Structural basis of phosphatidylinositol 3-kinase C2α function.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis, angiogenesis, viral replication, platelet formation and a role in mitosis. The molecular basis of these activities of PI3KC2α is poorly understood. Here, we report high-resolution crystal structures as well as a 4.

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi.

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding.

A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide.

Cyclotriazadisulfonamide (CADA) inhibits the cotranslational translocation of type I integral membrane protein human CD4 (huCD4) across the endoplasmic reticulum in a signal peptide (SP)-dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared with huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA.

The glucose-sensing transcription factor ChREBP is targeted by proline hydroxylation.

Cellular energy demands are met by uptake and metabolism of nutrients like glucose. The principal transcriptional regulator for adapting glycolytic flux and downstream pathways like lipogenesis to glucose availability in many cell types is carbohydrate response element-binding protein (ChREBP). ChREBP is activated by glucose metabolites and post-translational modifications, inducing nuclear accumulation and regulation of target genes.

Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs.

Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking , , and genes, either individually or in combination.

Mussel-Inspired Polymerization of Peptides: The Chemical Activation Route as Key to Broaden the Sequential Space of Artificial Mussel-Glue Proteins.

A previously introduced tyrosinase-activated polymerization of Tyr- and Cys-bearing peptides yielding artificial mussel-glue proteins is realized without the need of the specific enzyme by a chemical activation route. This decouples the sequence of polymerizable peptides (unimers) from the constraints of tyrosinase substrates and enables the polymerization of minimal motifs such as Dopa-Lys-Cys (U ) or Dopa-Gly-Cys (U ). In the polymerization procedure, sodium periodate is used to oxidize Dopa residues of the unimers to Dopa-quinones to which the thiol of a Cys residue is added in a Michael-type reaction.

Fish and Clips: A Convenient Strategy to Identify Tyrosinase Substrates with Rapid Activation Behavior for Materials Science Applications.

Peptides with suitable substrate properties for a specific tyrosinase are selected by combinatorial means from a one-bead-one-compound (OBOC) peptide library. The identified sequences exhibit tyrosine residues that are rapidly oxidized to 3,4-dihydroxyphenylalanine (Dopa), making the peptides interesting for enzyme-activated adhesives. The selection process of peptides involves tyrosinase oxidation of tyrosine-bearing sequences on a solid support, yielding dopaquinone residues (fish from the sequence pool), to which thiol-functional fluorescent probes attach by Michael-reaction (clip to mark).

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results.

Identification of functional lipid metabolism biomarkers of brown adipose tissue aging.

Objective: Aging is accompanied by loss of brown adipocytes and a decline in their thermogenic potential, which may exacerbate the development of adiposity and other metabolic disorders. Presently, only limited evidence exists describing the molecular alterations leading to impaired brown adipogenesis with aging and the contribution of these processes to changes of systemic energy metabolism.

Methods: Samples of young and aged murine brown and white adipose tissue were used to compare age-related changes of brown adipogenic gene expression and thermogenesis-related lipid mobilization.

Structural changes of TasA in biofilm formation of .

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography.

Oxidative inactivation of the endogenous antioxidant protein DJ-1 by the food contaminants 3-MCPD and 2-MCPD.

3-Chloro-1,2-propanediol (3-MCPD) and 2-chloro-1,3-propanediol (2-MCPD) are heat-induced food contaminants being present either as free substances or as fatty acid esters in numerous foods. 3-MCPD was classified to be possibly carcinogenic to humans (category 2B) with kidney and testis being the primary target organs according to animal studies. A previous 28-day oral feeding study with rats revealed that the endogenous antioxidant protein DJ-1 was strongly deregulated at the protein level in kidney, liver, and testis of the experimental animals that had been treated either with 3-MCPD, 2-MCPD or their dipalmitate esters.

Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells.

Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/βC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry.

A Complex of Htm1 and the Oxidoreductase Pdi1 Accelerates Degradation of Misfolded Glycoproteins.

A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal.

