The effect of tunicamycin on the protease activity of GP63 from Leishmania major

The protease activity of gp63 from L. major was studied in relation to tunicamycin induced N-deglycosylation. It was found that after tunicamycin treatment, a N-deglycosylated product of gp63 with protease activity is present at the cell surface of Leishmania promastigote.     

Insights from the analysis of a predicted model of gp63 in Leishmania donovani

Leishmaniasis is a protozoal disease of human that occurs in most parts of the world. By considering the progress of bioinformatics in molecular modeling, major surface glycoprotein of Leishmania donovani (gp63) structure was modeled using homology modeling with high accuracy based on the X-ray crystal structure of the Leishmania major gp63 as a template, and then analyzed 3D structure of gp63 which can reveal exact facts about its structure and interaction. The objective of this study was to find folding and three dimensional structure of the gp63 as potent antigen for human. In this project, we applied the theory of evolution method, including comparative modeling and threading. This study presented a simple protocol for rapid and precise finding 3D structure of gp63 and investigation of its structural properties. The translated amino acid sequence showed that Leishmania donovani gp63 contains 590 amino acids precursor protein consisting of an NH₂-terminal signal peptide of 39 amino acids for membrane targeting, a pro region of 48 amino acids, the mature protein of 478 amino acids containing glycosylation and putative catalytic sites, and a COOH-terminal signal peptide of 25 amino acids for GPI attachment. Based on our model, the protein consists of three domains: the N-terminal, central and C-terminal domains. Additionally, these results could guide future structure-function analyses of gp63 protein.     

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

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Sperm protein (sp50) binds to acromosome and plasma membranes in a Ca2+-dependent manner: Possible role in acrosome reaction

Annexins are a family of Ca²⁺-binding proteins involved in the exocytotic process. The presence and the role of annexins in mammalian spermatozoa have not been well established. Two annexin-like proteins were obtained from guinea pig testis, a doublet of Mr 31–33 kD (p31/33) and a protein of Mr 50 kD (p50). Both proteins were able to bind to erythrocyte ghosts in a Ca²⁺-dependent fashion. Polyclonal antibodies against p31/33 reacted with two major proteins, Mrs 50 kD (sp50) and 42 kD (sp42), from mature and immature guinea pig spermatozoa. p50 and sp50 are likely the native proteins from testis and spermatozoa, respectively, and they are seemingly related. By immunofluorescence, sp50 was only found in the apical acrosome region of immature and capacitated and noncapacitated spermatozoa, and its location was intracellular. In spermatozoa undergoing acrosome reaction, sp50 was detected in the whole acrosome, while in spermatozoa that had undergone acrosome reaction sp50 was not detected. However, in the protein pattern of acrosome reaction vesicles, anti-p31/33 antibody revealed diffuse bands of Mr 35–38 kD. sp50 was able to bind to plasma membrane fragments and acrosome outer membrane from demembranated sperm in a Ca²⁺-dependent fashion. The presence of sp50 in the acrosome region, its distribution throughout the acrosome membrane just before the acrosome reaction, and its ability to bind both plasma and outer acrosome membranes in a Ca²⁺-dependent manner suggest that sp50 may participate in the acrosome reaction mechanism in guinea pig spermatozoa. © 1996 Wiley-Liss, Inc.     

NAK is recruited to the TNFR1 complex in a TNFalpha-dependent manner and mediates the production of RANTES: identification of endogenous TNFR-interacting proteins by a proteomic approach

Tumor necrosis factor alpha (TNFalpha) is a proinflammatory cytokine with pleiotropic immunological and biological activities. TNFalpha signaling is triggered by the engagement of soluble TNFalpha to two types of cell surface receptors, TNFR1 and TNFR2. This recruits cytosolic proteins to the intracellular domains of the receptors and initiates signaling to downstream effectors. In this study, we used a proteomic approach to identify these cytosolic proteins from affinity-purified, endogenous TNFalpha.TNFR complexes in human myelomonocytic U937 cells. Seven proteins were identified, including TRADD, TRAP2, and TRAF2, which are three proteins known to be recruited to TNFalpha receptors. NAK, RasGAP3, TRCP1, and TRCP2 were also identified. We further showed that NAK is recruited to TNFR1 in a temporally regulated and TNFalpha-dependent manner and that it mediates the TNFalpha-induced production of the chemokine RANTES (regulated on activation normal T cell expressed and secreted). These data demonstrate that NAK is a component of the TNFalpha.TNFR1 signaling complex and confirm the physiological role of NAK in the TNFalpha-mediated response.     

