Identification of a novel consensus sequence at the cleavage site of the Lassa virus glycoprotein

The Lassa virus glycoprotein consists of an amino-terminal and a carboxy-terminal cleavage fragment designated GP-1 and GP-2, respectively, that are derived by proteolysis from the precursor GP-C. The membrane-anchored GP-2 obtained from purified virions of the Josiah strain revealed the N-terminal tripeptide GTF(262) when analyzed by Edman degradation. Upstream of this site, GP-C contains the tetrapeptide sequence RRLL(259), which is conserved in all Lassa virus isolates published to date. Systematic mutational analysis of vector-expressed GP-C revealed that the motif R-X (L/I/V)-L(259) (where X stands for L, I, or V) is essential for cleavage of the peptide bond between leucine(259) and glycine(260). This cleavage motif is homologous to the consensus sequence recognized by a novel class of cellular endoproteases which have so far not been implicated in the processing of viral glycoproteins.     

Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage.

Products generated by the digestion of human aggrecan with recombinant human stromelysin have been purified and analyzed by N-terminal sequencing and C-terminal peptide isolation. N-terminal analysis of chondroitin sulfate-bearing fragments revealed a clearly identifiable sequence initiating at residue Phe342 of human aggrecan, providing evidence for a cleavage site at the Asn341-Phe342 bond located within the interglobular domain. This cleavage site, which separates the G1 domain from the remainder of the molecule, was confirmed by isolation from the liberated G1 domain of a C-terminal tryptic peptide with the sequence YDAICYTGEDFVDIPEN (in which the C-terminal residue is Asn341). This peptide was also isolated from tryptic digests of hyaluronan-binding proteins (A1D4 samples) prepared by CsCl gradient centrifugation of extracts of mature human articular cartilages. Since these A1D4 samples contain G1 domain which accumulates as a result of aggrecan catabolism in vivo, these results clearly indicate that stromelysin cleaves the Asn341-Phe342 bond of human aggrecan in situ.     

TargetFinder: a software for antisense oligonucleotide target site selection based on MAST and secondary structures of target mRNA

SUMMARY: TargetFinder is a PC/Windows program for interactive effective antisense oligonucleotide (AO) selection based on mRNA accessible site tagging (MAST) and secondary structures of target mRNA. To make MAST result intuitive, both the alignment result and tag frequency profile is illustrated. As theoretical reference, secondary structure and single strand probability profile of target mRNA is also represented. All of these sequences and profiles are displayed in aligned mode, which facilitates identification of the accessible sites in target mRNA. Graphical, user-friendly interface makes TargetFinder a useful tool in AO target site selection. AVAILABILITY: The software is freely available at http://www.bioit.org.cn/ao/targetfinder.htm CONTACT: sqwang@nic.bmi.ac.cn.     

Selected non-steroidal anti-inflammatory drugs and their derivatives target gamma-secretase at a novel site. Evidence for an allosteric mechanism

Gamma-secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-beta (Abeta) peptides from processing intermediates of the beta-amyloid precursor protein. Because Abeta peptides are thought to be causative for Alzheimer's disease, inhibiting gamma-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of gamma-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Abeta(1-42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Abeta(1-42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective gamma-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the gamma-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of gamma (42)-cleavage over alternative cleavages in the beta-amyloid precursor protein and, more importantly, alternative substrates.     

New twists in understanding the fate of antisense oligodeoxynucleotide mRNA targets.

In this issue of Molecular Cell, Thoma et al. show that after a target mRNA is cleaved, upon treatment with an antisense oligodeoxynucleotide, the 3' cleavage product persists and is translated to produce an N-terminally truncated version of the protein encoded by the target mRNA.     

Identification and analysis of antisense RNA target regions of the human immunodeficiency virus type 1.

Antisense RNA, transcribed intracellularly from constitutive expression cassettes, inhibits the replication of the human immunodeficiency virus type 1 (HIV-1) as demonstrated by a quantitative microinjection assay in human SW480 cells. Infectious proviral HIV-1 DNA was co-microinjected together with a fivefold molar excess of plasmids expressing antisense RNA complementary to a set of ten different HIV-1 target regions. The most inhibitory antisense RNA expression plasmids were targeted against a 1 kb region within the gag open reading frame and against a 562 base region containing the coding sequences for the regulatory viral proteins tat and rev. Experimental evidence is presented that the antisense principle is the inhibitory mechanism in this assay system.     

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.     

Identification of a novel phosphorylation site in ataxin-1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease resulting from an expanded CAG repeat in the SCA1 gene that leads to an expanded polyglutamine tract in the gene product. Previous studies have demonstrated that serine at site 776 is phosphorylated [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Studies of ataxin-1 S776 and serine mutated to an alanine, A776, have also shown differential protein-protein interactions and reduced neurodegeneration [H.K. Chen, P. Fernandez-Funez, S.F. Acevedo, Y.C. Lam, M.D. Kaytor, M.H. Fernandez, A. Aitken, E.M. Skoulakis, H.T. Orr, J. Botas, H.Y. Zoghbi, Interaction of Akt_phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.]. However, mutation of the site serine 776 to an alanine did not abolish all phosphorylation of the protein ataxin-1, suggesting the presence of additional phosphorylation sites [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mutational analysis demonstrated a novel phosphorylation site at serine 239 of ataxin-1.     

