Peroxicretion: a novel secretion pathway in the eukaryotic cell

Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. Results: Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extracellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. Conclusion: Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

Systems approaches to predict the functions of glycoside hydrolases during the life cycle of Aspergillus niger using developmental mutants ∆brlA and ∆flbA

Background The filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis. Results In this study, we used developmental mutants (ΔflbA and ΔbrlA) which are characterized by an aconidial phenotype when grown on a plate, but also in bioreactor-controlled submerged cultivations during carbon starvation. By comparing the transcriptomes, proteomes, enzyme activities and the fungal cell wall compositions of a wild type A. niger strain and these developmental mutants during carbon starvation, a global overview of the function of carbohydrate-active enzymes is provided. Seven genes encoding carbohydrate-active enzymes, including cfcA, were expressed during starvation in all strains; they may encode enzymes involved in cell wall recycling. Genes expressed in the wild-type during starvation, but not in the developmental mutants are likely involved in conidiogenesis. Eighteen of such genes were identified, including characterized sporulation-specific chitinases and An15g02350, member of the recently identified carbohydrate-active enzyme family AA11. Eight of the eighteen genes were also expressed, independent of FlbA or BrlA, in vegetative mycelium, indicating that they also have a role during vegetative growth. The ΔflbA strain had a reduced specific growth rate, an increased chitin content of the cell wall and specific expression of genes that are induced in response to cell wall stress, indicating that integrity of the cell wall of strain ΔflbA is reduced. Conclusion The combination of the developmental mutants ΔflbA and ΔbrlA resulted in the identification of enzymes involved in cell wall recycling and sporulation-specific cell wall modification, which contributes to understanding cell wall remodeling mechanisms during development

Targeting proline in (phospho)proteomics

Mass spectrometry-based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome. Here, we argue that there may be further room for improvement and explore the protease EndoPro. For optimal peptide identification rates, we explored multiple peptide fragmentation techniques (HCD, ETD, and EThcD) and employed Byonic as search algorithm. We obtain peptide IDs for about 40% of the MS2 spectra (66% for trypsin). EndoPro cleaves with high specificity at the C-terminal site of Pro and Ala residues and displays activity in a broad pH range, where we focused on its performance at pH = 2 and 5.5. The proteome coverage of EndoPro at these two pH values is rather distinct, and also complementary to the coverage obtained with trypsin. As about 40% of mammalian protein phosphorylations are proline-directed, we also explored the performance of EndoPro in phosphoproteomics. EndoPro extends the coverable phosphoproteome substantially, whereby both the, at pH = 2 and 5.5, acquired phosphoproteomes are complementary to each other and to the phosphoproteome obtained using trypsin. Hence, EndoPro is a powerful tool to exploit in (phospho)proteomics applications

Targeting proline in (phospho)proteomics

Mass spectrometry-based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome. Here, we argue that there may be further room for improvement and explore the protease EndoPro. For optimal peptide identification rates, we explored multiple peptide fragmentation techniques (HCD, ETD, and EThcD) and employed Byonic as search algorithm. We obtain peptide IDs for about 40% of the MS2 spectra (66% for trypsin). EndoPro cleaves with high specificity at the C-terminal site of Pro and Ala residues and displays activity in a broad pH range, where we focused on its performance at pH = 2 and 5.5. The proteome coverage of EndoPro at these two pH values is rather distinct, and also complementary to the coverage obtained with trypsin. As about 40% of mammalian protein phosphorylations are proline-directed, we also explored the performance of EndoPro in phosphoproteomics. EndoPro extends the coverable phosphoproteome substantially, whereby both the, at pH = 2 and 5.5, acquired phosphoproteomes are complementary to each other and to the phosphoproteome obtained using trypsin. Hence, EndoPro is a powerful tool to exploit in (phospho)proteomics applications

Is Proteomics a Reliable Tool to Probe the Oxidative Folding of Bacterial Membrane Proteins?

<p>The oxidative folding of proteins involves disulfide bond formation, which is usually catalyzed by thiol-disulfide oxidoreductases (TDORs). In bacteria, this process takes place in the cytoplasmic membrane and other extracytoplasmic compartments. While it is relatively easy to study oxidative folding of water-soluble proteins on a proteome-wide scale, this has remained a major challenge for membrane proteins due to their high hydrophobicity. Here, we have assessed whether proteomic techniques can be applied to probe the oxidative folding of membrane proteins using the Gram-positive bacterium Bacillus subtilis as a model organism. Specifically, we investigated the membrane proteome of a B. subtilis bdbCD mutant strain, which lacks the primary TDOR pair BdbC and BdbD, by gel-free mass spectrometry. In total, 18 membrane-associated proteins showed differing behavior in the bdbCD mutant and the parental strain. These included the ProA protein involved in osmoprotection. Consistent with the absence of ProA, the bdbCD mutant was found to be sensitive to osmotic shock. We hypothesize that membrane proteomics is a potentially effective approach to profile oxidative folding of bacterial membrane proteins. Antioxid. Redox Signal. 18, 1159-1164.</p>

Morphology of N402 (A), ∆<i>flbA</i> (B) and ∆<i>brlA</i> (C) during growth on agar plates.

<p>The wild-type strain N402 forms aerial hyphae and conidiophores with black conidia, clearly visible from the top view (top) and schematic representation (bottom). The mutant strain ∆<i>flbA</i> forms only aerial hyphae that collapse in the colony center. Mutant strain ∆<i>brlA</i> lacks sporulation and forms conidial stalks that extend into the air, as clearly visible in the side view (middle).</p

Enzyme activities detected in culture filtrate of batch cultures of strains N402 (squares), ∆<i>flbA</i> (triangles) and ∆<i>brlA</i> (dots).

<p>Protease activity (A), total hydrolytic activity releasing galactose (B) and glucose (C) from the isolated <i>A</i>. <i>niger</i> cell walls and GH exo-activity releasing N-acetyl-glucosamine from GlcNAc-β-pNP (D). Values are given as mean ± SE of measurements performed for two biological duplicates.</p

Normalized gene expression levels of genes discussed in the manuscript.

<p>Mean gene expression levels given as % of actA expression during exponential growth.</p><p>^a Exp; exponential growth, Day 1,3 or 6: carbon starvation day 1, 3, 6</p><p>^b SP: predicted signal peptide presence (1) or absence (0)</p><p>^c GPI: predicted GPI-anchor presence (1), absence (0) or ambivalence (A)</p><p>Normalized gene expression levels of genes discussed in the manuscript.</p