts was 9 times higher in SPARCnull fibroblasts and their stability was lower than wild-type fibroblasts, consistent with a reduction in excision repair cross-complementing 1 the nucleotide excision repair enzyme in these cells. The SPARCnull mice showed an increase in both the speed and number of papillomas forming after topical administration of DMBA/TPA to the skin. These papillomas showed reduced growth and reduced progression to a more malignant phenotype, indicating that the effect of SPARC on tumorigenesis depends upon the transformation stage and/or tissue context. These data reinforce a growing number of observations in which SPARC has shown opposite effects on different tumor types/stages.

Sertoli cells as paracrine modulators of DNA synthesis in rat peritubular myoid cells in culture

To determine whether Sertoli cells influence DNA synthesis by rat peritubular myoid cells in vitro, the effects of Sertoli cells on [3H]thymidine incorporation by peritubular myoid cells in a coculture situation were examined. Incubation of testicular peritubular myoid cells with Sertoli cells in coculture induced a significant increase in [3H]thymidine incorporation by peritubular myoid cells. This indicates a cell-cell cooperation between Sertoli and peritubular myoid cells in the testis in terms of DNA synthesis. Secreted factors from Sertoli cells, as tested in a parabiotic culture situation, also increased [3H]thymidine incorporation by peritubular myoid cells. Moreover, in terms of total cellular protein, cocultures of Sertoli cells and peritubular myoid cells resulted in a significant increase when compared with the monocultures, and this coculture effect substituted for the stimulatory response of serum on peritubular myoid cell monoculture. This study investigated the cooperative role of Sertoli cells and peritubular myoid cells in paracrine regulation of testicular functions.

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.

Development of reverse phase protein microarrays for clinical applications and patient-tailored therapy

While genomics provide important information about the somatic genetic changes, and RNA transcript profiling can reveal important expression changes that correlate with outcome and response to therapy, it is the proteins that do the work in the cell. At a functional level, derangements within the proteome, driven by post-translational and epigenetic modifications, such as phosphorylation, is the cause of a vast majority of human diseases. Cancer, for instance, is a manifestation of deranged cellular protein molecular networks and cell signaling pathways that are based on genetic changes at the DNA level. Importantly, the protein pathways contain the drug targets in signaling networks that govern overall cellular survival, proliferation, invasion and cell death. Consequently, the promise of proteomics resides in the ability to extend analysis beyond correlation to causality. A critical gap in the information knowledge base of molecular profiling is an understanding of the ongoing activity of protein signaling in human tissue: what is activated and “in use” within the human body at any given point in time. To address this gap, we have invented a new technology, called reverse phase protein microarrays, that can generate a functional read-out of cell signaling networks or pathways for an individual patient obtained directly from a biopsy specimen. This “wiring diagram” can serve as the basis for both, selection of a therapy and patient stratification.

Drosophila 3′ UTRs are more complex than protein-coding sequences

The 3′ UTRs of eukaryotic genes participate in a variety of post-transcriptional (and some transcriptional) regulatory interactions. Some of these interactions are well characterised, but an undetermined number remain to be discovered. While some regulatory sequences in 3′ UTRs may be conserved over long evolutionary time scales, others may have only ephemeral functional significance as regulatory profiles respond to changing selective pressures. Here we propose a sensitive segmentation methodology for investigating patterns of composition and conservation in 3′ UTRs based on comparison of closely related species. We describe encodings of pairwise and three-way alignments integrating information about conservation, GC content and transition/transversion ratios and apply the method to three closely related Drosophila species: D. melanogaster, D. simulans and D. yakuba. Incorporating multiple data types greatly increased the number of segment classes identified compared to similar methods based on conservation or GC content alone. We propose that the number of segments and number of types of segment identified by the method can be used as proxies for functional complexity. Our main finding is that the number of segments and segment classes identified in 3′ UTRs is greater than in the same length of protein-coding sequence, suggesting greater functional complexity in 3′ UTRs. There is thus a need for sustained and extensive efforts by bioinformaticians to delineate functional elements in this important genomic fraction. C code, data and results are available upon request.