Functional screening pipeline to uncover laccase-like multicopper oxidase enzymes that transform industrial lignins.

Laccase-like multicopper oxidases are recognized for their potential to alter the reactivity of lignins for application in value-added products. Typically, model compounds are employed to discover such enzymes; however, they do not represent the complexity of industrial lignin substrates. In this work, a screening pipeline was developed to test enzymes simultaneously on model compounds and industrial lignins. A total of 12 lignin-active fungal multicopper oxidases were discovered, including 9 enzymes active under alkaline conditions (pH 11.0). Principal component analysis revealed the poor ability of model compounds to predict enzyme performance on industrial lignins. Additionally, sequence similarity analyses grouped these enzymes with Auxiliary Activity-1 sub-families with few previously characterized members, underscoring their taxonomic novelty. Correlation between the lignin-activity of these enzymes and their taxonomic origin, however, was not observed. These are critical insights to bridge the gap between enzyme discovery and application for industrial lignin valorization.

A Combinatorial Approach To Study Cytochrome P450 Enzymes for De Novo Production of Steviol Glucosides in Baker's Yeast.

Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPH-dependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologues were tested in batch growth. Among the CYPs, novel KO75 (CYP701) and novel KAH82 (CYP72) outperformed their respective functional homologues from Stevia rebaudiana, SrKO (CYP701A5) and SrKAH (CYP81), in assays where substrate was supplemented to culture broth. With kaurene produced from glucose in the cell, SrCPR1 from S. rebaudiana supported highest turnover for KO-KAH combinations, besting two other CPRs isolated from S. rebaudiana, the Arabidopsis thaliana ATR2, and a new class I CPR12. Some coexpressions of ATR2 with a second CPR were found to diminish KAH activity, showing that coexpression of CPRs can lead to competition for CYPs with possibly adverse effects on catalysis.

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass.

BACKGROUND: Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails. RESULTS: It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition. CONCLUSIONS: These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.

Global analysis of protein phosphorylation networks in insulin signaling by sequential enrichment of phosphoproteins and phosphopeptides.

Although the important role of protein phosphorylation in insulin signaling networks is well recognized, its analysis in vivo has not been pursued in a systematic fashion through proteome-wide studies. Here we undertake a global analysis of insulin-induced changes in the rat liver cytoplasmic and endosomal phosphoproteome by sequential enrichment of phosphoproteins and phosphopeptides. After subcellular fractionation proteins were denatured and loaded onto iminodiacetic acid-modified Sepharose with immobilized Al³âº ions (IMAC-Al resin). Retained phosphoproteins were eluted with 50 mM phosphate and proteolytically digested. The digest was then loaded onto an IMAC-Al resin and phosphopeptides were eluted with 50 mM phosphate, and resolved by 2-dimensional liquid chromatography, which combined offline weak anion exchange and online reverse phase separations. The peptides were identified by tandem mass spectrometry, which also detected the phosphorylation sites. Non-phosphorylated peptides found in the flow-through of the IMAC-Al columns were also analyzed providing complementary information for protein identification. In this study we enriched phosphopeptides to ~85% purity and identified 1456 phosphopeptides from 604 liver phosphoproteins. Eighty-nine phosphosites including 45 novel ones in 83 proteins involved in vesicular transport, metabolism, cell motility and structure, gene expression and various signaling pathways were changed in response to insulin treatment. Together these findings could provide potential new markers for evaluating insulin action and resistance in obesity and diabetes.

Erythropoietin-dependent endothelial proteins: potential use against erythropoietin resistance.

The endothelium was the first non-hematopoietic tissue to be identified as a physiological target for erythropoietin (EPO). EPO is involved in recruitment and mobilization of endothelial progenitors and stimulates the production of erythroid cell regulatory factors in endothelial cells. Production of these EPO-dependent factors is inhibited by IL-3 in vitro. Furthermore, EPO-dependent red cell formation in anemic mice is equally repressed by IL-3. The number of IL-3 receptors on endothelial cells increases in chronic inflammation and IL-3 may be one of the inflammatory cytokines, together with TNF-alpha, IFN-gamma or IL-6, which prevents optimal red cell formation in many patients with kidney failure receiving high doses of EPO. These patients could benefit from the administration of some of the EPO-stimulated endothelial factors, such as C21 (the C-terminal segment thrombospondin-4), thrombospondin-1 and chaperonin 10, because these proteins bypass EPO receptors and signaling pathways that are usually compromised in EPO resistance. C21 stimulates red cell formation in anemic mice, increases human hematopoietic cell proliferation in vitro and could eventually fight inflammation, because it is an osteopontin antagonist. Thrombospondin-1 prevents inflammation, stimulates erythroblast proliferation and counteracts IGFBP-3-mediated erythroid inhibition. Finally, chaperonin 10 stimulates hemoglobin synthesis and has anti-inflammatory properties through the inhibition of Toll-like receptor signaling pathways.

