Mass spectrometric revival of an l-rhamnose- and d-galactose-specific lectin from a lost strain of Streptomyces.

Blood type B-specific Streptomyces sp. 27S5 hemagglutinin (SHA) was discovered and characterized in the 1970s. Although strain 27S5 has been lost, the purified SHA protein survived intact under frozen conditions and retained its activity. Using modern techniques, here we further characterized SHA. Fourier-transform ion cyclotron resonance MS analysis determined the average molecular mass of SHA as 13,314.67 Da. MS of digested SHA peptides, Streptomyces genomic database matching, and N-terminal sequencing solved the 131-residue amino acid sequence of SHA. We found that SHA is homologous to N-terminally truncated hypothetical proteins encoded by the genomes of Streptomyces lavendulae, Streptomyces sp. Mg1, and others. The gene of the closest homologue in S. lavendulae, a putative polysaccharide deacetylase (PDSL), encodes 68 additional N-terminal amino acids, and its C terminus perfectly matched the SHA sequence, except for a single Ala-to-Glu amino acid difference. We expressed recombinant SHA(PDSL-A108E) (rSHA) as an enzymatically cleavable fusion protein in Escherichia coli, and glycan microarray analyses indicated that refolded rSHA exhibits the blood type B- and l-rhamnose-specific characteristics of authentic SHA, confirming that rSHA is essentially identical with SHA produced by Streptomyces sp. 27S5. We noted that SHA comprises three similar domains, representing 70% of the protein, and that these SHA domains partially overlap with annotated clostridial hydrophobic with conserved W domains. Furthermore, examination of GFP-tagged SHA revealed binding to microbial surfaces. rSHA may be useful both for studying the role of SHA/clostridial hydrophobic with conserved W domains in carbohydrate binding and for developing novel diagnostics and therapeutics for l-rhamnose-containing microorganisms.

Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity.

Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisin-like protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates.

CD4+ T cells mediate the protective effect of the recombinant Asp f3-based anti-aspergillosis vaccine.

The mortality and morbidity caused by invasive aspergillosis present a major obstacle to the successful treatment of blood cancers with hematopoietic cell transplants. Patients who receive hematopoietic cell transplants are usually immunosuppressed for extended periods, and infection with the ubiquitous mold Aspergillus fumigatus is responsible for most cases of aspergillosis. Previously, we demonstrated that vaccination with recombinant forms of the A. fumigatus protein Asp f3 protected cortisone acetate-immunosuppressed mice from experimentally induced pulmonary aspergillosis. Here, we investigated the vaccine's protective mechanism and evaluated in particular the roles of antibodies and T cells. After vaccination, Asp f3-specific preinfection IgG titers did not significantly differ between surviving and nonsurviving mice, and passive transfer of anti-Asp f3 antibodies did not protect immunosuppressed recipients from aspergillosis. We experimentally confirmed Asp f3's predicted peroxisomal localization in A. fumigatus hyphae. We found that fungal Asp f3 is inaccessible to antibodies, unless both cell walls and membranes have been permeabilized. Antibody-induced depletion of CD4+ T cells reduced the survival of recombinant Asp f3 (rAsp f3)-vaccinated mice to nonimmune levels, and transplantation of purified CD4+ T cells from rAsp f3-vaccinated mice into nonimmunized recipients transferred antifungal protection. In addition, residues 60 to 79 and 75 to 94 of Asp f3 contain epitopes that induce proliferation of T cells from vaccinated survivors. Vaccine-primed CD4+ T cells are not expected to clear the fungal pathogen directly; however, they may locally activate immunosuppressed phagocytes that elicit the antifungal effect.

Vaccine progress.

Despite the recent development of new anti-mould agents, there remains a significant incidence of invasive aspergillosis in the most immunocompromised hosts and the response to these agents is still dismal. There is a need for a different approach: prevention by vaccination. We have demonstrated that a hyphal sonicate of Aspergillus fumigatus was capable of conferring protection against subsequent invasive pulmonary aspergillosis in corticosteroid immunosuppressed mice. Subcutaneous vaccination was superior to nasal vaccination. Mice exposed intranasally to viable conidia were noted to respond serologically to a 19 kDa protein. This protein was identified as the allergen Asp f 3 by mass spectrometry. Vaccination with recombinant Asp f 3 was protective. Truncated forms of Asp f 3 that lacked either one of the two known IgE binding sites were cloned and also demonstrated protection against aspergillosis. Although all of these recombinant proteins required an adjuvant (TiterMax) for efficacy, a particulate preparation of rAsp f 3 was also found to be protective without requiring adjuvant. At least two T-cell epitopes (11-mer and 13-mer) have been identified in Asp f 3. There are homologues of Asp f 3 in other Aspergillus species as well as in other moulds (Coccidioides posadasii, Penicillium citrinum) and yeasts (Candida albicans, C. boidinii, Saccharomyces cerevisiae). Asp f 3, truncated non-allergenic versions of Asp f 3, and T-cells epitopes of Asp f 3 are potential candidates for vaccines potentially capable of protecting immunocompromised hosts against invasive aspergillosis.

