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The food protein ingredient market is dominated by dairy and egg proteins. Both milk whey and egg proteins are challenging proteins to replace, e.g. with plant proteins, due to the unique structural features of the animal proteins that render them highly functional. Thus, to provide a non-animal source of these important proteins the fungal host Trichoderma reesei was utilized for the biotechnical production of recombinant hen ovalbumin (TrOVA) and bovine beta lactoglobulin (TrBLG). These food proteins were investigated using two different promoter systems to test the concept of effectively expressing them in a fungal host. Both proteins were successfully produced in 24 well plate and bioreactor scale. The production level of TrBLG and TrOVA were 1 g/L and 2 g/L, respectively. Both proteins were further purified and characterized, and their functional properties were tested. TrBLG and TrOVA secondary structures determined by circular dichroism corresponded to the proteins of bovine and hen. The T. reesei produced proteins were found to be N-glycosylated, mostly with Man 5. TrBLG had emulsification properties matching to corresponding bovine protein. TrOVA showed excellent foaming characteristics and heat-induced gelation, although the strength of the gel was somewhat lower than with hen ovalbumin, possibly due to the partial degradation of TrOVA or presence of other host proteins. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming.Industrial relevance: The food protein ingredient market is dominated by dairy (largely whey proteins) and egg proteins. Whey proteins are valuable and versatile food ingredients due to their functional and nutritional quality. They are largely used in meat and milk products, low fat products, bakery, confectionary, infant formulas and sports nutrition. Similarly, egg white protein ovalbumin is a highly functional protein ingredient that facilitates structure formation and high nutritional quality in most food products. Together they comprise 40 - 70% of the revenue in the animal protein ingredients market. Both whey and egg proteins are extremely challenging proteins to replace, e.g., by plant proteins due to their unique structural features that render them with high functionality. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming.

In vitro studies on HIV (HIV-1) replication and neutralization are usually performed in human cell cultures supplemented with FBS instead of human serum (HS). Here we show that in contrast to FBS, addition of increasing amounts of human serum from noninfected donors to the cell culture directly correlates with an increase in HIV-1 replication in vitro. This effect is independent of cell line, virus strain, or batch of pooled human serum used. We found that human serum affects viral transcription in a dose-dependent manner by activating the activator protein-1 (AP-1) member proteins c-FOS, JunD, and JunB in TZM-bl cells. Analysis of the human serum component responsible for this effect indicates that it is a protein having a molecular mass between 250 and 300 kDa. This serum protein, HIV-1 enhancing serum protein (HESP), might promote viral transcription in vivo and consequently play a role in disease progression.

In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.

We present a versatile user-friendly software tool, PolyAlign, for the alignment of multiple LC-MS signal maps with the option of manual landmark setting or automated alignment. One of the spectral images is selected as a reference map, and after manually setting the landmarks, the program warps the images using either polynomial or Hermite transformation. The software provides an option for automated landmark finding. The software includes a very fast zoom-in function synchronized between the images, which facilitate detecting correspondences between the adjacent images. Such an interactive visual process enables the analyst to decide when the alignment is satisfactory and to correct known irregularities. We demonstrate that the software provides significant improvements in the alignment of LC-MALDI data, with 10–15 landmark pairs, and it is also applicable to correcting electrospray LC-MS data. The results with practical data show substantial improvement in peak alignment compared to MZmine, which was among the best analysis packages in a recent assessment. The PolyAlign software is freely available and easily accessible as an integrated component of the popular MZmine software, and also as a simpler stand-alone Perl implementation to preview data and apply landmark directed polynomial transformation.