Biological activities and molecular interactions of the C-terminal residue of thrombospondin-4, an epitome of acidic amphipathic peptides.

C21, the C-terminal residue of thrombospondin-4 (TSP-4), was identified as a peptide growth factor during an investigation concerning erythropoietin-dependent, erythroid stimulating factors of endothelial origin. It is active in cultures of several human hematopoietic stem cells, skin fibroblasts and kidney epithelial cells and stimulates red cell formation in anemic mice. A method of affinity chromatography in the presence of high concentrations of Triton X-100, previously developed for identifying proteins associated with the TSP-1 receptor CD47, was utilized for the detection of C21 binding molecules and their detergent-resistant, associated partners. These experiments helped to delineate two different mechanisms of C21 action, which are compatible with its cell proliferating activity. As a cell matrix peptide, C21 binds to the osteopontin receptor CD44 and could act as an osteopontin antagonist, preventing the inhibition of primitive hematopoietic stem cell proliferation. TSP-1, another matrix protein, binds to C21 and could indirectly act as an antagonist, by shunting C21-CD44 interactions. The second mechanism is a direct effect of C21 on cell proliferation. The extremely rapid internalization and nuclear localization of the peptide could be explained by CD44-mediated internalization, followed by a microtubule-mediated transport towards the nucleus, or, eventually, direct membrane insertion. These alternative hypotheses are supported by previously observed membrane insertion of similar synthetic and viral acidic amphipathic peptides, the presence of microtubule-associated protein 1B (MAP1B) and dynactin in the triton-soluble complexes associated with C21 and the presence in such complexes of dual compartment proteins for nuclei and plasma membranes, such as MAP1B, AHNAK and CD44.

Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

Quantitative proteomics of nasal mucus in chronic sinusitis with nasal polyposis.

BACKGROUND: Proteomics has been used as a tool for identification of the protein content of nasal mucus in diseased and healthy subjects. Thirty-five proteins in both chronic rhinosinusitis (CRS) and control groups were identified in a previous study by our group using conventional mass spectrometry analysis. Ten of these proteins were related to innate and acquired immunity and showed differences in expression between the two groups. OBJECTIVE: To investigate the quantitative differential expression of specific nasal mucus proteins previously identified by our group using multiple reaction monitoring (MRM) mass spectrometry in patients with CRS with nasal polyposis compared with normal subjects. METHODS: In a prospective case control study, nasal mucus from patients and control subjects was collected, desalted, resolubilized, and digested using proteolytic enzymes. Previously identified nasal mucus proteins with differential expression in CRS patients were targeted and quantitatively measured using MRM mass spectrometry. RESULTS: Analysis of 12 samples (6 patients and 6 controls) identified 7 of the 10 targeted proteins, many of which were related to innate and acquired immunity. Quantitative analysis showed differential expression in CRS patients compared with control subjects. A detailed analysis and characterization of the protein isolates is outlined. CONCLUSION: This is the first proteomics study of nasal mucus in CRS with polyposis using the MRM technique. The findings suggest that innate and acquired immunity may play a role in the pathophysiology of CRS. Future steps in evaluating the protein characteristics of the mucus of CRS patients are aimed at developing biomarkers and potentially targeted therapies.

Proteomics of nasal mucus in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is among the three most common chronic diseases in North America. The area of proteomics research is providing tremendous insight into the mechanisms of a variety of physiological processes and disease states. The purpose of this study was to evaluate qualitative and quantitative differences in protein content of nasal mucus in patients with chronic hypertrophic sinusitis with nasal polyposis when compared with control subjects. METHODS: A case-control study was performed in a tertiary academic center. Nasal mucus was collected from four patients with CRS and nasal polyposis as well as four control subjects. The protein content was digested using proteolytic enzymes, labeled with iTRAQ reagents, and subjected to mass spectrometry (MS) analysis. RESULTS: A total of 35 proteins were identified, many of which were related to innate and acquired immunity. Lysozyme C precursor was found to be down-regulated by a ratio (R) of 0.65 (p = 0.016) in CRS patients, as was Clara cell phospholipid-binding protein (R = 0.23; p = 0.0018), and antileukoproteinase 1 (R = 0.47; p < 0.0001). A detailed analysis and characterization of the protein isolates is outlined. CONCLUSION: The field of proteomics has great potential in leading to a better understanding of the mechanism of the disease process in CRS. Differences in the expression of proteins related to regulation of immune cells and mediators merit additional investigation.

