Traditional GFP-type cyclization and unexpected fragmentation site in a purple chromoprotein from Anemonia sulcata, asFP595

The purple chromoprotein (asFP595) from Anemonia sulcata belongs to the family of green fluorescent protein (GFP). Absorption and emission spectra of asFP595 are similar to those of a number of recently cloned GFP-like red proteins of the DsRed subfamily. The earlier proposed asFP595 chromophore structure [Martynov, V. I.; et al. (2001) J. Biol. Chem. 276, 21012-21016] was postulated to result from an "alternative cyclization" giving rise to a pyrazine-type six-membered heterocycle. Here we report that the asFP595 chromophore is actually very close in chemical structure to that of zFP538, a yellow fluorescent protein [Zagranichny, V. E.; et al. (2004) Biochemistry 43, 4764-4772]. NMR spectroscopic studies of four chromophore-containing peptides (chromopeptides) isolated under mild conditions from enzymatic digests of asFP595 and one chromopeptide obtained from DsRed revealed that all of them contain a p-hydroxybenzylideneimidazolinone moiety formed by Met-65/Gln-66, Tyr-66/67, and Gly-67/68 of asFP595/DsRed, respectively. Two asFP595 chromopeptides are proteolysis products of an isolated full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other asFP595 chromopeptides were isolated as proteolysis products of the purified chromophore-containing C-terminal fragment. One of these has an oxo group at Met-65 C(alpha) and is a hydrolysis product of another one, with the imino group at Met-65 C(alpha). The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain cleavage at a very unexpected site, the former peptide bond between Cys-64 C' and Met-65 N(alpha). Our data strongly suggest that both zFP538 and asFP595 could be attributed to the DsRed subfamily of GFP-like proteins.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

Scanning cytometry with a LEAP: laser-enabled analysis and processing of live cells in situ.

BACKGROUND: Scanning cytometry now has many of the features (and power) of multiparameter flow cytometry while keeping its own advantages as an imaging technology. Modern instruments combine capabilities of scanning cytometry with the ability to manipulate cells. A new technology, called LEAP (laser-enabled analysis and processing), offers a unique combination of capabilities in cell purification and selective macromolecule delivery (optoinjection). METHODS: LEAP-mediated cell purification and optoinjection effects were assessed in model experiments using adherent and suspension cell types and cell mixtures plated and processed at different densities. Optoinjection effects were visualized by delivering fluorescent dextrans into cells. Results were analyzed using the LEAP instrument's own imaging system as well as by fluorescence and confocal microscopy. RESULTS: Live cell samples (adherent and suspension) could be purified to 90-100% purity with 50-90% yield, causing minimal cell damage depending on the cell type and plating density. Nearly one hundred percent of the targeted cells of all cell types examined could be successfully optoinjected with dextrans of 3-70 kDa, causing no visual damage to the cells. Indirect optoinjection effects were observed on untargeted cells within 5-60 microm to targeted areas under conditions used here. CONCLUSIONS: LEAP provides solutions in cell purification and targeted macromolecule delivery for traditional and challenging applications where other methods fall short.     

The brain is getting ready for dinner

Every evening, as we get ready for dinner, in addition to the routine behaviors of preparing the meal itself, we also prepare our bodies to cope with the upcoming meal. This could take the form of making restaurant reservations, changing into appropriate attire, washing hands, priming ourselves with an aperitif, or even consciously avoiding snacks as the meal approaches. A study by Johnstone and colleagues in this issue of Cell Metabolism (Johnstone et al., 2006) provides evidence that in parallel to our learned preparatory behaviors, our central nervous system is going through comparable motions as it gets ready for the anticipated meal.     

An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands

Oxidative cell damage is involved in the pathogenesis of atherosclerosis, cancer, diabetes and other diseases. Uptake of fruit juice with especially high content of antioxidant flavonoids/polyphenols, might reduce oxidative cell damage. Therefore, an intervention study was performed with a red mixed berry juice [trolox equivalent antioxidative capacity (TEAC): 19.1 mmol/L trolox] and a corresponding polyphenol-depleted juice (polyphenols largely removed, TEAC 2.4 mmol/L trolox), serving as control. After a 3-week run-in period, 18 male probands daily consumed 700 mL juice, and 9 consumed control juice, in a 4-week intervention, followed by a 3-week wash-out. Samples were collected weekly to analyze DNA damage (comet assay), lipid peroxidation (plasma malondialdehyde: HPLC/fluorescence; urinary isoprostanes: GC-MS), blood glutathione (photometrically), DNA-binding activity of nuclear factor-kappaB (ELISA) and plasma carotenoid/alpha-tocopherol levels (HPLC-DAD). During intervention with the fruit juice, a decrease of oxidative DNA damage (p<5x10(-4)) and an increase of reduced glutathione (p<5x10(-4)) and of glutathione status (p<0.05) were observed, which returned to the run-in levels in the subsequent wash-out phase. The other biomarkers were not significantly modulated by the juice supplement. Intervention with the control juice did not result in reduction of oxidative damage. In conclusion, the fruit juice clearly reduces oxidative cell damage in healthy probands.     

