39 resultados para Gfp-like Proteins
Resumo:
The schistosome blood flukes are some of the largest global causes of parasitic morbidity. Further study of the specific antibody response during schistosomiasis may yield the vaccines and diagnostics needed to combat this disease. Therefore, for the purposes of antigen discovery, sera and antibody-secreting cell (ASC) probes from semi-permissive rats and sera from susceptible mice were used to screen a schistosome protein microarray. Following Schistosoma japonicum infection, rats had reduced pathology, increased antibody responses and broader antigen recognition profiles compared with mice. With successive infections, rat global serological reactivity and the number of recognized antigens increased. The local antibody response in rat skin and lung, measured with ASC probes, increased after parasite migration and contributed antigen-specific antibodies to the multivalent serological response. In addition, the temporal variation of anti-parasite serum antibodies after infection and reinfection followed patterns that appear related to the antigen driving the response. Among the 29 antigens differentially recognized by the infected hosts were numerous known vaccine candidates, drug targets and several S. japonicum homologs of human schistosomiasis resistance markers-the tegument allergen-like proteins. From this set, we prioritized eight proteins that may prove to be novel schistosome vaccine and diagnostic antigens.
Resumo:
Synucleins are small proteins that are highly expressed in brain tissue and are localised at presynaptic terminals in neurons. alpha-Synuclein has been identified as a component of intracellular fibrillar protein deposits in several neurodegenerative diseases, and two mutant forms of alpha-synuclein have been associated with autosomal-dominant Parkinson's Disease. A fragment of alpha-synuclein has also been identified as the non-Abeta component of Alzheimer's Disease amyloid. In this review we describe some structural properties of alpha-synuclein and the two mutant forms, as well as alpha-synuclein fragments, with particular emphasis on their ability to form beta-sheet on ageing and aggregate to form amyloid-like fibrils. Differences in the rates of aggregation and morphologies of the fibrils formed by alpha-synuclein and the two mutant proteins are highlighted. Interactions between alpha-synuclein and other proteins, especially those that are components of amyloid or Lewy bodies, are considered. The toxicity of alpha-synuclein and related peptides towards neurons is also discussing in relation to the aetiology of neurodegenerative diseases.
Resumo:
Little is known about the origin of basal-like breast cancers, an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues, and p63 expression may be a marker of basal-like breast cancers. Here we report that Delta Np63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless, we found that BRCA1 interacts with the specific p63 isoform Delta Np63 gamma along with transcription factor isoforms AP-2 alpha and AP-2 gamma. BRCA1 required Delta Np63 gamma and AP-2 gamma to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the Delta Np63 isoforms. In mammary stem/progenitor cells, siRNA- mediated knockdown of Delta Np63 expression resulted in genomic instability, increased cell proliferation, loss of DNA damage checkpoint control, and impaired growth control. Together, our findings establish that transcriptional upregulation of Delta Np63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-Delta Np63 signaling are key events in the pathogenesis of basal-like breast cancer. Cancer Res; 71( 5); 1933-44. (c) 2011 AACR.
Resumo:
Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.
Resumo:
An alternative method for monitoring protein-protein interactions in Saccharomyces cerevisiae has been developed. It relies on the ability of two fragments of enhanced green fluorescent protein (EGFP) to reassemble and fluoresce when fused to interacting proteins. Since this fluorescence can be detected in living cells, simultaneous detection and localisation of interacting pairs is possible. DNA sequences encoding N- and C-terminal EGFP fragments flanked by sequences from the genes of interest were transformed into S. cerevisicie JPY5 cells and homologous recombination into the genome verified by PCR. The system was evaluated by testing known interacting proteins: labelling of the phosphofructokinase subunits, Pfk1p and Pfk2p, with N- and C-terminal EGFP fragments, respectively, resulted in green fluorescence in the cytoplasm. The system works in other cellular compartments: labelling of Idh1p and Idh2p, (mitochondrial matrix), Sdh3p and Sdh4p (mitochondrial membrane) and Pap2p and Mtr4p (nucleus) all resulted in fluorescence in the appropriate cellular compartment. (c) 2008 Elsevier Inc. All rights reserved.