Different cytokines are required for induction and maintenance of the Th2 type response in DBA/2 mice resistant to infection with Leishmania major

Experimental cutaneous leishmaniasis is a useful model in studying the mechanism regulating immune responses between T helper type 1 (Th1) and Th2. Mice susceptible to Leishmania major infection such as BALB/c (H-2(d)) are associated with the induction of the disease-promoting Th2 response, while the resistant mice such as DBA/2 (H-2(d)) develop the protective Th1 response. To understand the induction mechanism of Th1 and Th2 responses, it is necessary to establish an immunization scheme by which the induction of each Th response can be easily and experimentally controlled. Adjuvants are known to enhance the immune responses through the combined effect of several factors: prolonged release of antigen, migration of cells, mitogenic effect and so forth. When the genetically resistant DBA/2 mice were immunized twice with soluble leishmanial antigen (SLA), emulsified in incomplete Freund's adjuvant (IFA) before L. major inoculation, these mice mounted a Th2 cell response and suffered from progressive infection. While IL-4 and IL-13 were upregulated early after the infection in both healer and non-healer groups of mice, IL-5 and IL-10 were upregulated only in non-healer mice. From these results, IL-5 and IL-10 appear to have an important role, at least in the early phases of the infection, rather than IL-4 and IL-13 in establishing the disease-promoting Th2 response in leishmaniasis. Further, IL-9 was found to be expressed in both BALB/c and DBA/2 mice immunized with IFA/SLA. This cytokine may support the establishment of a Th2 response in these mice. Therefore it is suggested that Th2 cytokines play different roles between priming and maintaining the Th2 immune response after the infection.     

Peptidases and gp63-like proteins in Herpetomonas megaseliae: possible involvement in the adhesion to the invertebrate host

The cell-associated and extracellular peptidases of Herpetomonas megaseliae grown in brain-heart infusion and in modified Roitman's complex media were analyzed by measuring peptidase activity on gelatin, casein and hemoglobin in zymograms. Casein was the best proteinaceous substrate for the peptidase detection on both growth conditions. However, no proteolytic activity was detected when hemoglobin was used. Our results showed that cellular cysteine peptidase (115-100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities were detected on both media in casein and gelatin zymograms. Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. Irrespective of the culture medium composition, H. megaseliae released metallopeptidases exclusively in the extracellular environment. The presence of gp63-like molecules on the H. megaseliae surface was shown by flow cytometry using anti-gp63 antibody raised against recombinant gp63 from Leishmania mexicana. The pre-treatment of parasites with phospholipase C reduced the number of gp63-positive cells, suggesting that these molecules were glycosylphosphatidylinositol-anchored to the surface. Additionally, the supernatant obtained from phospholipase C-treated cells and probed with anti-cross-reacting determinant confirmed that at least a 52 kDa gp63-like molecule is glycosylphosphatidylinositol-anchored. Furthermore, we assessed a possible function for the gp63-like molecules in H. megaseliae on the interaction with explanted guts of its original host, Megaselia scalaris, and with an experimental model employing Aedes aegypti. Parasites pre-treated with either anti-gp63 antibody or phospholipase C showed a significant reduction in the adhesion to M. scalaris and A. aegypti guts. Similarly, the pre-treatment of the explanted guts with purified gp63 diminished the interaction process. Collectively, these results corroborate the ubiquitous existence of gp63 homologues in insect trypanosomatids and the potential adhesion of these molecules to invertebrate host tissues.     