Thrombospondin 1, produced by endothelial cells under the action of erythropoietin, stimulates thymidine incorporation into erythroid cells and counteracts the inhibitory action of insulin-like growth factor binding protein 3.

The nature of erythropoietin (EPO)-dependent, erythroid cell regulatory factors secreted by endothelial cells is largely unknown. The production of thrombospondin 1 (TSP-1) and insulin-like growth factor binding protein 3 (IGFBP-3) is increased in cultures of human umbilical vein endothelial cells (HUVEC) incubated with erythropoietin (EPO). Simultaneous incubation of HUVEC with EPO and interleukin 3 (IL-3) resulted in a decreased production, suggesting that both TSP-1 and IGFBP-3 belong to the EPO- and IL-3-dependent erythroid regulatory factors previously described in cultures of bone marrow endothelial cells. TSP-1 and TSP-1 derived synthetic peptides based on the CD36 and CD47 binding sites of TSPs increased thymidine incorporation into bovine erythroid cells of fetal liver. IGBBP-3 inhibited thymidine incorporation in the same cells. Preincubation of erythroid cells with TSP-1 eliminated the inhibitory activity of IGFBP-3. We suggest that EPO-dependent, endothelial-derived TSP-1 may play a positive role in red cell production by acting directly on erythroid cells, stimulating DNA synthesis and preventing the inhibitory action of IGFBP-3.

The C-terminal peptide of thrombospondin-4 stimulates erythroid cell proliferation.

Erythropoietin (EPO) stimulates the production of small erythroid cell stimulating factors (molecular weight <5 kDa) in cultures of bone marrow endothelial cells. We identified a fragment of thrombospondin-4 (TSP-4) as an EPO-stimulated protein in endothelial cell lysates. Pre-incubation of the low molecular weight fractions from supernatants of EPO-treated umbilical cord endothelial cells (HUVEC) with antibodies against the C-terminal residues of TSP-1,2 and TSP-4 decreased the erythroid cell stimulating activity. The C-terminal TSP-1 section corresponding to a molecular weight lower than 6 kDa has the integrin-associated protein binding motif VVM. The corresponding TSP-4 fragment, lacking the three residue sequence VVM, has a distinctive acidic peptide comprising the last 21 amino acids (C21) with the characteristics of an amphipathic helix. C21 stimulated thymidine incorporation into bovine erythroid cells, increased cell numbers in cultures of cord blood CD36+ erythroid precursors and skin fibroblasts, and decreased HUVEC proliferation. SC21, a homologous peptide of identical amino acid composition but with interchanged residues, was non-amphipathic and had no erythroid cell stimulating activity.

The fusion of IGF I with stromal cell-derived factor I or alpha1 proteinase inhibitor alters their mitogenic or chemotactic activities while keeping their ability to inhibit HIV-1-gp120 binding.

It has been previously reported that insulin-like growth factor I (IGF I) decreases in AIDS patients with wasting, a condition that is partially prevented by combined IGF I growth hormone therapy. By generating bifunctional proteins of IGF I and stromal cell-derived factor 1alpha (SDF-1alpha) or alpha1 proteinase inhibitor (API), two proteins known to prevent HIV infection, it may be possible to improve the therapeutic effectiveness of these compounds for the treatment of AIDS-mediated wasting. SDF-1alpha or the M351E-M358L mutant of API were attached at the C-terminal end of IGF I and synthesized by a stable insect cell expression technique. The IGF I-SDF-1alpha chimera reduced the enhancement of thymidine incorporation into bovine fetal erythroid cells observed in the presence of insect cell produced IGF I alone. It also decreased the SDF-1 and IGF I-stimulated hematopoietic cell migration, without losing the capacity to compete with the binding of HIV-1 (IIIB)-surface glycoprotein gp120. The IGF I-API chimera displayed the same mitogenic activity and a similar, but lower chemotactic activity than IGF I in the assays mentioned above. It had a comparable anti-elastase activity to that observed with a previously described IGF II-API fusion protein with the single mutation M351E. The binding of gp120 to a murine hematopoietic cell line was stimulated by human neutrophil elastase (25-100 nM) and inhibited by IGF I-API. In conclusion, the linkage of IGF I with SDF-1 or API can alter some biological functions of the single components of the chimera while keeping their ability to compete with HIV-1-gp120 binding.