Vitamin K epoxide reductase regulation of androgen receptor activity.

Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in γ-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be γ-carboxylated, and mapped the γ-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is γ-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPARγ and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer.

The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus.

Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target.

Protein targets for broad-spectrum mycosis vaccines: quantitative proteomic analysis of Aspergillus and Coccidioides and comparisons with other fungal pathogens.

Aspergillus species are responsible for most cases of fatal mold infections in immunocompromised patients, particularly in those receiving hematopoietic stem cell transplants. Experimental vaccines in mouse models have demonstrated a promising avenue of approach for the prevention of aspergillosis, as well as infections caused by other fungal pathogens, such as Coccidioides, the etiological agent of valley fever (coccidioidomycosis). Here, we investigated the hyphal proteomes of Aspergillus fumigatus and Coccidioides posadasii via quantitative MS(E) mass spectrometry with the objective of developing a vaccine that cross-protects against these and other species of fungi. Several homologous proteins with highly conserved sequences were identified and quantified in A. fumigatus and C. posadasii. Many abundant proteins from the cell wall of A. fumigatus present themselves as possible cross-protective vaccine candidates, due to the high degree of sequence homology to other medically relevant fungal proteins and low homologies to human or murine proteins.

Alternative mechanisms by which mediator subunit MED1/TRAP220 regulates peroxisome proliferator-activated receptor gamma-stimulated adipogenesis and target gene expression.

Mediator is a general coactivator complex connecting transcription activators and RNA polymerase II. Recent work has shown that the nuclear receptor-interacting MED1/TRAP220 subunit of Mediator is required for peroxisome proliferator-activated receptor gamma (PPARgamma)-stimulated adipogenesis of mouse embryonic fibroblasts (MEFs). However, the molecular mechanisms remain undefined. Here, we show an intracellular PPARgamma-Mediator interaction that requires the two LXXLL nuclear receptor recognition motifs on MED1/TRAP220 and, furthermore, we show that the intact LXXLL motifs are essential for optimal PPARgamma function in a reconstituted cell-free transcription system. Surprisingly, a conserved N-terminal region of MED1/TRAP220 that lacks the LXXLL motifs but gets incorporated into Mediator fully supports PPARgamma-stimulated adipogenesis. Moreover, in undifferentiated MEFs, MED1/TRAP220 is dispensable both for PPARgamma-mediated target gene activation and for recruitment of Mediator to a PPAR response element on the aP2 target gene promoter. However, PPARgamma shows significantly reduced transcriptional activity in cells deficient for a subunit (MED24/TRAP100) important for the integrity of the Mediator complex, indicating a general Mediator requirement for PPARgamma function. These results indicate that there is a conditional requirement for MED1/TRAP220 and that a direct interaction between PPARgamma and Mediator through MED1/TRAP220 is not essential either for PPARgamma-stimulated adipogenesis or for PPARgamma target gene expression in cultured fibroblasts. As Mediator is apparently essential for PPARgamma transcriptional activity, our data indicate the presence of alternative mechanisms for Mediator recruitment, possibly through intermediate cofactors or other cofactors that are functionally redundant with MED1/TRAP220.

Vaccinations with recombinant variants of Aspergillus fumigatus allergen Asp f 3 protect mice against invasive aspergillosis.

A vaccine that effectively protects immunocompromised patients against invasive aspergillosis is a novel approach to a universally fatal disease. Here we present a rationale for selection and in vivo testing of potential protein vaccine candidates, based on the modification of an immunodominant fungal allergen for which we demonstrate immunoprotective properties. Pulmonary exposure to viable Aspergillus fumigatus conidia as well as vaccination with crude hyphal extracts protects corticosteroid-immunosuppressed mice against invasive aspergillosis (J. I. Ito and J. M. Lyons, J. Infect. Dis. 186:869-871, 2002). Sera from the latter animals contain antibodies with numerous and diverse antigen specificities, whereas sera from conidium-exposed mice contain antibodies predominantly against allergen Asp f 3 (and some against Asp f 1), as identified by mass spectrometry. Subcutaneous immunization with recombinant Asp f 3 (rAsp f 3) but not with Asp f 1 was protective. The lungs of Asp f 3-vaccinated survivors were free of hyphae and showed only a patchy low-density infiltrate of mononuclear cells. In contrast, the nonimmunized animals died with invasive hyphal elements and a compact peribronchial infiltrate of predominantly polymorphonuclear leukocytes. Three truncated versions of rAsp f 3, spanning amino acid residues 15 to 168 [rAsp f 3(15-168)], 1 to 142, and 15 to 142 and lacking the known bipartite sequence required for IgE binding, were also shown to be protective. Remarkably, vaccination with either rAsp f 3(1-142) or rAsp f 3(15-168) drastically diminished the production of antigen-specific antibodies compared to vaccination with the full-length rAsp f 3(1-168) or the double-truncated rAsp f 3(15-142) version. Our findings point to a possible mechanism in which Asp f 3 vaccination induces a cellular immune response that upon infection results in the activation of lymphocytes that in turn enhances and/or restores the function of corticosteroid-suppressed macrophages to clear fungal elements in the lungs.