The aggregation of full-length (residues 1-40) amyloid beta-peptide (A beta) and fragments corresponding to residues 1-23 and 24-40 was studied by electrospray mass spectrometry, using gramicidin as a non-aggregating reference. Following a lag period, A beta(1-40) at 140 microM concentration aggregates with apparent first-order kinetics. Under acidic conditions A beta(1-40) undergoes spontaneous cleavage between Asp23-Val24 and to a lesser extent also at two other Asp-X motifs. Incubation in acidic H(2)18O showed incorporation of 18O in fragment A beta(1-23), confirming that the Asp23-Val24 peptide bond had been hydrolyzed. Incubation of synthetic A beta(1-23) and A beta(24-40) peptides with A beta(1-40) showed that A beta(24-40) remained in solution for several months, that A beta(1-23) partly disappeared from solution, whereas A beta(1-40) completely disappeared. Further, treatment of sedimentable aggregates formed after co-incubation of the three peptides with hexafluoro-2-propanol or formic acid recovered the intensity of A beta(1-40). These data support previous studies showing that the region of A beta encompassing residues 16-24 is necessary for aggregation into amyloid fibrils.

The tetrapeptide KFFE is one of the shortest amyloid fibril-forming peptides described. Herein, we have investigated how the structural environment of this motif affects polymerization. Using a turn motif (YNGK) or a less rigid sequence (AAAK) to fuse two KFFE tetrapeptides, we show by several biophysical methods that the amyloidogenic properties are strongly dependent on the structural environment. The dodecapeptide KFFEAAAKKFFE forms abundant thick fibril bundles. Freshly dissolved KFFEAAAKKFFE is monomeric and shows mainly disordered secondary structure, as evidenced by circular dichroism, NMR spectroscopy, hydrogen/deuterium exchange measurements, and molecular modeling studies. In sharp contrast, the dodecapeptide KFFEYNGKKFFE does not form fibrils but folds into a stable beta-hairpin. This structure can oligomerize into a stable 12-mer and multiples thereof, as shown by size exclusion chromatography, sedimentation analysis, and electrospray mass spectrometry. These data indicate that the structural context in which a potential fibril forming sequence is present can prevent fibril formation by favoring self-limiting oligomerization over polymerization.

The lung surfactant-associated protein C (SP-C) consists mainly of a polyvaline α-helix, which is stable in a lipid membrane. However, in agreement with the predicted β-strand conformation of a polyvaline segment, helical SP-C unfolds and transforms into β-sheet aggregates and amyloid fibrils within a few days in aqueous organic solvents. SP-C fibril formation and aggregation have been associated with lung disease. Here, we show that in a recently isolated biosynthetic precursor of SP-C (SP-Ci), a 12 residue N-terminal propeptide locks the metastable polyvaline part in a helical conformation. The SP-Ci helix does not aggregate or unfold during several weeks of incubation, as judged by hydrogen/deuterium exchange and mass spectrometry. Hydrogen/deuterium exchange experiments further indicate that the propeptide reduces exchange in parts corresponding to mature SP-C. Finally, in an acidic environment, SP-Ci unfolds and aggregates into amyloid fibrils like SP-C. These data suggest a direct role of the N-terminal propeptide in SP-C biosynthesis.

Amyloid fibrils in which specific proteins have polymerized into a cross-beta-sheet structure are found in about 20 diseases. In contrast to the close structural similarity of fibrils formed in different amyloid diseases, the structures of the corresponding native proteins differ widely. We show here that peptides as short as 4 residues with the sequences KFFE or KVVE can form amyloid fibrils that are practically identical to fibrils formed in association with disease, as judged by electron microscopy and Congo red staining. In contrast, KLLE or KAAE do not form fibrils. The fibril-forming KFFE and KVVE show partial beta-strand conformation in solution, whereas the non-fibril-forming KLLE and KAAE show random structure only, suggesting that inherent propensity for beta-strand conformation promotes fibril formation. The peptides KFFK or EFFE do not form fibrils on their own but do so in an equimolar mixture. Thus, intermolecular electrostatic interactions, either between charged dipolar peptides or between complementary charges of co-fibrillating peptides favor fibril formation.