Pressure response of amide one-bond J-couplings in model peptides and proteins

The pressure dependence of the one-bond indirect spin–spin coupling constants ¹ J _N–H was studied in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH₂ (with Xxx being one of the 20 proteinogenic amino acids). The response of the ¹ J _N–H coupling constants is amino acid type specific, with an average increase of its magnitude by 0.6 Hz at 200 MPa. The variance of the pressure response is rather large, the largest pressure effect is observed for asparagine where the coupling constant becomes more negative by ?2.9 Hz at 200 MPa. The size of the J-coupling constant at high pressure is positively correlated with its low pressure value and the β-propensity, and negatively correlated with the amide proton shift and the first order nitrogen pressure coefficient and the electrostatic solvation free energy.     

Aryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitors

Synthesis and SAR are described for a structurally distinct class of DPP–IV inhibitors based on aminobenzo[a]quinolizines bearing (hetero-)aromatic substituents in the S1 specificity pocket. The m-(fluoromethyl)-phenyl derivative (S,S,S)-2g possesses the best fit in the S1 pocket. However, (S,S,S)-2i, bearing a more hydrophilic 5-methyl-pyridin-2-yl residue as substituent for the S1 pocket, displays excellent in vivo activity and superior drug-like properties.     

Genetic Dissection of Differential Signaling Threshold Requirements for the Wnt/β-Catenin Pathway In Vivo

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/β-catenin pathway, we challenged the allele combinations by genetically restricting intracellular β-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/β-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/β-catenin signaling capacity similar to that in the germline of the Apc^min mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc^min mice arise independently of intestinal tumorigenesis. Together, the present genotype–phenotype analysis suggests tissue-specific response levels for the Wnt/β-catenin pathway that regulate both physiological and pathophysiological conditions.     

Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics

Proteomic analyses of different subcellular compartments, so-called organellar proteomics, facilitate the understanding of cellular functions on a molecular level. In this work, various orthogonal multidimensional separation techniques both on the protein and on the peptide level are compared with regard to the number of identified proteins as well as the classes of proteins accessible by the respective methodology. The most complete overview was achieved by a combination of such orthogonal techniques as shown by the analysis of the yeast mitochondrial proteome. A total of 851 different proteins (PROMITO dataset) were identified by use of multidimensional LC-MS/MS, 1D-SDS-PAGE combined with nano-LC-MS/MS and 2D-PAGE with subsequent MALDI-mass fingerprinting. Our PROMITO approach identified the 749 proteins, which were found in the largest previous study on the yeast mitochondrial proteome, and additionally 102 proteins including 42 open reading frames with unknown function, providing the basis for a more detailed elucidation of mitochondrial processes. Comparison of the different approaches emphasizes a bias of 2D-PAGE against proteins with very high isoelectric points as well as large and hydrophobic proteins, which can be accessed more appropriately by the other methods. While 2D-PAGE has advantages in the possible separation of protein isoforms and quantitative differential profiling, 1D-SDS-PAGE with nano-LC-MS/MS and multidimensional LC-MS/MS are better suited for efficient protein identification as they are less biased against distinct classes of proteins. Thus, comprehensive proteome analyses can only be realized by a combination of such orthogonal approaches, leading to the largest dataset available for the mitochondrial proteome of yeast.     

Genome sequence of Aeromonas hydrophila ATCC 7966T: jack of all trades

The complete genome of Aeromonas hydrophila ATCC 7966(T) was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966(T). Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments.     

Applications of highly sensitive phosphopeptide derivatization methods without the need for organic solvents

Detection and identification of phosphorylated amino acid residues is still one of the most challenging tasks in contemporary protein analysis. Especially serine- and threonine-phosphorylation is very often involved in the regulation of enzyme activities and regulates key functions in cellular processes.     

Human acid sphingomyelinase.

Human acid sphingomyelinase (haSMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to ceramide and phosphorylcholine. An inherited haSMase deficiency leads to Niemann-Pick disease, a severe sphingolipid storage disorder. The enzyme was purified and cloned over 10 years ago. Since then, only a few structural properties of haSMase have been elucidated. For understanding of its complex functions including its role in certain signaling and apoptosis events, complete structural information about the enzyme is necessary. Here, the identification of the disulfide bond pattern of haSMase is reported for the first time. Functional recombinant enzyme expressed in SF21 cells using the baculovirus expression system was purified and digested by trypsin. MALDI-MS analysis of the resulting peptides revealed the four disulfide bonds Cys120-Cys131, Cys385-Cys431, Cys584-Cys588 and Cys594-Cys607. Two additional disulfide bonds (Cys221-Cys226 and Cys227-Cys250) which were not directly accessible by tryptic cleavage, were identified by a combination of a method of partial reduction and MALDI-PSD analysis. In the sphingolipid activator protein (SAP)-homologous N-terminal domain of haSMase, one disulfide bond was assigned as Cys120-Cys131. The existence of two additional disulfide bridges in this region was proved, as was expected for the known disulfide bond pattern of SAP-type domains. These results support the hypothesis that haSMase possesses an intramolecular SAP-type activator domain as predicted by sequence comparison [Ponting, C.P. (1994) Protein Sci., 3, 359-361]. An additional analysis of haSMase isolated from human placenta shows that the recombinant and the native human protein possess an identical disulfide structure.