SORLA regulates calpain-dependent degradation of synapsin.

Introduction: Sorting-related receptor with A-type repeats (SORLA) is an intracellular sorting receptor in neurons and a major risk factor for Alzheimer disease.

Methods: Here, we performed global proteome analyses in the brain of SORLA-deficient mice followed by biochemical and histopathologic studies to identify novel neuronal pathways affected by receptor dysfunction.

Results: We demonstrate that the lack of SORLA results in accumulation of phosphorylated synapsins in cortex and hippocampus.

Glycoprotein B of equine herpesvirus type 1 has two recognition sites for subtilisin-like proteases that are cleaved by furin.

Glycoprotein B (gB) of equine herpesvirus type 1 (EHV-1) is predicted to be cleaved by furin in a fashion similar to that of related herpesviruses. To investigate the contribution of furin-mediated gB cleavage to EHV-1 growth, canonical furin cleavage sites were mutated. Western blot analysis of mutated EHV-1 gB showed that it was cleaved at two positions, 518RRRR521 and 544RLHK547, and that the 28 aa between the two sites were removed after cleavage.

In-cell NMR in mammalian cells: part 3.

Irrespective of how isotope-labeled proteins are delivered into mammalian cells, laboratory routines are needed to assess the quality of the resulting in-cell NMR samples. These include methods to evaluate overall cell viability, protein transduction efficiency, intracellular protein concentration, localization, and stability. In addition, quality control experiments to assess protein leakage from manipulated cells are of particular importance for in-cell NMR experiments.

Improved two-dimensional reversed phase-reversed phase LC-MS/MS approach for identification of peptide-protein interactions.

Quantitative mass spectrometry (MS) in combination with affinity purification approaches allows for an unbiased study of protein-protein and peptide-protein interactions. In shotgun approaches that are based on proteolytic digestion of complex protein mixtures followed by two-dimensional liquid-phase chromatography, the separation effort prior to MS analysis is focused on tryptic peptides. Here we developed an improved offline 2-D liquid chromatography-MS/MS approach for the identification and quantification of binding proteins utilizing reversed-phase capillary columns with acidic acetonitrile-containing eluents in both chromatographic dimensions.

Effects of thawing, refreezing and storage conditions of tissue samples and protein extracts on 2-DE spot intensity.

We report that reliable quantitative proteome analyses can be performed with tissue samples stored at -80 degrees C for up to 10 years. However, storing protein extracts at 4 degrees C for 24 h and freezing protein extracts at -80 degrees C and thawing them significantly altered 41.6 and 17.

In-Gel 18O labeling for improved identification of proteins from 2-DE Gel spots in comparative proteomic experiments.

The reliability of 2-DE gel-based comparative proteomics is severely impaired by the potential presence of overlapping proteins. We describe a methodological procedure which may solve this problem. Corresponding protein spots from two experimental groups are digested in the presence of 16O and 18O, respectively.

The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry.

Qualitative and quantitative analysis of post-translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well-characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined.

The whereabouts of transmembrane proteins from rat brain synaptosomes during two-dimensional gel electrophoresis.

Little is known about what happens to transmembrane proteins (TMP) in 2-DE. In order to obtain more insight into the whereabouts of these proteins we prepared membrane-enriched synaptosomes from rat frontal cortex and washed them with 7 M urea or Na(2)CO(3). From each preparation, 200 microg protein was loaded on 2-DE gels covering the 4-7 and 6-11 pH ranges, respectively.

Development of a hyphenated microanalytical system for the investigation of leaching kinetics of heavy metals in environmental samples.

A fast and convenient method for on-line monitoring of the extraction of heavy metals from solid (environmental) matrixes was developed. By the incorporation of microcartridges filled with dried and pulverized solid samples into the conduits of a flow system and appropriate selection of the liquid flowing through the cartridge, information about the degree of leaching and in particular of the kinetics of the leaching process are obtained. The method was optimized for determination of different metals of environmental concern using in-line detection by FAAS and ICPMS.