Probing the structure of the infectious amyloid form of the prion-forming domain of HET-s using high resolution hydrogen/deuterium exchange monitored by mass spectrometry

The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218-289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218-289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218-289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218-289) sequence. The HET-s-(218-289) fibrils are thus likely to be organized as a succession of beta-sheet segments interrupted by short turns or short loops.     

Structural analysis of deacylated lipopolysaccharide of Escherichia coli strains 2513 (R4 core-type) and F653 (R3 core-type).

Lipopolysaccharide (LPS) of Escherichia coli strain 2513 (R4 core-type) yielded after alkaline deacylation one major oligosaccharide by high-performance anion-exchange chromatography (HPAEC) which had a molecular mass of 2486.59 Da as determined by electrospray ionization mass spectrometry. This was in accordance with the calculated molecular mass of a tetraphosphorylated dodecasaccharide of the composition shown below. NMR-analyses identified the chemical structure as where l-alpha-d-Hep is l-glycero-alpha-d-manno-heptopyranose and Kdo is 3-deoxy-alpha-d-manno-oct-2-ulopyranosylonic acid and all hexoses are present as d-pyranoses. We have also isolated the complete core-oligosaccharides of E. coli F653 LPS for which only preliminary data were available and investigated the deacylated LPS by NMR and MS. The proposed structure determined previously by methylation analysis was confirmed and is shown below. In addition we have quantified the side-chain heptose substitution of the inner core with GlcpN ( approximately 30%) and confirmed that this sugar is only present when the phosphate at the second l,d-Hepp residue is absent.     

Characterization of very high gravity ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-saccharification and yeast strain

Ethanol was produced from very high gravity mashes of dry milled corn (35% w/w total dry matter) under simultaneous saccharification and fermentation conditions. The effects of glucoamylase dosage, pre-saccharification and Saccharomyces cerevisiae strain on the growth characteristics such as the ethanol yield and volumetric and specific productivity were determined. It was shown that higher glucoamylase doses and/or pre-saccharification accelerated the simultaneous saccharification and fermentation process and increased the final ethanol concentration from 106 to 126 g/kg although the maximal specific growth rate was decreased. Ethanol production was not only growth related, as more than half of the total saccharides were consumed and more than half of the ethanol was produced during the stationary phase. Furthermore, a high stress tolerance of the applied yeast strain was found to be crucial for the outcome of the fermentation process, both with regard to residual saccharides and final ethanol concentration. The increased formation of cell mass when a well-suited strain was applied increased the final ethanol concentration, since a more complete fermentation was achieved.     

Alternative protein secretion: the Mam1 ABC transporter supports secretion of M-factor linked GFP in fission yeast

To examine whether the fission yeast Mam1 ABC transporter can be used for secretion of heterologous proteins, thereby bypassing the classical secretion pathway, we have analyzed chimeric forms of the M-factor precursor. It was demonstrated that GFP can be exported when fused to both the amino-terminal prosequence from mfm1 and a CaaX motif. This secretion was dependent on the Mam1 transporter and not the classical secretion pathway. The secretion efficiency of GFP, however, was relatively low and most of the reporter protein was trapped in the vacuolar membranes. Our findings suggest that the Mam1 ABC protein is a promiscuous peptide transporter that can accommodate globular proteins of a relatively large size. Furthermore, our results help in defining the sequences required for processing and secretion of natural M-factor.     

Non-mendelian inheritance of the HET-s prion or HET-s prion domains determines the het-S spore killing system in Podospora anserina

Two alleles of the het-s/S locus occur naturally in the filamentous fungus Podospora anserina, het-s and het-S. The het-s encoded protein can form a prion that propagates a self-perpetuating amyloid aggregate, resulting in two phenotypes for the het-s strains. The prion-infected [Het-s] shows an antagonistic interaction to het-S whereas the prion-free [Het-s*] is neutral in interaction to het-S. The antagonism between [Het-s] and het-S is seen as heterokaryon incompatibility at the somatic level and as het-S spore killing in the sexual cycle. Two different domains of the HET-s and HET-S proteins have been identified, and a structure-function relationship has been established for interactions at the somatic level. In this study, we correlate accumulation of the HET-s and HET-S proteins (visualized using GFP) during the sexual cycle with timing of het-S spore abortion. Also, we present the structure-function relationship of the HET-s domains for interactions in the sexual cycle. We show that the constructs that ensure het-s incompatibility function in somatic mycelium are also active in het-S spore killing in the sexual cycle. In addition, paternal prion transmission and het-S spore killing has been found with the HET-s(157-289) truncated protein. The consequences of the unique transition from a coenocytic to a cellular state in the sexual phase and the timing, and localization of paternal and maternal HET-s and HET-S expression that are pertinent to prion transmission, and het-S spore killing are elaborated. These data further support our previously proposed model for het-S spore killing.     

Detection of low level genomic alterations by comparative genomic hybridization based on cDNA micro-arrays

MOTIVATION: The accumulation of genomic alterations is an important process in tumor formation and progression. Comparative genomic hybridization performed on cDNA arrays (cDNA aCGH) is a common method to investigate the genomic alterations on a genome-wide scale. However, when detecting low-level DNA copy number changes this technology requires the use of noise reduction strategies due to a low signal to noise ratio. RESULTS: Currently a running average smoothing filter is the most frequently used noise reduction strategy. We analyzed this strategy theoretically and experimentally and found that it is not sensitive to very low level genomic alterations. The presence of systematic errors in the data is one of the main reasons for this failure. We developed a novel algorithm which efficiently reduces systematic noise and allows for the detection of low-level genomic alterations. The algorithm is based on comparison of the biological relevant data to data from so-called self-self hybridizations, additional experiments which contain no biological information but contain systematic errors. We find that with our algorithm the effective resolution for +/-1 DNA copy number changes is about 2 Mb. For copy number changes larger than three the effective resolution is on the level of single genes.     

Advances in biocatalytic synthesis of pharmaceutical intermediates

Biocatalytic synthesis of enantiomerically pure compounds for pharmaceutical intermediates is gaining momentum. This is the result of advances in genomics, screening and evolution technologies leading to the increased availability of new and robust biocatalysts suited for industrial-scale application, and is stimulated by an increased demand for catalysts that are able to address the increased complexity of active pharmaceutical ingredients. The vast majority of biotransformation reactions for the manufacturing of optically active pharmaceutical intermediates are still based on enantioselective ketone reductions and enantiospecific hydrolyses. This review aims to point at alternative reaction types and integrated multi-enzymatic steps that are emerging in large-scale applications.