RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

Characterization of the Entamoeba histolytica Ornithine Decarboxylase-Like Enzyme

Background: The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme. Methodology and Results: To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of similar to 1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size similar to 46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids-aspartate-332, aspartate-361, and tyrosine-323-by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity. Conclusion: To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.

A beta-adrenergic receptor kinase-like enzyme is involved in olfactory signal termination.

We have previously shown that second-messenger-dependent kinases (cAMP-dependent kinase, protein kinase C) in the olfactory system are essential in terminating second-messenger signaling in response to odorants. We now document that subtype 2 of the beta-adrenergic receptor kinase (beta ARK) is also involved in this process. By using subtype-specific antibodies to beta ARK-1 and beta ARK-2, we show that beta ARK-2 is preferentially expressed in the olfactory epithelium in contrast to findings in most other tissues. Heparin, an inhibitor of beta ARK, as well as anti-beta ARK-2 antibodies, (i) completely prevents the rapid decline of second-messenger signals (desensitization) that follows odorant stimulation and (ii) strongly inhibits odorant-induced phosphorylation of olfactory ciliary proteins. In contrast, beta ARK-1 antibodies are without effect. Inhibitors of protein kinase A and protein kinase C also block odorant-induced desensitization and phosphorylation. These data suggest that a sequential interplay of second-messenger-dependent and receptor-specific kinases is functionally involved in olfactory desensitization.

Cotranscriptional role for Arabidopsis DICER-LIKE 4 in transcription termination.

Transcription termination is emerging as an important component of gene regulation necessary to partition the genome and minimize transcriptional interference. We have discovered a role for the Arabidopsis RNA silencing enzyme DICER-LIKE 4 (DCL4) in transcription termination of an endogenous Arabidopsis gene, FCA. DCL4 directly associates with FCA chromatin in the 3' region and promotes cleavage of the nascent transcript in a domain downstream of the canonical polyA site. In a dcl4 mutant, the resulting transcriptional read-through triggers an RNA interference–mediated gene silencing of a transgene containing the same 3' region. We conclude that DCL4 promotes transcription termination of the Arabidopsis FCA gene, reducing the amount of aberrant RNA produced from the locus.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification and characterization of jararassin-I, a thrombin-like enzyme from Bothrops jararaca snake venom

A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bbeta chain of fibrinogen while the Aalpha chain and gammachain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bbeta chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mmx0.2 mmx0.2 mm) and used for X-ray diffraction experiments.

Toll-like receptor 2 knockdown modulates Interleukin (IL)-6 and IL-8 but not Stromal Derived Factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Functional Diversification of Dicer-like Proteins and Small RNAs Required for Genome Sculpting.

In eukaryotes, small RNAs (sRNAs) have key roles in development, gene expression regulation, and genome integrity maintenance. In ciliates, such as Paramecium, sRNAs form the heart of an epigenetic system that has evolved from core eukaryotic gene silencing components to selectively target DNA for deletion. In Paramecium, somatic genome development from the germline genome accurately eliminates the bulk of typically gene-interrupting, noncoding DNA. We have discovered an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later during Paramecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs ("scan" RNAs [scnRNAs]) in targeting DNA for elimination. We show that whole-genome duplications have facilitated successive differentiations of Paramecium Dicer-like proteins, leading to cooperation between Dcl2 and Dcl3 to produce scnRNAs and to the production of iesRNAs by Dcl5. These innovations highlight the ability of sRNA systems to acquire capabilities, including those in genome development and integrity.

Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20), patients with other parasitic infections (n=87) and patients with malignancies (n=121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

STRUCTURE OF CUPIENNIUS SALEI VENOM HYALURONIDASE Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. FUNCTION OF VENOM HYALURONIDASES Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1–42 maturase

The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2’s subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin. was purified by DEAF A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH2-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg, The fibrinopeptides released, identified by HPLC consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it. indicating it is venom serine protease. (C) 2000 Elsevier Science Ltd. All rights reserved.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.