The improved survival of hematopoietic cells cultured with a fusion protein of insulin-like growth factor II (IGF-II) and interleukin 3 (IL-3) is associated with increases in Bcl-xL and phosphatidylinositol-3 kinase activity.

We compared the antiapoptotic activity of a recombinant chimera of insulin-like growth factor II (IGF-II) and interleukin (IL)-3 with the corresponding equimolar mixture of the individual components based on changes in several factors associated with survival in the CD34+ human hematopoietic cell line TF-1. Propidium iodide-stained cells analyzed by fluorescein-activated cell sorter indicated that the chimera was more effective than the corresponding equimolar mixture in decreasing the amounts of apoptotic cells and increasing the proportion of cells in the S-phase of the cell cycle. The chimera was more effective in increasing the antiapoptotic protein Bclx(L) and produced a significant increase in signal transducer and activator of transcription-5 phosphorylation and in phosphatidylinositol-3 kinase (PI-3K) activity. The PI-3K inhibitor LY294002 specifically inhibited cell survival in the presence of the chimera, suggesting a key role of this enzyme in the potentiation of survival caused by the linkage of IGF and IL-3. This potentiation of survival and its preferential inhibition by LY294002 were also observed in a nontransformed, primary culture of human umbilical cord endothelial cells.

Potentiation of hematopoietic cell migration with an IGF-interleukin-3 fusion protein.

A chimera of an N-terminally modified insulin growth factor (IGF)-II, NQPQMVHTY-hIGF-II(9-67) (BOMIGF), fused to interleukin-3 (IL-3) significantly improved the migration of CD34(+) human hematopoietic cells with respect to the effects observed during co-stimulation with BOMIGF and IL-3. A phosphatidylinositol-3 (PI-3) kinase inhibitor specifically inhibited migration in the presence of the chimera, while no significant difference in the inhibition of migration was observed in the presence of a Rho kinase inhibitor. These results suggest a key role of the PI-3 kinase pathway in the potentiation of migration caused by the linkage of BOMIGF and IL-3.

Antagonism between interleukin 3 and erythropoietin in mice with azidothymidine-induced anemia and in bone marrow endothelial cells.

Azidothymidine (AZT)-induced anemia in mice can be reversed by the administration of IGF-IL-3 (fusion protein of insulin-like growth factor II (IGF II) and interleukin 3). Although interleukin 3 (IL-3) and erythropoietin (EPO) are known to act synergistically on hematopoietic cell proliferation in vitro, injection of IGF-IL-3 and EPO in AZT-treated mice resulted in a reduction of red cells and an increase of plasma EPO levels as compared to animals treated with IGF-IL-3 or EPO alone. We tested the hypothesis that the antagonistic effect of IL-3 and EPO on erythroid cells may be mediated by endothelial cells. Bovine liver erythroid cells were cultured on monolayers of human bone marrow endothelial cells previously treated with EPO and IGF-IL-3. There was a significant reduction of thymidine incorporation into both erythroid and endothelial cells in cultures pre-treated with IGF-IL-3 and EPO. Endothelial cell culture supernatants separated by ultrafiltration and ultracentrifugation from cells treated with EPO and IL-3 significantly reduced thymidine incorporation into erythroid cells as compared to identical fractions obtained from the media of cells cultured with EPO alone. These results suggest that endothelial cells treated simultaneously with EPO and IL-3 have a negative effect on erythroid cell production.

Insect cell production of a secreted form of human alpha(1)-proteinase inhibitor as a bifunctional protein which inhibits neutrophil elastase and has growth factor-like activities.

alpha(1)-proteinase inhibitor (API) is a potential therapeutic agent in all diseases in which elastase released by neutrophils has to be effectively neutralized. We ligated the cDNA of human API to the C-terminal section of an insulin-like growth factor II analogue (BOMIGF), known to be properly folded and secreted in insect cells using the baculovirus expression system. The BOMIGF-API chimera was recovered from the incubation medium of the infected cells. It shared the properties of both IGFs and API. It inhibited neutrophil elastase and formed SDS-stable complexes with the enzyme. The attachment of the large API protein to the C-terminal end of the 10 kDa IGF analogue did not destroy the IGF-mediated stimulation of thymidine incorporation into bovine fetal erythroid cells. We tested the capacity of the chimera to affect fibronectin-dependent TF-1 cell migration. BOMIGF-API significantly restored TF-1 cell migration in the presence of elastase, which is the enzyme of burn wound fluid most probably involved in fibronectin degradation. Some of the beneficial uses for this chimera may include all instances for which inhibition of elastase-mediated extracellular matrix destruction as well as stimulation of cell migration and proliferation are required for tissue repair.