Identification of N-terminal autodigestion target site in subtilisin ALP I.

Autodigestion of subtilisin ALP I (ALP I), secreted from the alkalophilic Bacillus sp. NKS-21 and its predicted amino acid sequence having about 60% identity with other alkaline subtilisins, was examined under alkaline conditions. At the alkaline pH of 12, ALP I was rapidly degraded, and almost no breakdown products were detectable. However, by incubating ALP I at 5 degrees C for an extended time, a couple of specific peptides (26.7 kDa and 25.6 kDa) were accumulated. Each of them was purified and amino acid sequences of these fragments were found. Both peptides appeared to start at Gly-19 of the mature sequence of ALP I.     

Identification of a novel interleukin-6 response element containing an Ets-binding site and a CRE-like site in the junB promoter.

Interleukin-6 (IL-6) activation of the immediate-early gene junB has been shown to require both a tyrosine kinase and an unknown 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7)-sensitive pathway. Here we report the identification and characterization of an IL-6 immediate-early response element in the junB promoter (designated JRE-IL6) in HepG2 cells. The JRE-IL6 element, located at -149 to -124, contains two DNA motifs, an Ets-binding site (EBS) (CAGGAAGC) and a CRE-like site (TGACGCGA). Functional studies using variously mutated JRE-IL6 elements showed that both motifs were necessary and sufficient for IL-6 response of the promoter. The EBS of the JRE-IL6 element (JEBS) appears to bind a protein in the Ets family or a related protein which could also form a major complex with the EBSs of the murine sarcoma virus long terminal repeat or human T-cell leukemia virus type 1 long terminal repeat. The CRE-like site appears to weakly bind multiple CREB-ATF family proteins. Despite the similarity in the structure between the JRE-IL6 element and the polyomavirus enhancer PyPEA3, composed of an EBS and an AP1-binding site and known to be activated by a variety of oncogene signals, JRE-IL6 could not be activated by activated Ha-Ras, Raf-1, or 12-O-tetradecanoylphorbol-13-acetate. We show that IL-6 activates JRE-IL6 through an H7-sensitive pathway that does not involve protein kinase C, cyclic AMP-dependent kinase, Ca(2+)- or calmodulin-dependent kinases, Ras, Raf-1, or NF-IL6 (C/EBP beta). The combination of JEBS and the CRE-like site appears to form the basis for the selective and efficient response of JRE-IL6 to IL-6 signals, but not to signals generated by activated Ha-Ras, Raf-1, or protein kinase C.     

Comparative study of two ribozymes and DNA-enzyme against the same RNA target.

Two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme were designed to cleave a same RNA target, the same site of the rat complement regulatory factor 512 antigen mRNA. The kinetic properties of these RNA-cleaving enzymes were measured and compared under the same conditions, using multiple turnover kinetics and competition kinetics. The catalytic efficiencies of these enzymes, and also the order of these enzymes will be discussed.     

Selective inhibition of normal murine myelopoiesis in vitro by a Hox 2.3 antisense oligodeoxynucleotide.

Multiple homeobox genes are expressed in haematopoietic cell lineages and their expression is cell-type specific. Thus we hypothesized that certain homeobox genes may play an important role in the process of haematopoiesis. To prove that issue, normal murine bone marrow cells were stimulated with appropriate Colony Stimulating Factors in the presence of mouse homeobox gene (Hox 2.3) sense or antisense oligodeoxynucleotides and the effects on the haematopoietic colony formation were examined. Treatment of the cells to Hox 2.3 antisense oligodeoxynucleotides led to a selective inhibition of myeloid colony formation, both in size and in numbers, but without significant effect on erythroid and megakaryocytic haematopoiesis. Exposure to Hox 2.3 sense oligodeoxynucleotides (no-oligomers), had no such effect. It was further showed that inhibition of myelopoiesis by Hox 2.3 antisense oligodeoxynucleotides was dependent on the differentiation stage of target cells. These findings demonstrated that Hox 2.3 gene plays a critical role in regulating normal murine myelopoiesis.     

Identification of ATP-synthase as a novel intracellular target for microcystin-LR

Microcystins (MCs) are a group of closely related cyclic heptapeptides produced by a variety of common cyanobacteria. These are potent and highly specific hepatotoxins, the toxicity of which is based upon their inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases. Apart from protein phosphatases, it is not known whether these phosphatase-inhibiting peptides could bind any other cellular proteins. We wanted to determine whether any possible unknown MC-adducts could explain the apoptotic effects observed at high concentrations of MCs. The question of other possible cellular proteins binding to MCs is also relevant when these compounds are employed for affinity purification of protein phosphatases. In MC-treated cell lysates, antibodies to MC recognized three protein adducts of 35-37 and 55 kD. By immunochemical and proteomics approaches, these proteins were identified as the catalytic subunits of type-1 and type-2A protein phosphatases and the ATP-synthase beta-subunit. The latter target could be associated with the suggested apoptosis-inducing potential of MCs.     

Identification of the most accessible sites to ribozymes on the hepatitis C virus internal ribosome entry site

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.