A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICP0 is modified in a cell-type-specific manner and Is recruited to the nucleus after infection

Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0). ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (gamma2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells. We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22. This protein (apparent Mr of 60,000) has not been previously described and has no known motifs. Analyses of p60 revealed the following. (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R. Bruni and B. Roizman, J. Virol. 72:8525-8531, 1998) bound all forms of ICP22. The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms. (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22. (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm. In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent Mr but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase. (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei. Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0. Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent Mr. The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases. The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22.     

p63 is a prosurvival factor in the adult mammary gland during post-lactational involution,affecting PI-MECs and ErbB2 tumorigenesis

In embryogenesis, p63 is essential to develop mammary glands. In the adult mammary gland, p63 is highly expressed in the basal cell layer that comprises myoepithelial and interspersed stem/progenitor cells, and has limited expression in luminal epithelial cells. In adult skin, p63 has a crucial role in the maintenance of epithelial stem cells. However, it is unclear whether p63 also has an equivalent role as a stem/progenitor cell factor in adult mammary epithelium. We show that p63 is essential in vivo for the survival and maintenance of parity-identified mammary epithelial cells (PI-MECs), a pregnancy-induced heterogeneous population that survives post-lactational involution and contain multipotent progenitors that give rise to alveoli and ducts in subsequent pregnancies. p63+/− glands are normal in virgin, pregnant and lactating states. Importantly, however, during the apoptotic phase of post-lactational involution p63+/− glands show a threefold increase in epithelial cell death, concomitant with increased activation of the oncostatin M/Stat3 and p53 pro-apoptotic pathways, which are responsible for this phase. Thus, p63 is a physiologic antagonist of these pathways specifically in this regressive stage. After the restructuring phase when involution is complete, mammary glands of p63+/− mice again exhibit normal epithelial architecture by conventional histology. However, using Rosa^LSL-LacZ;WAP-Cre transgenics (LSL-LacZ, lox-stop-lox β-galactosidase), a genetic in vivo labeling system for PI-MECs, we find that p63+/− glands have a 30% reduction in the number of PI-MEC progenitors and their derivatives. Importantly, PI-MECs are also cellular targets of pregnancy-promoted ErbB2 tumorigenesis. Consistent with their PI-MEC pool reduction, one-time pregnant p63+/− ErbB2 mice are partially protected from breast tumorigenesis, exhibiting extended tumor-free and overall survival, and reduced tumor multiplicity compared with their p63+/+ ErbB2 littermates. Conversely, in virgin ErbB2 mice p63 heterozygosity provides no survival advantage. In sum, our data establish that p63 is an important survival factor for pregnancy-identified PI-MEC progenitors in breast tissue in vivo.     

Design of a polytopic construct of LACK,TSA and GP63 proteins for the diagnosis of cutaneous leishmaniasis: An in silico strategy

Cutaneous leishmaniasis (CL), which is caused by different species of the genus Leishmania, including L. major, is a significant parasitic disease worldwide. Commercial leishmaniasis diagnostic kits frequently target the entire parasite antigen, although this approach can reduce the specificity of the tests. The present study sought to increase the specificity of antigen detection tests by identifying the B-cell epitopes on the surface of the protozoa and developing an antigenic multiepitope construct to react with the system of B-cell epitopes unique to L. major. More specifically, the linear and conformational B-cell epitopes for three L. major proteins, namely, GP63, LACK, and TSA, were predicted using the ABCprd, IEDB, LBtope, CBTOPE, and DiscoTope servers. The three-dimensional structures of these proteins, as well as of the complete multiepitope, were predicted using the I-TASSER server. The multiepitope structure models were validated by means of Ramachandran plots and the Prosa and Verify3D servers. The multiepitope construct was evaluated using the ProtParam and SOLpro servers. The synthesized multiepitope was then successfully expressed into a pET28a plasmid. The expression was confirmed using SDS-PAGE and dot blot techniques. Moreover, the expressed protein was evaluated by means of Western blotting. A band of approximately 39 kDa was observed by SDS-PAGE. The rGLT-pET-28a showed a high response with the 1:1000 dilution of the anti-His-tag monoclonal antibody by Western blot. The results of this study indicated that the integrated GP63, LACK, and TSA multiepitope antigens of L. major represent a potential target for a novel diagnostic kit for CL.     