All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding

The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.Hepatitis C virus (HCV) is a human pathogen which chronically infects nearly 3% of the world''s population (36, 37). Persistent infection, in 80% of cases, leads to chronic hepatitis which can progress to liver cirrhosis and, in the worst cases, hepatocellular carcinoma (37). Current therapies lack specificity and efficacy due largely to an incomplete understanding of the complex molecular mechanisms of virus infectivity, RNA replication, and assembly (4, 36). HCV is a member of the Flaviviridae family of enveloped viruses (30), with a positive-sense RNA genome of ∼9.6 kb consisting of a single open reading frame (ORF) that encodes 10 structural and nonstructural viral proteins (3, 16, 25). Cap-independent translation of the ORF (29) yields a large polyprotein of approximately 3,000 amino acid residues that is cleaved co- and posttranslationally by host and viral proteases into 10 mature virus proteins; these cleavage products are ordered from the amino to the carboxy terminus as follows: core (C), envelope proteins 1 and 2 (E1 and E2), p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (3, 16, 25). At the flanking ends of the genome are two highly conserved untranslated regions (UTRs). The 5′ UTR is highly structured and consists of the internal ribosome entry site (IRES), which is important for the initiation of cap-independent translation of the polyprotein (29). The 3′ UTR consists of a short genotype-specific variable region, a tract of variable length comprising solely pyrimidine residues (predominantly U), and a conserved 98-nucleotide sequence, known as the X region, containing three stem-loops (13, 23) (Fig. ​(Fig.1A).1A). The 3′ UTR is the initiation site for the synthesis of the negative-strand RNA during viral replication (13) and is involved in translational regulation.Open in a separate windowFIG. 1.The HCV 3′ UTR RNA. (A) The positive-strand 3′ UTR consists of three distinct regions, i.e., a short genotype-specific variable region, a polypyrimidine tract [poly(U/UC)] of variable length, and a conserved 98-nucleotide sequence known as the X region containing three stable stem-loops. The predicted structure of the genotype 1b 3′ UTR is shown. (B) Left panel, the integrities of in vitro-transcribed radiolabeled full-length 3′ UTR RNAs of genotypes 1b (nucleotides 9375 to 9595) and 2a (nucleotides 9443 to 9678) and the poly(U/UC) (nucleotides 9406 to 9497) and X region (nucleotides 9498 to 9595) of genotype 1b are shown on denaturing polyacrylamide gels. Right panel, the integrities of in vitro-transcribed radiolabeled RNAs comprising the 3′-terminal NS5B-coding region plus the 3′ UTR RNAs of genotypes 1b (nucleotides 9136 to 9595) and 2a (nucleotides 9204 to 9678) (KL-3′ UTR) are shown on denaturing polyacrylamide gels.HCV RNA replication occurs on membranous structures derived from the endoplasmic reticulum (ER) in a complex that includes host cell factors as well as viral nonstructural proteins, including NS5B, the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome in vivo and in vitro (2, 25, 30). Initiation of the synthesis of the negative-strand RNA is thought to occur upon recognition and specific binding of the NS5B polymerase to the 3′ UTR of the genomic RNA (2, 16, 26). This replication activity and template specificity of NS5B in vivo are dependent, however, on the presence of the other nonstructural proteins, such as the proteases NS2 and NS3, which are required for polyprotein processing and helicase activity, and the multifunctional protein NS5A (16).NS5A is a proline-rich phosphoprotein that is absolutely required for viral replication and is also involved in virus particle assembly (9, 10, 20, 22, 35). Its specific function in the latter process is, however, still unknown. NS5A is membrane associated due to the presence of an N-terminal amphipathic helix that serves as a membrane anchor allowing association with ER-derived membranes (Fig. ​(Fig.2)2) (24, 27). The cytoplasmic portion of NS5A is organized into three domains that are separated by low-complexity sequences (Fig. ​(Fig.2A)2A) (20). The X-ray crystal structure of domain I has revealed that it is a zinc binding domain which forms a homodimer with contacts at the N-terminal ends of the molecules; the resultant large, basic groove at the dimeric interface has been proposed to be involved in RNA binding during viral replication (17, 33). NS5A has also been shown to interact with uridylate and guanylate-rich RNA and to bind to the 3′ ends of the HCV positive- and negative-strand RNAs (8). These observations suggest that NS5A may specifically interact with the large U/G stretches in the IRES of the 5′ UTR, implying a role in HCV translation and genome multiplication, while its interactions with the polypyrimidine tract of the 3′ UTR suggest that NS5A may affect the efficiency of RNA synthesis by NS5B (8, 28, 32). The reported interactions with both flanking regions of the HCV genome imply that NS5A may play a role in the switch between translation and replication that must occur during the viral life cycle (8).Open in a separate windowFIG. 2.Domain structure and expression of HCV NS5A. (A) Schematic diagram of the functional domains of NS5A and design of the constructs used in the study (genotype 1b NS5A protein numbering). The N-terminal amphipathic helix of NS5A (black box) is responsible for the interaction of NS5A with membranes. NS5A is organized into three domains that are separated by low-complexity sequences, indicated by black boxes. The NS5A constructs used all lacked the N-terminal amphipathic helix and were designed to include an N-terminal Strep tag and a C-terminal hexahistidine tag. (B and C) SDS-PAGE and Western blot analysis of the NS5A(ΔAH) and NS5A domain constructs purified by nickel affinity and Streptactin tag affinity chromatography. Coomassie brilliant blue-stained gels and Western blots (WB) using anti-NS5A antibodies for NS5A proteins of genotype 1b strain J4 (B) and genotype 2a strain JFH-1 (C) are shown.Among HCV genotypes, domains II and III are less well conserved than domain I (34). By mutational analysis, domain II, along with domain I, has been attributed to the replicase activity of NS5A (12). Contrastingly, domain III has been shown to be dispensable for RNA replication, and large heterologous insertions and deletions in this region can be tolerated, maintaining RNA replication (34). It has been shown, however, that these insertions and deletions within domain III do have an impact on virus particle assembly, highlighting the critical role of domain III NS5A in the viral life cycle (1, 10). Recent nuclear magnetic resonance (NMR) studies of domains II and III of NS5A revealed that they both adopt a natively unfolded state (6, 14, 15). The high degree of disorder and flexibility observed in these domains may contribute to the promiscuity of NS5A, which has been shown to interact with a variety of biological partners essential for NS5A function and virus persistence (11, 18, 19, 21, 31). In addition, regions within domains I and II of NS5A interact with NS5B, stimulating the in vitro activity of the polymerase and supporting the hypothesis that NS5A has a role in the modulation of RNA replication (28, 32).In this study, we have investigated in detail the RNA binding properties of NS5A. We have mapped the RNA binding regions of NS5A using bacterially expressed deletion constructs of NS5A and have assayed their binding affinity for HCV positive-strand 3′ UTR RNA. In addition, we provide evidence that the RNA binding activity of NS5A is specific and that NS5A interacts preferentially with the polypyrimidine region of the 3′ UTR.     