Resumo:
Glucagon-like peptide-1(7-36)amide (tGLP-1) is an important insulin-releasing hormone of the enteroinsular axis which is secreted by endocrine L-cells of the small intestine following nutrient ingestion. The present study has evaluated tGLP-1 in the intestines of normal and diabetic animal models and estimated the proportion present in glycated form. Total immunoreactive tGLP-1 levels in the intestines of hyperglycaemic hydrocortisone-treated rats, streptozotocin-treated mice and ob/ob mice were similar to age-matched controls. Affinity chromatographic separation of glycated and non-glycated proteins in intestinal extracts followed by radioimmunoassay using a fully crossreacting anti-serum demonstrated the presence of glycated tGLP-1 within the intestinal extracts of all control animals (approximately 19%., of total tGLP-1 content). Chemically induced and spontaneous animal models of diabetes were found to possess significantly greater levels of glycated tGLP-1 than controls, corresponding to between 24-71% of the total content. These observations suggest that glycated tGLP-1 may be of physiological significance given that such N-terminal modification confers resistance to DPP IV inactivation and degradation, extending the very short half-life (
Resumo:
In common with other terrestrial flatworms, the mucus produced by Artioposthia triangulata may have a number of functions, including protection from environmental factors and from predators, and it provides the flatworm with lubrication for movement and adhesion. No previous work has been carried out on the characterization of proteins present in the mucus of A. triangulata and this study was a preliminary investigation of the mucus. Mucus was analysed by SDS-polyacrylamide gel electrophoresis, biotinylated peptide affinity probes and the API ZYM enzyme detection kit. Results have revealed the presence of at least 40 polypeptides in the mucus and further studies with biotinylated probes have characterised one of them as a chymotrypsin-like serine protease. (C) 1998 Elsevier Science Inc.
Resumo:
Anthrax lethal toxin (LeTx) induces rapid cell death of RAW246.7 macrophages. We recently found that a small population of these macrophages is spontaneously and temporally refractory to LeTx-induced cytotoxicity. Analysis of genome-wide transcripts of a resistant clone before and after regaining LeTx sensitivity revealed that a reduction of two closely related mitochondrial proteins, Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) and Bnip3-like (Bnip3L), correlates with LeTx resistance. Down-regulation of Bnip3 and Bnip3L was also found in "toxin-induced resistance" whereby sublethal doses of LeTx induce resistance to subsequent exposure to cytolytic toxin doses. The role of Bnip3 and Bnip3L in LeTx-induced cell death was confirmed by showing that overexpression of either Bnip3 or Bnip3L rendered the resistant cells susceptible to LeTx, whereas down-regulation of Bnip3 and Bnip3L in wild-type macrophages conferred resistance. The down-regulation of Bnip3 and Bnip3L mRNAs by LeTx occurred at both transcriptional and mRNA stability levels. Inhibition of the p38 pathway by lethal factor was responsible for the destabilization of Bnip3/Bnip3L mRNAs as confirmed by showing that p38 inhibitors stabilized Bnip3 and Bnip3L mRNAs and conferred resistance to LeTx cytotoxicity. Therefore, Bnip3/Bnip3L play a crucial role in LeTx-induced cytotoxicity, and down-regulation of Bnip3/Bnip3L is a mechanism of spontaneous or toxin-induced resistance of macrophages.
Resumo:
An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.
Resumo:
We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4(+) T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.-Robinson, M. W., Alvarado, R., To, J., Hutchinson, A. T., Dowdell, S. N., Lund, M., Turnbull, L., Whitchurch, C. B., O'Brien, B. A., Dalton, J. P., Donnelly, S. A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase.
Resumo:
To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.
Resumo:
Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IkappaBalpha-dependent NF-kappaBeta pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.
Resumo:
actin-depolymerising factor (ADF)/cofilin group of proteins are stimulus-responsive actin-severing proteins, members of which are regulated by reversible phosphorylation. The phosphorylation site on the maize ADF, ZmADF3, is Ser-6 but the kinase responsible is unknown [Smertenko et al,, Plant J. 14 (1998) 187-193]. We have partially purified the ADF kinase(s) and found it to be calcium-regulated and inhibited by N-(6-aminohesyl)-[H-3]5-chloro-1-naphthalenesulphonamide. Immunoblotting reveals that calmodulin-like domain protein kinase(s) (CDPK) are enriched in the purified preparation and addition of anti-CDPK to in vitro phosphorylation assays results in the inhibition of ADF phosphorylation, These data strongly suggest that plant ADP is phosphorylation by CDPK(s), a class of protein kinases unique to plants and protozoa. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.