Selenium and mercury are redistributed to the brain during viral infection in mice

As part of the general host response to coxsackievirus B3 (CB3) infection, the concentration of essential and nonessential trace elements changes in different target organs of the infection. Essential (e.g., Se) and nonessential (e.g., Hg) trace elements are known to interact and affect inflammatory tissue lesions induced by CB3 infection. However, it is unknown whether these changes involve the brain. In the present study, the brain Hg and Se contents were measured through inductively coupled plasma-mass spectrometry and their distribution investigated by means of nuclear microscopy in the early phase (d 3) of CB3 infection in normally fed female Balb/c mice. Because of the infection, the concentration of Hg (4.07±0.46 ng/g wet wt) and Se (340±16 ng/g wet wt) in the brain increased twofold for Hg (8.77±1.65 ng/g wet wt, p<0.05) and by 36% for Se (461±150 ng/g wet wt, ns). Nuclear microscopy of brain sections from mice having elevated Se and Hg concentrations failed to find localized levels of the elements high enough to make detection possible, indicating approximately homogeneous tissue distribution. Although the pathophysiological interpretation of these findings requires further research, the increase of Hg in the brain during infection might have an influence on the pathogenesis of the disease.     

Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro

The Bet v. 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belong to a group of conserved intracellular defense-related proteins that have been termed 'ribonuclease-like' PR proteins, on the basis of the partial sequence homology observed between PR1, a Bet v 1-homologue from parsley, and a recently characterized ginseng ribonuclease. However, this enzymatic activity has not yet been demonstrated, not for any of the members of this family of PR proteins, nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v 1 using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro, but show no activity on single or double-stranded DNA.     

The development of a Th1-type response and resistance to Leishmania major infection in the absence of CD40-CD40L costimulation.

CD40-CD40L interactions have been shown to be essential for the production of IL-12 and IFN-gamma and control of L. major infection. In contrast, C57BL/6 mice deficient in CD28 develop a dominant Th1-type response and heal infection. In this study, we investigate the effects of a deficiency in both CD40L and CD28 molecules on the immune response and the course of L. major infection. We compared infection in mice genetically lacking CD40L (CD40L(-/-)), CD28 (CD28(-/-)), or both (CD40L(-/-)CD28(-/-)), and in C57BL/6 mice, all on a resistant background. Although CD40L(-/-) mice failed to control infection, CD28(-/-) and CD40L(-/-)CD28(-/-) mice, as well as C57BL/6 mice, spontaneously resolved their infections. Healing mice had reduced numbers of lesion parasites compared with nonhealing CD40L(-/-) mice. At wk 9 of infection, we detected similar levels of IL-4, IFN-gamma, IL-12p40, and IL-12Rbeta2 mRNA in draining lymph nodes of healing C57BL/6, CD28(-/-), and CD40L(-/-)CD28(-/-) mice, whereas CD40L(-/-) mice had increased mRNA levels for IL-4 but reduced levels for IFN-gamma, IL-12p40, and IL-12Rbeta2. In a separate experiment, blocking of the CD40-CD40L pathway using Ab to CD40L led to an exacerbation of infection in C57BL/6 mice, but had little or no effect on infection in CD28(-/-) mice. Together, these results demonstrate that in the absence of CD28 costimulation, CD40-CD40L interaction is not required for the development of a protective Th1-type response. The expression of IL-12p40, IL-12Rbeta2, and IFN-gamma in CD40L(-/-)CD28(-/-) mice further suggests the presence of an additional stimulus capable of regulating IL-12 and its receptors in absence of CD40-CD40L interactions.