A new DEAD-box helicase ATP-binding protein (OsABP) from rice is responsive to abiotic stress

The DEAD-box RNA helicase family comprise enzymes that participate in every aspect of RNA metabolism, associated with a diverse range of cellular functions including response to abiotic stress. In the present study, we report on the identification of a new DEAD-box helicase ATP-binding protein (OsABP) from rice which is upregulated in response e to multiple abiotic stress treatments  including NaCl, dehydration, ABA, blue and red light. It possesses an ORF of 2772 nt, encoding a protein of 923 aa, which contains the DEAD and helicase C-terminal domains, along with the nine conserved motifs specific to DEAD-box helicases. The in silico putative interaction with other proteins showed that OsABP interacts with proteins involved in RNA metabolism, signal transduction or stress response. These results imply that OsABP might perform important functions in the cellular response to specific abiotic stress.     

A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response

Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.     

Using or abusing: viruses and the cellular DNA damage response

During infection, viruses attempt to hijack the cell while the host responds with various defense systems. Traditional defenses include the interferon response and apoptosis, but recent work suggests that this antiviral arsenal also includes the cellular DNA damage response machinery. The observation of interactions between viruses and cellular DNA repair proteins has not only uncovered new complexities of the virus-host interaction but is also reinforcing the view that viruses can reveal key regulators of cellular pathways through the proteins they target.     

A proteome analysis of the cadmium response in Saccharomyces cerevisiae

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.     

Alterations of A549 lung cell gene expression in response to biochemical toxins

Health risks associated with the inhalation of potentially toxic materials have been a topic of great public concern. In vitro cellular analyses can provide mechanistic information on the molecular-level responses of lung-derived cell lines to a variety of these hazards. This understanding may be used to develop methods to reduce the damage from such toxins or to detect early stages of their effects. Here we describe an evaluation of the alterations in gene expression of an immortalized lung cell line (A549, human type II epithelia) to a variety of inhalation health hazards including etoposide, gliotoxin, streptolysin O, methyl methansesulfonate (MMS), and Triton X-100. The A549 cells display a dose–response relationship to each toxin with initial responses including alterations in metabolic activity, increases in membrane permeability, and initiation of response genes. In general, membrane-damaging agents (streptolysin O and Triton X-100) induce production of new ion channel proteins, structural proteins, and metabolic enzymes. Gliotoxin impacted the metabolic machinery, but also altered ion channels. Etoposide and MMS caused alterations in the cell cycle, induced DNA repair enzymes, and initiated apoptotic pathways, but MMS also induced immune response cascades. The mechanism of cell response to each toxin is supported by physiological analyses that indicated a fairly slow initiation of cell response to all compounds tested, except for Triton, which caused rapid decline in cell function due to solubilization of the cell membrane. However, Triton does induce production of a number of cell membrane-associated proteins and so its effects at low concentrations are likely translated throughout the cell. Together these results indicate a broader array of cellular responses to each of the test toxins than have previously been reported.     

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.     

Genetically encoded optical sensors of neuronal activity and cellular function

Fluorescent proteins (FPs) have been engineered to produce an optical report in response to cellular signals. FP fluorescence can be made directly sensitive to the chemical environment, via specific mutations of or around the chromophore. Alternatively, FPs can be made indirectly sensitive to cellular signals by their fusion to 'detector' proteins that respond to specific cellular signals with structural rearrangements that act on the FP to alter fluorescence. These optical sensors of membrane voltage, neurotransmitter release, and intracellular messengers, including powerful new sensors of Ca(2+), cyclic nucleotides and nitric oxide, are likely to provide new insights into the workings of cellular signals and of information processing in neural circuits.     

Protein profile in HBx transfected cells: A comparative iTRAQ-coupled 2D LC-MS/MS analysis

The x protein of HBV (HBx) has been involved in the development of hepatocellular carcinoma (HCC), with a possible link to individual genotypes. Nevertheless, the underlying mechanism remains obscure. In this study, we aim to identify the HBx-induced protein profile in HepG2 cells by LC-MS/MS proteomics analysis. Our results indicated that proteins were differentially expressed in HepG2 cells transfected by HBx of various genotypes. Proteins associated with cytoskeleton were found to be either up-regulated (MACF1, HMGB1, Annexin A2) or down-regulated (Lamin A/C). These may in turn result in the decrease of focal adhesion and increase of cell migration in response to HBx. Levels of other cellular proteins with reported impact on the function of extracellular matrix (ECM) proteins and cell migration, including Ca²⁺-binding proteins (S100A11, S100A6, and S100A4) and proteasome protein (PSMA3), were affected by HBx. The differential protein profile identified in this study was also supported by our functional assay which indicated that cell migration was enhanced by HBx. Our preliminary study provided a new platform to establish a comprehensive cellular protein profile by LC-MS/MS proteomics analysis. Further downstream functional assays, including our reported cell migration assay, should provide new insights in the association between HCC and HBx.     

A new Smurf in the village.

TGF-beta signaling is modulated by Smurfs, E3-ubiquitin ligases that selectively target the receptors and Smad proteins for degradation. New evidence from Drosophila suggests that Smurfs regulate the amplitude and the duration of the cellular response to signaling in vivo.     

Mass spectrometry reveals specific and global molecular transformations during viral infection

Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.     

Interferon Induction by Viruses

Interferons are proteins of cellular origin capable of conferring virus resistance to vertebrate cells. Most cells do not produce interferons except in response to proper stimulation. Clearly, the stimulation of interferon production encompasses two phenomena. When stimulated, some cell systems produce their interferons by synthesizing new proteins. Other cell systems do not require the synthesis of new proteins to produce interferons, and still other cell systems may produce interferons by both means. Before much can be learned from the detailed study of the nature of the molecules which stimulate interferons, the type of phenomenon which the stimulus induces must be identified. Chick embryo tissues apparently make interferons by synthesizing new proteins. Many viruses stimulate interferon production in chick embryo tissues. Data available suggest that neither the protein nor nucleic acid moieties of the added virions act as inducing molecules. Also, double-stranded replicative form is probably not responsible. It is suggested that the inducer molecule may be cellular in nature and may be produced in response to a wide variety of insults among which are viral infections.     

A genetic screening strategy identifies novel regulators of the proteostasis network

A hallmark of diseases of protein conformation and aging is the appearance of protein aggregates associated with cellular toxicity. We posit that the functional properties of the proteostasis network (PN) protect the proteome from misfolding and combat the proteotoxic events leading to cellular pathology. In this study, we have identified new components of the proteostasis network that can suppress aggregation and proteotoxicity, by performing RNA interference (RNAi) genetic screens for multiple unrelated conformationally challenged cytoplasmic proteins expressed in Caenorhabditis elegans. We identified 88 suppressors of polyglutamine (polyQ) aggregation, of which 63 modifiers also suppressed aggregation of mutant SOD1(G93A). Of these, only 23 gene-modifiers suppressed aggregation and restored animal motility, revealing that aggregation and toxicity can be genetically uncoupled. Nine of these modifiers were shown to be effective in restoring the folding and function of multiple endogenous temperature-sensitive (TS) mutant proteins, of which five improved folding in a HSF-1-dependent manner, by inducing cytoplasmic chaperones. This triage screening strategy also identified a novel set of PN regulatory components that, by altering metabolic and RNA processing functions, establish alternate cellular environments not generally dependent on stress response activation and that are broadly protective against misfolded and aggregation-prone proteins.