Hans Christian Beck

Quantitative proteomic analysis of post-translational modifications of human histones.

Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the “histone code” that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys57 from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.

Catabolism of leucine to branched‐chain fatty acids in Staphylococcus xylosus

Aims:  Staphylococcus xylosus is an important starter culture in the production of flavours from the branched‐chain amino acids leucine, valine and isoleucine in fermented meat products. The sensorially most important flavour compounds are the branched‐chain aldehydes and acids derived from the corresponding amino acids and this paper intends to perspectivate these flavour compounds in the context of leucine metabolism.

Novel pyrazine metabolites found in polymyxin biosynthesis by Paenibacillus polymyxa

A complex mixture of methyl-branched alkyl-substituted pyrazines was found in the growth medium of the polymyxin-producing bacterium Paenibacillus polymyxa, and of these, seven are new natural compounds. A total of 19 pyrazine metabolites were identified. The dominant metabolite was 2,5-diisopropylpyrazine as identified using a combination of high-resolution mass spectrometry, (1)H- and (13)C-nuclear magnetic resonance, gas chromatography-mass spectrometry as well as co-elution with an authentic standard. Its biosynthesis was correlated with growth and production was strongly stimulated by valine supplementation. The other pyrazine metabolites, all related pyrazines with either one, two or three alkyl substituents, were identified by means of their mass spectral data and/or co-elution with authentic standards.

Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum

In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Gel bands were excised and in-gel digested with trypsin. The resulting peptides were analysed by capillary-LC-ESI-MS/MS. The peptide sequences were used for a database search and allowed identification of a total of 29 proteins, many of which could potentially be involved in the action of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics.

Proteomic analysis of osteogenic differentiation of dental follicle precursor cells.

Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network of differentiation. In the present study we applied 2‐DE combined with capillary‐LC‐MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B proteins, plastin 3 T‐isoform, beta‐actin, superoxide dismutases, and transgelin were found to be highly up‐regulated, whereas cofilin‐1, pro‐alpha 1 collagen, destrin, prolyl 4‐hydrolase and dihydrolipoamide dehydrogenase were found to be highly down‐regulated. The group of up‐regulated proteins is associated with actin‐bundling and defence against oxidative cellular stress, whereas down‐regulated proteins were associated with collagen biosynthesis. Bioinformatic analyses of the entire data set confirmed these findings that represent significant steps towards the understanding of DFPC differentiation. The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down‐regulated and proteins involved in catabolism, cell motility and biological quality were up‐regulated. These results display the general physiological state of DFPCs before and after osteogenic differentiation. We also identified regulatory proteins, such as the transcription factors TP53 and Sp‐1, associated with the differentiation process. Further studies will investigate the impact of identified regulatory proteins for cell proliferation and osteogenic differentiation in DFPCs.

Cloning and Inactivation of a Branched-Chain-Amino-Acid Aminotransferase Gene from Staphylococcus carnosus and Characterization of the Enzyme

ABSTRACT Staphylococcus carnosus and Staphylococcus xylosus are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the ilvE gene from S. carnosus by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical to that of the corresponding enzyme in Staphylococcus aureus and that the ilvE gene was constitutively expressed as a monocistronic transcript. To study the influence of ilvE on BCAA catabolism, we constructed an ilvE deletion mutant by gene replacement. The IlvE protein from S. carnosus was shown mainly to catalyze the transamination of isoleucine, valine, leucine, and, to some extent, methionine using pyridoxal 5′-phosphate as a coenzyme. The ilvE mutant degraded less than 5% of the BCAAs, while the wild-type strain degraded 75 to 95%. Furthermore, the mutant strain produced approximately 100-fold less of the methyl-branched carboxy acids, 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which derived from the BCAA catabolism, clearly emphasizing the role of IlvE in aroma formation. In contrast to previous reports, we found that IlvE was the only enzyme that catalyzed the deamination of BCAAs in S. carnosus. The ilvE mutant strain showed remarkably lower growth rate and biomass yield compared to those of the wild-type strain when grown in rich medium. Normal growth rate and biomass yield were restored by addition of the three BCAA-derived α-keto acids, showing that degradation products of BCAAs were essential for optimal cell growth.

Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins.

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.

Proteomic Analysis of Human Blastocoel Fluid and Blastocyst Cells

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required on a large scale. In the undifferentiated state, hESCs appear to possess an unlimited potential for proliferation, but optimal, defined, and safe culture conditions remain a challenge. The aim of the present study was to identify proteins in the natural environment of undifferentiated hESCs, namely, the blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass spectrometry, 286 proteins were identified in the blastocoel fluid and 1,307 proteins in the corresponding cells of the blastocyst. Forty-two were previously uncharacterized proteins-8 of these originated from the blastocoel fluid. Furthermore, several heat shock proteins (Hsp27, Hsp60, Hsc70, and Hsp90) were identified in blastocoel fluid together with zona pellucida proteins (ZP2-4), Vitamin D-binding protein, and Retinol-binding protein 4. Proteins that regulate ciliary assembly and function were also identified, including Bardet-Biedl syndrome protein 7. This study has identified numerous proteins that cells from the ICM of the human blastocyst are exposed to via the blastocoel fluid. These results can be an inspiration for the development of improved culture conditions for hESCs.

Evidence of non-canonical NOTCH signaling: Delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals

Canonical NOTCH signaling, known to be essential for tissue development, requires the Delta-Serrate-LAG2 (DSL) domain for NOTCH to interact with its ligand. However, despite lacking DSL, Delta-like 1 homolog (DLK1), a protein that plays a significant role in mammalian development, has been suggested to interact with NOTCH1 and act as an antagonist. This non-canonical interaction is, however controversial, and evidence for a direct interaction, still lacking in mammals. In this study, we elucidated the putative DLK1-NOTCH1 interaction in a mammalian context. Taking a global approach and using Dlk1(+/+) and Dlk1(-/-) mouse tissues at E16.5, we demonstrated that several NOTCH signaling pathways indeed are affected by DLK1 during tissue development, and this was supported by a lower activation of NOTCH1 protein in Dlk1(+/+) embryos. Likewise, but using a distinct Dlk1-manipulated (siRNA) setup in a mammalian cell line, NOTCH signaling was substantially inhibited by DLK1. Using a mammalian two-hybrid system, we firmly established that the effect of DLK1 on NOTCH signaling was due to a direct interaction between DLK1 and NOTCH1. By careful dissection of this mechanism, we found this interaction to occur between EGF domains 5 and 6 of DLK1 and EGF domains 10-15 of NOTCH1. Thus, our data provide the first evidence for a direct interaction between DLK1 and NOTCH1 in mammals, and substantiate that non-canonical NOTCH ligands exist, adding to the complexity of NOTCH signaling.

Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries From Patients With Type 2 Diabetes Mellitus and Lower Levels Among Metformin Users

Background—The increased risk of cardiovascular diseases in type 2 diabetes mellitus has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes mellitus and moreover hypothesized that metformin intake influences the protein composition. Methods and Results—We analyzed nonatherosclerotic repair arteries gathered at coronary bypass operations from 30 patients with type 2 diabetes mellitus and from 30 age- and sex-matched nondiabetic individuals. Quantitative proteome analysis was performed by isobaric tag for relative and absolute quantitation-labeling and liquid chromatography–mass spectrometry, tandem mass spectrometry analysis on individual arterial samples. The amounts of the basement membrane components, &agr;1-type IV collagen and &agr;2-type IV collagen, &ggr;1-laminin and &bgr;2-laminin, were significantly increased in patients with diabetes mellitus. Moreover, the expressions of basement membrane components and other vascular proteins were significantly lower among metformin users when compared with nonusers. Patients treated with or without metformin had similar levels of hemoglobin A1c, cholesterol, and blood pressure. In addition, quantitative histomorphometry showed increased area fractions of collagen-stainable material in tunica intima and media among patients with diabetes mellitus. Conclusions—The distinct accumulation of arterial basement membrane proteins in type 2 diabetes mellitus discloses a similarity between the diabetic macroangiopathy and microangiopathy and suggests a molecular explanation behind the alterations in vascular remodeling, biomechanical properties, and aneurysm formation described in diabetes mellitus. The lower amounts of basement membrane components in metformin-treated individuals are compatible with the hypothesis of direct beneficial drug effects on the matrix composition in the vasculature.