Posttranslational Modification of the NH2-terminal Region of CXCL5 by Proteases or Peptidylarginine Deiminases (PAD) Differently Affects Its Biological Activity

Posttranslational modifications, e.g. proteolysis, glycosylation, and citrullination regulate chemokine function, affecting leukocyte migration during inflammatory responses. Here, modification of CXCL5/epithelial cell-derived neutrophil-activating protein-78 (ENA-78) by proteases or peptidylarginine deiminases (PAD) was evaluated. Slow CXCL5(1–78) processing by the myeloid cell marker aminopeptidase N/CD13 into CXCL5(2–78) hardly affected its in vitro activity, but slowed down the activation of CXCL5 by the neutrophil protease cathepsin G. PAD, an enzyme with a potentially important function in autoimmune diseases, site-specifically deiminated Arg⁹ in CXCL5 to citrulline, generating [Cit⁹]CXCL5(1–78). Compared with CXCL5(1–78), [Cit⁹]CXCL5(1–78) less efficiently induced intracellular calcium signaling, phosphorylation of extracellular signal-regulated kinase, internalization of CXCR2, and in vitro neutrophil chemotaxis. In contrast, conversion of CXCL5 into the previously reported natural isoform CXCL5(8–78) provided at least 3-fold enhanced biological activity in these tests. Citrullination, but not NH₂-terminal truncation, reduced the capacity of CXCL5 to up-regulate the expression of the integrin α-chain CD11b on neutrophils. Truncation nor citrullination significantly affected the ability of CXCL5 to up-regulate CD11a expression or shedding of CD62L. In line with the in vitro results, CXCL5(8–78) and CXCL5(9–78) induced a more pronounced neutrophil influx in vivo compared with CXCL5(1–78). Administration of 300 pmol of either CXCL5(1–78) or [Cit⁹]CXCL5(1–78) failed to attract neutrophils to the peritoneal cavity. Citrullination of the more potent CXCL5(9–78) lowers its chemotactic potency in vivo and confirms the tempering effect of citrullination in vitro. The highly divergent effects of modifications of CXCL5 on neutrophil influx underline the potential importance of tissue-specific interactions between chemokines and PAD or proteases.     

System xc? and Thioredoxin Reductase 1 Cooperatively Rescue Glutathione Deficiency

GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in γ-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation.