Eukaryote DNA is wrapped around nucleosomes that are comprised of octamers of core histones (two heterodimers of histones H2A and H2B and a heterotetramer of histones H3 and H4). Nucleosomes are joined by linker DNA and histone H1 to form chromatin. A central mechanism for regulating chromatin activity is the (reversible) covalent modification of histones by enzymes. A complex interplay between posttranslational modifications (PTMs) 1 either represses or activates transcription in a site-specific and cooperative manner (1). The NH 2 -and COOH-terminal tails of the core histones are subjected to multisite modifications by methylation, acetylation, phosphorylation, ADP-ribosylation, ubiquitination (2, 3), and sumoylation (4). A "histone code" model has been hypothesized where the cell uses a combinatorial system of (clustered) covalent modifications to regulate specific genomic functions (5-7). The current hypothesis is that the pattern and types of modifications modulate protein-protein and protein-DNA interactions, e.g. for recruitment of transcription factors, as a function of the cellular state and environment (8).Acetylation of lysine residues is a major mediator of the histone code. Hyperacetylation of histone tails is usually a sign of high transcriptional activity, whereas hypoacetylation indicates transcriptional silencing (9). Reversible acetylation/ deacetylation of lysine residues is a dynamic balance between the activities of histone acyltransferases and histone deacetylases (HDACs). Inhibition of HDAC activity results in hyperacetylation of lysine residues in the NH 2 tails of histones H3 and H4 (10), and it leads to the expression of genes that induce growth arrest, cell differentiation, and apoptotic cell death in cultured tumor cells (11,12). Non-toxic levels of several HDACis have shown promising pharmacological properties by inhibiting tumor growth in animal models (13)(14)(15). Consequently there is a great interest in developing HDACis as anticancer drugs, and some of these compounds are currently being evaluated in clinical trails (16,17).Mass spectrometry is rapidly becoming a key analytical technology in molecular cell biology and proteomics. Mass spectrometry is particularly suited for the examination of protein primary structure and for determining post-translational modifications of proteins as it reveals covalent modifications From the ‡Danish Technological Institute,

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. Methods and Results: GC and GC/MS analysis combined with stable isotope labelling was used to study leucine catabolism. This amino acid together with valine and isoleucine was used as precursors for the production of branched-chain fatty acids for cell membrane biosynthesis during growth. A 83AE3% of the cellular fatty acids were branched. The dominating fatty acid was anteiso-C 15:0 that constituted 55% of the fatty acids. A pyridoxal 5¢-phosphate and a-ketoacid dependent reaction catalysed the deamination of leucine, valine and isoleucine into their corresponding a-ketoacids. As a-amino group acceptor a-keto-b-methylvaleric acid and a-ketoisovaleric acid was much more efficient than a-ketoglutarate. The sensorially and metabolic key intermediate on the pathway to the branched-chain fatty acids, 3-methylbutanoic acid was produced from leucine at the onset of the stationary growth phase and then, when the growth medium became scarce in leucine, from the oxidation of glucose via pyruvate. Conclusions: This paper demonstrates that the sensorially important branched-chain aldehydes and acids are important intermediates on the metabolic route leading to branched-chain fatty acids for cell membrane biosynthesis. Significance and Impact of the Study: The metabolic information obtained is extremely important in connection with a future biotechnological design of starter cultures for production of fermented meat.

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.

Triple-negative breast cancer (TNBC) is a heterogeneous subtype with varying disease outcomes. Tumor-infiltrating lymphocytes (TILs) are frequent in TNBC and have been shown to correlate with outcome, suggesting an immunogenic component in this subtype. However, other factors intrinsic to the cancer cells may also influence outcome. To identify proteins and molecular pathways associated with recurrence in TNBC, 34 formalin-fixed paraffin-embedded (FFPE) primary TNBC tumors were investigated by global proteomic profiling using mass spectrometry. Approximately, half of the patients were lymph node-negative and remained free of local or distant metastasis within 10 y follow-up, while the other half developed distant metastasis. Proteomic profiling identified >4,000 proteins, of which 63 exhibited altered expression in primary tumors of recurrence versus recurrence-free patients. Importantly, downregulation of proteins in the major histocompatibility complex (MHC) class I antigen presentation pathways were enriched, including TAP1, TAP2, CALR, HLA-A, ERAP1 and TAPBP, and were associated with significantly shorter recurrence-free and overall survival. In addition, proteins involved in cancer cell proliferation and growth, including GBP1, RAD23B, WARS and STAT1, also exhibited altered expression in primary tumors of recurrence versus recurrence-free patients. The association between the antigen-presentation pathway and outcome were validated in a second sample set of 10 primary TNBC tumors and corresponding metastases using proteomics and in a large public gene expression database of 249 TNBC and 580 basal-like breast cancer cases. Our study demonstrates that downregulation of antigen presentation is a key mechanism for TNBC cells to avoid immune surveillance, allowing continued growth and spread.

Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.